self antigens. Though there are more than just this negative selection…" /> self antigens. Though there are more than just this negative selection…" />
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Autoimmunity - negative selection of T-cells - APC

Autoimmunity - negative selection of T-cells - APC



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Through negative selection of T-cell in the thymus T-cells lose the possibility to react to antigens which are "body own" -> self antigens. Though there are more than just this negative selection for preventing T-cells recognizing own antigens some T-cell become self reacting. This T-cell in our case cd4 T-cells can recognize antigens presented on MHC II complexes by APC cells.

And the question is:

Who presents the self-antigens to the wrong selected CD4 T-cell, which leads to autoimmunity? How does the APC cell get this self-antigen in the first place?


Since you already know that the repertoire of T cell receptors is developed in the thymus, the missing half of the story in the context of antigen-presenting cells is that MHC complexes do not discriminate between self, foreign and mutated self peptides. The take home point is that selection in the thymus is meant to produce a repertoire of T cells which can overcome this fact.

In all healthy tissues, nucleated cells express class I MHC molecules loaded with self-peptides. A T cell probably isn't going to respond to it, however, because trafficking of T cells into tissues requires signals such as cytokines and chemokines. The T cells circulate to and from the lymph node T cell zones.

In the context of inflammation and cellular injury, self proteins may be released into the extracellular space where they're regularly cleared by macrophages via endocytosis. The extrinsic antigen presentation pathway loads peptides onto class II MHC molecules. In this very context, T cells are trafficked into distressed tissues by activated endothelium, where they interact with tissue APCs. Autoreactive clones that escaped thymic selection have the opportunity, then, to react to self antigens, ignoring regulatory or suppressor cells (ref).

Not just self-peptides, however, but also potential self-antigen mimics that take well meaning T cells directed against bacterial peptides, which happen to closely resemble a self antigen, and pit them against your own tissues (example). Unlike autoimmune T cells that have escaped selection and attack your cells, this invites T cells that have gone through the proper channels to do so as well.

See Cellular and Molecular Immunology, 8th ed. for an even better overview of T cell-based autoimmunity.


Immune homeostasis enforced by co-localized effector and regulatory T cells

FOXP3(+) regulatory T cells (Treg cells) prevent autoimmunity by limiting the effector activity of T cells that have escaped thymic negative selection or peripheral inactivation. Despite the information available about molecular factors mediating the suppressive function of Treg cells, the relevant cellular events in intact tissues remain largely unexplored, and whether Treg cells prevent activation of self-specific T cells or primarily limit damage from such cells has not been determined. Here we use multiplex, quantitative imaging in mice to show that, within secondary lymphoid tissues, highly suppressive Treg cells expressing phosphorylated STAT5 exist in discrete clusters with rare IL-2-positive T cells that are activated by self-antigens. This local IL-2 induction of STAT5 phosphorylation in Treg cells is part of a feedback circuit that limits further autoimmune responses. Inducible ablation of T cell receptor expression by Treg cells reduces their regulatory capacity and disrupts their localization in clusters, resulting in uncontrolled effector T cell responses. Our data thus reveal that autoreactive T cells are activated to cytokine production on a regular basis, with physically co-clustering T cell receptor-stimulated Treg cells responding in a negative feedback manner to suppress incipient autoimmunity and maintain immune homeostasis.

Figures

Extended Data Figure 1. Role of IL-2…

Extended Data Figure 1. Role of IL-2 in induction of pSTAT5 in Tregs

Extended Data Figure 2. Spatial correlation between…

Extended Data Figure 2. Spatial correlation between IL-2 + cells and pSTAT5 + Treg clusters

Extended Data Figure 3. Phenotypic characterization of…

Extended Data Figure 3. Phenotypic characterization of IL-2-producing cells in the steady state in IL-2…

Extended Data Figure 4. Histo-cytometry analysis of…

Extended Data Figure 4. Histo-cytometry analysis of DC subsets associated with Treg clusters

Extended Data Figure 5. CD86 expression on…

Extended Data Figure 5. CD86 expression on DCs associated with Treg clusters

Extended Data Figure 6. The correlation between…

Extended Data Figure 6. The correlation between CD73/CTLA-4 expression on Tregs and their distance to…

Extended Data Figure 7. Role of TCR…

Extended Data Figure 7. Role of TCR signaling in high expression of CD73 and CTLA-4…


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T-lymphocytes (T cells)

Ontogeny

The process of development and maturation of the T Cells in mammals begins with the haematopoietic stem cells (HSC) in the fetal liver and later in the bone marrow where HSC differentiate into multipotent progenitors. A subset of multipotent progenitors initiates the transcription of recombination activating gene 1 and 2 (RAG 1 and RAG2) and become lymphoid-primed multipotent progenitors and then common lymphoid progenitors (CLP). Only a small subset of pluripotent cells migrates to the thymus and differentiates into early thymic progenitors (ETP). The thymus does not contain self-renewing progenitors and therefore, long-term thymopoiesis depends on the recruitment of thymus-settling progenitors throughout the life of the individual (1). These progenitors must enter the thymus to become gradually reprogrammed into fully mature and functional T Cells. The T Cell’s distinct developmental steps, as illustrated in Figure 1, are coordinated with the migration of the developing thymocytes towards specific niches in the thymus that provide the necessary stage-specific factors that are needed for further differentiation.

Figure 1

Overview of T Cell development and maturation. Adapted from Rothenberg et al. (4). Abbreviations. HSC: Haematopoietic stem cells, CLP: Common lymphoid progenitors, ETP: Early thymic progenitors, DN: Double negative DP: Double positive, SP: Single positive, (more. )

The ETP are multipotent and can generate T Cells, B Cells, Natural killer cells (NK), myeloid cells, and dendritic cells (DC). ETP represent a small and heterogenous subset, have the ability to proliferate massively, and can be identified by the phenotype Lin low , CD25 − , Kit high as well as by their expression of Flt3, CD24, and CCR9 (1). These cells, which are attracted by the chemokines CCL19 and CCL21, enter the thymus via the corticomedullar junction. In the stroma of the thymus, the ETP encounter a large number of ligands for the Notch receptors as well as growth factors such as Kit-ligand and IL-7 which trigger and support the differentiation and proliferation of these cells in the initial stages of T Cell development (2). Moreover, the expression of Notch-1 receptors and their interaction with Delta-like ligands is essential for the differentiation of the T Cells in the thymus and for the inhibition of the non-T Cell lineage development (3).

Within the thymic cortex, ETP differentiate into double negative (DN) cells that do not express either CD4 or CD8 (i.e., CD4 − and CD8 − ). Some authors consider the ETP a DN1 cell that later differentiates into DN2 when it acquires the CD25 + and CD44 + receptors. At this stage of development, the cells lose the B potential and begin to express proteins that are critical for the subsequent T Cell receptor (TCR) gene rearrangement such as RAG1 and RAG2. They also begin to express proteins necessary for TCR assembly and signaling as CD3 chains, kinases, and phosphatases such as LCK, ZAP70, and LAT (4). DN3 cells can take two divergent routes of differentiation. A cell can either express the αβ chains of the TCR and follow the process of selection to generate CD4 + or CD8 + T Cells or express the γδ chains to generate a subpopulation of γδ lymphocytes with special functional characteristics (5,6) (Table 1).

Table 1

Characteristics of αβ T cells and γδ T cells.

The expression of the β chain of TCR, at the DN3 stage, cascades the simultaneous expression of the CD4 and CD8 molecules and thus, the cells convert into double positives (DP), which constitutes the largest population of cells in the thymus (4,7). At this stage of maturation, the DP cells enter a control point known as positive selection to select the cells with functional TCRs that bind to self-peptides with intermediate affinity and avidity. For this, the epithelial cells of the thymic cortex “put the DP cells to the test” by presenting their own peptides in the context of the class I (HLA-I) and class II (HLA-II) HLA molecules. Only a fraction (1%-5%) of the DP cells, that express a TCR with intermediate affinity for these Ags persists by survival signals. DP cells incapable of binding HLA-I or HLA-II undergo apoptosis. Positive selection allows the differentiation of the DP thymocytes towards a single positive (SP) population that is restricted to HLA (i.e., DP cells that recognize HLA-I differentiate into CD4 − CD8 + and those that recognize HLA-II differentiate into CD4 + CD8 − ) (8, 9). Subsequently, SP cells enter the medulla of the thymus where a second control point known as negative selection takes place. At the medulla, positively selected thymocytes are exposed to a diverse set of self-antigens presented by medullary thymic epithelial cells (mTEC) and DC. mTECs use a special epigenetic mechanism to give rise to what is often referred to as promiscuous gene expression which contributes to the low expression of many genes including tissue-restricted self-antigens. SP cells with a high affinity or avidity for binding self peptides presented on HLA-I or HLA-II are eliminated by apoptosis, thus assuring the destruction of potentially autoreactive cells (9). Cells that survive negative selection mature and become naïve T Cells given the fact that they have not been primed by Ag for which they express a specific TCR. Naïve T Cells leave the thymus and migrate continuously to the secondary lymphoid organs to be primed and differentiate into effector cells with specialized phenotypes.

T cell receptor (TCR) complex

During the maturation process, T Cells acquire a receptor called TCR that recognizes a specific Ag. TCR is a multiprotein complex composed of two variable antigen-binding chains, αβ or γδ, which are associated with invariant accessory proteins (CD3γε, CD3δε, and CD247 ζζ chains) that are required for initiating signaling when TCR binds to an Ag (10).

The αβ-TCR does not recognize Ag in its natural form but recognizes linear peptides which have been processed and presented in the HLA-I or HLA-II context. The peptides presented by HLA-I molecules are small (8� aminoacids) and have an intracellular origin while those presented by HLA-II molecules are longer (13� aminoacids) and are generally of extracellular origin. Nevertheless, the αβ-TCR of NKT cells and the γδ-TCR can recognize glycolipids and phospholipids presented by CD1 molecules.

TCR α and β chains are very polymorphic, which favors the recognition of a great diversity of peptides. Each chain has a variable (V) and a constant domain (C) with a joining segment (J) that lies between them. The β chain also has an additional diversity segment (D). Each (V) domain has three hypervariable sectors known as CDR-1, -2, and -3 (complementarity-determining regions) and is capable of generating an inmense pool of combinations to produce different TCR specific for an Ag. CDR3α and β regions bind to the central region of the peptide presented. This region represents the most diverse region of the TCR and is considered to be the main determinant of specificity in Ag recognition. CDR1α and β also contribute to peptide recognition and bind to it through the amino and carboxy-terminal motifs respectively. TCR regions that come into contact with HLA mainly correspond to CDR-1 and CDR-2 (10). TCR associates with a molecule called CD3, which is composed of three different chains: gamma, delta, and epsilon γδε. These chains are associated as heterodimers γε and δε. TCR is also associated with a homodimer of δε chains (CD247) that has a long intracytoplasmic portion and participates in the downstream transductional activation signals. Both the CD3 chains and the δε chains that associate with TCR possess tyrosine-based activation motifs (ITAMs) in their intracytoplasmic moeities, which are phosphorylated to initiate T Cell activation (11).

TCR gene rearrangement is essential during T Cell development. Multiple gene segments dispersed in the genomic DNA must bind and transcribe to produce a functional TCR. This process occurs independently for each chain beginning with the recombination of genes for the β chain (12). Genes that code for the TCR chains in humans map to four loci: TCRA and TCRD on chromosome 14 and TCRB and TCRG on chromosome 7. The locus for the β chain has 42 gene segments for the region (V), 2 for (D), 12 for (J), and 2 for (C) while the locus for the α chain has 43 gene segments for the region (V) and 58 for (J) (13) (Figure 2). Somatic recombination of these gene segments occurs at the DN2 and DN3 stages of T Cell development and is mediated by the gene products RAG-1 and RAG-2. Nuclease and ligase activity, as well as the addition or elimination of nucleotides, generates the great variety of TCR present in our organism at the moment of birth. It is estimated that the diversity of TCR in humans may reach 2󗄇 (13).

Figure 2

TCR generation by somatic recombination. Adapted from Turner et al. (13). Abbreviations. TCR: T Cell receptor C: constant gene segment, V: variable gene segment, D: diversity gene segment, J: junctional gene segment, N: addition of non-template-encoded (more. )

Activation of the naïve T cells

T Cell activation and differentiation will only be sucessfull if three signals are present: i) interaction of the TCR with the peptide presented by the HLA molecule, ii) signaling through co-stimulatory molecules, and iii) participation of cytokines that initiate clonal expansion (14).

Additionally, the cytokine microenvironment that accompanies the activation defines the type of response that will be generated later.

Ag recognition and signal transduction pathways in T cells

Constant migration of the naïve T Cells towards the secondary lymphoid organs is essential in order for each one to encounter its specific Ag presented by an antigen-presenting cell (APC) (15). For this to occur, the naïve T Cells constitutively express L-selectin, an adhesion molecule which acts on the initial binding of T Cells to the high endothelial postcapillary venules located in the lymph nodes, tonsils, and aggregated lymphatic follicles. Only the specialized endothelial cells in the post-capillary veins allow constant passage of the T Cells from the blood towards the lymph nodes or Peyer’s patches given that the latter two constitutively express the addressins PNAd (peripheral node addressin) or MAdCAM-1 (mucosal addressin cell adhesion molecule-1) respectively. Both interact with the L-selectin of the lymphocytes. The endothelial cells of the rest of the vasculature restrict or impede binding of lymphocytes unless their receptors are induced by inflammation mediators (16).

In the lymph nodes, T Cells establish temporary contact with a great number of dendritic cells (DC) but only halt and bind to those which present an Ag which is compatible and specific to their receptor (15).

T Cells within lymph nodes migrate at high speeds of about 11� μ per minute. This is in contrast to DCs which transit through lymph nodes at speeds of about 3𠄶 μ per minute and then stop. This allows DCs to constantly establish new contacts with T Cells. In the absence of Ag, T Cells do not stop, but in the presence of an Ag, the duration of the interaction with the DC may be transitory (3 - 11 min) or stable (several hours) depending on the affinity for the Ag (15). Stable unions are favored by the high presence of peptides in the DC, highly antigenic ligands, mature DC, and expression of molecules such as ICAM-1 (15).

Antigen recognition by TCR induces the formation of several “TCR microclusters” that accompany the reorganization and approach of other membrane molecules and signaling proteins towards the contact zone with the DC. This contact zone between the T Cell and DC membranes is known as an immunological synapse and consists of a highly organized and dynamic molecular complex divided into three concentric zones known as the central, peripheral, and distal supramolecular activation clusters. The central region is composed of the TCR complex, co-stimulatory and co-inhibitory molecules, and co-receptors. These co-receptors are known as primary and secondary activation signals. The peripheral zone is mainly made up of the adhesion molecules LFA-1-ICAM-1 and CD2-LFA-3 that, due to their affinity, maintain and stabilize binding between the cells. The distal zone consists of F-actin and phosphatase CD45 (17).

After Ag recognition, a complex signaling process is initiated on the internal side of the membrane and in the cytoplasm for the subsequent activation of three essential transcription factors: NFAT, AP-1, and NF-㮫. These signaling pathways are shown in a simplified diagram in Figure 3 and start when phosphatase CD45 activates the tyrosine-kinases, Fyn and Lck, which are associated with the ε chains of the CD3 and the co-receptors CD4/CD8 respectively (18). Once activated, these kinases autophosphorylate and phosphorylate the ITAM moieties of the δε chains and CD3. Phosphorylated ITAMs attract the ZAP-70 molecule. Then, the binding of ZAP-70 to phospholipase C 㬱 (PLC 㬱) or LAT initiates two different cascades.

Figure 3

Overview of TCR signalling pathways. Adapted from Brownlie et al. (18). Abbreviations. LCK: lymphocyte-specific protein tyrosine kinase, FYN: a member of Src tyrosine kinases, ZAP70: ζ-chain associated protein kinase of 70 kDa, LAT: Linker for (more. )

A first cascade is initiated when PLC- 㬱 converts the phosphatidylinositol biphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). The IP3 diffuses into the cytoplasm and binds to the receptors of the endoplasmic reticulum, where it induces the release of Ca 2+ deposits to the cytosol. The intracellular increase of Ca 2+ stimulates the enzyme calmodulin, which is a serine/threonine-kinase. The activated calmodulin, in turn, activates calcineurin, a phosphatase that catalyzes the desphosphorylation of the nuclear transcription factor NF-AT to allow entry into the nucleus and activate the expression of several genes (e.g., IL-2, etc.) (19).

A second signaling cascade is initiated when ZAP-70 phosphorylates an adaptor protein known as LAT. LAT recruits several proteins that allow transference of guanine nucleotides from GDP to GTP for the activation of some proteins called Ras. These initiate a cascade of phosphorylations resulting in the activation of mitogen activated protein kinases (MAPK). These MAPK are tasked with activating the transcription factor AP-1 which is composed of the proteins c-fos and c-jun. MAPKs allow dimerization of those proteins to initiate the transcription of genes (18).

The third signaling pathway is initiated with the production of DAG, which activates protein kinase C (PKC). Later, it gives rise to recruitment of the IKK complex which requires the proteins Carma1, Bcl10, and MALT1 for its activation. Activation of the IKK kinases permits the phosphorylation of the I㮫 inhibitors, which are then ubiquitinated and degraded. This releases NF-㮫 dimers that translocate to the nucleus and activate transcription of their target genes (20).

The transcription factors NF-AT, AP-1, and NF-㮫 enter the nucleus and induce the transcription of genes to initiate the secretion of IL-2 the expression of its high affinity alpha receptor (IL-2Rα) the expression of integrins that promote cellular adhesion the expression of costimulatory molecules such as CD40L and the expression of anti-apoptotic proteins (19, 20).

Co-stimulation

A co-stimulatory molecule is defined as a surface molecule that is not itself able to activate T Cells but which can significantly amplify or reduce the signaling induced by the TCR complex (21,22). The main T Cell co-stimulatory molecules and their respective ligands for the profesional and non-professional APC are shown in Table 2.

Table 2

T Cell co-stimulatory molecules and their ligands.

Positive co-stimulatory signals are known as the second activation signal and are indispensable for potentiating the production of IL-2 due to the induction of a sustained activation of the nuclear transcription factor NF-㮫. Furthermore, interaction between these molecules initiates antiapoptotic signals that prolong T Cell life span and initiate the expression of adherence molecules as well as the production of growth factors and cytokines that promote their proliferation and differentiation.

Only CD28, CD27, and HVEM are expressed constitutively while the remaining co-stimulatory molecules are inducible and expressed only after activation. Constitutive co-stimulatory molecules have a positive regulatory effect (21, 22).

Although most of the co-stimulatory molecules have a monotypic binding (one ligand), some of them, e.g., CD28 and PD-1, interact with more than one ligand. Moreover, other molecules such as those of the SLAM family interact homotypically with identical molecules. Almost all of the T Cell co-stimulatory molecules belong to the CD28/B7 superfamily or the TNF/TNFR family (21, 22).

There is a hierarchy in the downstream activation of these co-stimulatory molecules. For example, it has been observed that co-stimulation with CD28 significantly increases the induction of ICOS and OX-40 on the surface of the T Cell (22).

Clonal expansion

In response to antigen recognition and co-stimulatory signals, T Cells initiate the synthesis of IL-2 and express the high affinity receptor for it (IL2Rα or CD25) transitorily. CD25 binds to the other chains of the IL2R which are the β chain (CD122) and common γ chain (CD132). However, it does not participate in the signaling, but increases the affinity for IL-2 from 10 to 100 times (23).

IL-2 acts as an autocrine and paracrine growth factor. IL-2 activates blastogenesis or clonal expansion which gives rise to large numbers of T Cells with receptors identical to the original, able to recognize only the Ag that initiated its activation. IL-15 and IL-21 also participate in this process of clonal expansion (23).

T Cell activation and clonal expansion is followed by a death phase during which 90% of the effector cells are eliminated by apoptosis. The mechanisms which induce this phase of contraction or death include interactions Fas-FasL, TNF, and TNFR I and II as well as CD40-CD40L. In addition, molecules such as perforins, IFN- γ, and IL-2 regulate the contraction phase of the T Cells (24).

CD4 + T cell subsets

The differentiation of a CD4 + T Cell into distinct subpopulations or cell phenotypes is determined by the nature and concentration of the Ag, the type of APC and its activation state, the cytokine microenvironment that accompanies the antigenic presentation, and the presence and quantity of co-stimulatory molecules, along with other variables.

If the T Cell expresses CD4, it is converted into a T-helper cell (Th) which has a double function: to produce cytokines and to stimulate B Cells to generate Abs. Until recently, only four distinct phenotypes had been identified: Th1, Th2, Th17, and T-regulatory cells (Treg) each of which secretes a different cytokine profile. However, in the last few years, new T-helper subsets such as Th9, Th22, and follicular helpers (Tfh) have been identified. Figure 4 summarizes the main characteristics of these T Cell subsets, the factors that induce them, and the cytokines they produce.

Figure 4

CD4 T Cell subsets. Adapted from Lloyd et al. (30). Abbreviations. TH: T-helper, IFNγ: interferon gamma, DTH: delayed type hypersensitivity, TNF: tumor necrosis factor, FGF: fibroblast growth factor, AHR: airway hyperresponsiveness.

Th1. The differentiation of the Th1 cells is induced by IL-12, IL-18, and type 1 IFNs (IFN-α and IFN-β) secreted by DC and macrophages after being activated by intracellular pathogens. IL-18 potentiates the action of IL-12 on the development of the Th1 phenotype. In general, the response mediated by Th1 depends on the T-bet transcription factor and the STAT4 molecule. These cells produce IFN- γ, IFN-α, IFN-β, and IL-2 and express CXCR3 and CD161 (25). They stimulate strong cell immunity to intracellular pathogens as well as participate in the pathogenesis of the autoinmune diseases and in the development of delayed type hyper-sensitivity. In Th1 cells, the IL-2 increases the expression of T-bet and IL-12R㬢 which then promotes the sustainability of this phenotype (23).

Th2. A Th2 response is induced by extracellular pathogens and allergens. It is generated by the effect of the IL-4, IL-25, IL-33, and IL-11 secreted by mast cells, eosinophils, and NKT cells. These cytokines induce the intracellular activation of STAT-6 and GATA-3, which initiates the secretion of cytokines of the Th2 phenotype such as IL-4, IL-5, IL-9, IL-13, IL-10, and IL-25, as well as, the expression of CCR4 and ICOS (26, 27). Th2 cells induce immunoglobulin class switching to IgE, through a mechanism mediated by IL-4. The IgE, in turn, activates cells of the innate immune system such as basophils and mast cells and induces their degranulation and the liberation of histamin, heparin, proteases, serotonin, cytokines, and chemokines. These molecules generate contraction of the smooth muscle, increase vascular permeability, and recruit more inflammatory cells. Th2 cells also migrate to the lung and intestinal tissue where they recruit eosinophils (through the secretion of IL-5) and mast cells (through IL-9). This leads to tissue eosinophilia and hyperplasia of mast cells. When acting upon epithelial cells and the smooth muscle (through IL-4 and IL-13), the Th2 cells induce production of mucus, metaplasia of the Goblet cells, and airway hyper-responsiveness as observed in allergic diseases (26). In the Th2 cells, IL-2 induces the expression of IL-4Rα and keeps the loci of the IL-4 and IL-13 genes in an accessible configuration during the final stages of the differentiation of these cells, which helps to conserve this phenotype (23).

Th9. This subset of T-helper cells arises through the effect of TGF-β and IL-4. Th9 cells produce IL-9 and IL-10 and do not express cytokines or transcription factors of the Th1, Th2, or Th17 subsets (28). IL-9 promotes the growth of mast cells and the secretion of IL-1β, IL-6, IL-13, and TGF-β. Nevertheless, IL-9 is not exclusive to this cell subpopulation. It is also produced by Th2, Th17, Treg, mast cells, and NKT cells (29). In allergic processes and infections by helminthes, the IL-9 stimulates the liberation of mast cell products and, through IL-13 and IL-5, indirectly induces the production of mucus, eosinophilia, hyperplasia of the epithelium, and muscular contraction (30).

Th17. These cells are induced by the combined action of IL-6, IL-21, IL-23, and TGF-β. The IL-6 activates the naïve T Cell resulting in the autocrine production of IL-21 which in synergy with TGF-β induces the nuclear transcription factor (ROR)c and the production of IL-17A and IL-17F. IL-23 is essential for the survival and activation of Th17 after its differentiation and selectively regulates the expression of IL-17 (31).

The Th17 cells are mainly located in the pulmonary and digestive mucosa. They produce IL-17A, IL-17F, IL-6, IL-9, IL-21, IL-22, TNF-α, and CCL20. IL-17, in synergy with TNF-α, promotes the expression of genes that amplify the inflammatory process. IL-17 binds to its receptor in mesenchymatous cells such as fibroblasts, epithelial cells, and endothelial cells to promote the liberation of chemokines and inflammation mediators such as IL-8, MCP-1, G-CSF, and GM-CSF (31). IL-17 and IL-22 also induce the production of defensins. The inflammatory environment generated by Th17 cells is associated with diseases that have an important inflammatory component such as rheumatoid arthritis, systemic lupus erythematosus (SLE), bronchial asthma, and transplant rejection (32).

Th22.This T Cell subset is generated by the combined action of the IL-6 and TNF-α with the participation of plasmacytoid DC. Th22 cells are characterized by the secretion of IL-22 and TNF-α. The transcripcional profile of these cells also includes genes that encode for FGF (fibroblast growth factor), IL-13, and chemokines implicated in angiogenesis and fibrosis. The main transcription factor associated with this phenotype is AHR. In the skin, IL-22 induces antimicrobial peptides, promotes the proliferation of keratinocytes, and inhibits their differentiation which suggests a role in the scarring of wounds and in natural defence mechanisms (33). The Th22 cells express CCR4, CCR6, and CCR10 which allows them to infiltrate the epidermis in individuals with inflammatory skin disorders. They participate in Crohn’s disease, psoriasis, and the scarring of wounds (34).

Follicular helper T Cells (Tfh). These cells were discovered just over a decade ago as germinal center T Cells that help B Cells to produce antibodies. The development of these cells depends on IL-6, IL-12, and IL-21. They are characterized by the sustained expression of CXCR5 and the loss of CCR7, which allows Tfh cells to relocate from the T Cell zone to the B Cell follicles that express CXCL13. There, they induce the formation of germinal centers, the transformation of B Cells into plasma cells, the production of antibodies with different isotypes, and the production of memory B Cells (35).

Among all the T-helper cell subsets, the Tfh express the TCR with the highest affinity for Ag and the greatest quantity of costimulatory molecules such as ICOS and CD40L. Furthermore, they express the transcription factor BCL-6 and cytokines such as IL-21, IL-4, and IL-10 which induce the differentiation of B Cells and the production of Ab (35).

Regulatory T Cells (Treg). Regulatory T Cells represent 5% to 10% of CD4+ T Cells in healthy adults. They constitutively express markers of activation such as CTLA-4 (CD152), α receptor of IL-2 (CD25), OX-40, and L-selectin (36). These are considered anergic in the absense of IL-2 which makes them dependent on the IL-2 secreted by other cells. By their mechanism of action and origin, they represent a heterogenous population of cells that can be divided into two: natural Treg cells of thymic origin and induced Treg cells differentiated on the periphery (37).

The natural Treg cells are CD4 + CD25 high and constitutively express the transcription factor FOXP3 + which is essential for their development. The CD4 + CD25 − FOXP3 − cells can differentiate into Treg cells in the presence of IL-10 and TGF-β and for interaction with immature DC. In contrast, the differentiation of Treg cells is inhibited when mature DC produces IL-6.

The production of Treg cells is essential in preventing autoimmune diseases and avoiding prolonged immunopathological processes and allergies. They are also essential for inducing tolerance to allogenic transplants as well as tolerance of the foetus during pregnancy. They supress the activation, proliferation, and effector function of a wide range of immune cells including autoreactive CD4 or CD8 T Cells which escape negative selection in the thymus, NK cells, NKT, LB, and APC. Like a double-edged sword, Treg cells also supress antitumoral responses, which favors tumor development (37).

Action mechanisms of Treg cells are depicted in Figure 5. These mechanisms can be broadly divided into those that target T Cells (regulatory cytokines, IL-2 consumption, and cytolysis) and those that primarily target APCs (decreased costimulation or decreased antigen presentation). Major mechanisms by which Treg cells exert their functions include (36, 38):

Figure 5

Mechanisms of action of T regulatory cells. Abbreviations. TGFβ: Transforming growth factor beta, CTLA4: cytotoxic T-lymphocyte antigen 4.


Peripheral T-Cell Selection

  • Central and Peripheral tolerance occur in tandem, in the case that central tolerance is not completely effective partly because not all autoantigens are expressed in the thymus
  • Several autoreactive clones are found in the peripheral blood of healthy people, and some lymphocytes from people without MS react in vitro to MBP (a target of the immune response in MS)
  • Autoreactive clones can potentially become activated and proliferate in the periphery when properly stimulated (e.g. sub-acute bacterial endocarditis can lead to emergence of self-reactive clones that damage the kidneys)
  • Peripheral mechanisms of tolerance eliminate or suppress autoreactive clones that escape to the periphery
  • Mechanisms of peripehral T-cell tolerance include:
    • A. Clonal deletion
    • B. Ignorance
    • C. Anergy
    • D. Immune regulation

    A. Clonal Deletion

    • The best-studied mechanism eliminating activated T-cell clones is activation-induced cell death (FIGURE 2)
    • T-cells activated by antigen-presenting cells express IL-2 and IL-2R for autocrine facilitation of proliferation
    • Activated T-cells also increase their expression of death receptors (e.g. Fas) and their ligands
    • Ligation of Fas leads to T-cell apoptosis via the caspase pathway, thereby ending the immune response

    Figure 2. Schematic representation of AICD. Upon uptake and processing of antigen by APCs (1) and subsequent presentation of the processed peptide to a CD4+ T cell (2), IL-2 production and expression of the IL-2R occurs followed by their autocrine binding (3), leading to T cell activation. Activated T cells express Fas (4) and FasL (5) on their surfaces or as soluble s-FasL after cleavage of the membrane-associated FasL (6). The interaction of Fas either with s-FasL (7) or with membrane-associated FasL (8) leads to apoptosis (9) by activation-induced cell death (AICD), thus ending the immune response [Reproduced with permission from Bellanti JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012].

    Mutations in Clonal Deletion Pathways

    • Mice whose T-cells do not express Fas, display expanded lymphocyte populations in their secondary lymphoid organs, leading to profound lymphadenopathy, significant self-reactivity (including many autoantibodies), and autoimmune damage to many organs (including kidneys)
    • Rare human diseases, ALPS 1a and 1b, are caused by similar mutations in Fas or FasL respectively, also leading to lymphadenopathy and autoantibodies
    • IL-2 affects the Fas pathway, and can eventually lead to AICD by increasing FasL expression
    • IL-2 also helps attenuate immunity by downregulating survival molecules (e.g. FLICE and FLIP) which would otherwise inhibit AICD by preventing assembly of the Fas death receptor complex

    B. Ignorance

    • Although peripherally, T-cells from healthy individuals can react with self-antigens in vitro, this does not commonly occur in vivo
    • It is thought that T-cells ignore certain self-antigens because they are located in immune-privileged sites or because they have low immunogencitiy (low levels of expression or low binding affinity)
    • Sympatheticautoimmuneophthalmia (Case 38 in Bellanti JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012 and related Case Study &ndash Case of eye injury and decreased vision) &ndash severe inflammatory damage to both eyes &ndash is caused by release of sequestered ocular self-antigens into circulation, where they can eventually activate peripheral autoreactive immune cells
    • the immune system is not normally exposed to ocular antigens, but trauma to a single eye releases autoantigens that activate autoreactive immune cells, leading to severe granulomatous inflammatory of both eyes

    C. Anergy

    • This is a major mechanism inactivating peripheral autoreactive T-cell clones
    • Anergic T-cell clones cannot respond to cognate antigenic stimuli: they do not produce IL-2 or IL-2R
    • Multiple proposed mechanisms explain this block in T-cell activation (FIGURE 3):
    • Disruption of the interaction between the T-cell co-receptor CD28 and APC co-stimualtory molecules CD80/86
    • Interaction of CTLA-4 with CD80/86, negatively regulating T-cell activation
    • T-cells displaying CD28 tend to be activated by APC, while T-cells displaying CTLA-4 tend to become anergic
    • Under physiologic conditions, T-cells express CD28 on initial encounter with APC
    • Shortly after such stimulation, they start displaying CTLA-4, which has a higher binding affinity than CD28 for CD80/86 than does CD28
    • CTLA-4 knockout mice develop profound lympho-proliferative disease

    Figure 3. Co-stimulation is important for the activation of T cells. Panel A: Following the activation of the 80/86 on the antigen-presenting cell provides the second signal, leading to T cell activation and proliferation. Panel B: Disruption of the CD28/CD80/86 signal or Panel C: Failure to express CD 80/86 can lead to anergy. Panel D: At the same time, the activated T cells upregulate the expression of CTLA-4, a molecule that also interacts with greater affinity to CD80/86, leading to disruption of the costimulatory signal and anergy. [Reproduced with permission from Bellanti JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012]

    Clinical Significance of CTLA-4

    • Rheumatoid arthritis is an autoimmune disease mainly affecting the joints
    • CTLA-4 is used as a biologic response modifier to treat these patients, significantly reducing joint inflammation
    • A fusion protein consisting of CTLA-4 and immunoglobulin (abatacept, belatacept) is used clinically in arthritis and after transplantation
    • There is also interest in blocking CTLA-4 using monoclonal antibodies (e.g. ipilimumab) to inhibit tumor tolerance
    • Insufficient production of key transcriptional activators (e.g. AP-1, NF-kB, NFAT-1) during T-cell activation reduces IL-2 production
    • Transcription suppressors (e.g. CREM) can also decrease transcriptional activity of the IL-2 gene promoter, leading to T-cell anergy
    • Alterations at multiple levels of regulation (e.g. activity of key kinases such as MAPK, or stability of IL-2 mRNA) can contribute to reduced IL-2 production in anergic T-cells

    D. Immune Regulation

    • Immune regulation is achieved by the action of Treg&rsquos
    • Treg are important in the maintenance of peripheral tolerance
    • When they are depleted from mice, autoimmunity results
    • A human patient with genetic Treg dysfunction develops lymphadenopathy and inflammatory infiltrates consisting of autoreactive T-cells in multiple organs
    • Naturally-occurring thymus-derived Treg display anergic properties in vitro, but can also suppress CD4+CD25- T-cells in vivo, via direct cell-cell contact, or secretion of cytokines (FIGURE 4)
    • During active inflammation (e.g. infection), Treg do not prevent protective immune function
    • Induced CD4+CD25+ Treg can also be activated in peripheral lymphoid organs (e.g. by TGF&beta) and suppress immune responses via anti-inflammatory cytokines (e.g. TGF&beta) rather than direct contact.

    Figure 4. Schematic representation of two mechanisms of immune regulation by Treg cells. Panel A: Self-antigen specific CD4+CD25+ T cells can directly suppress the activation of self-reactive T cells through cell-cell interaction. Panel B: Anti-inflammatory cytokines such as IL-10 and TGF-β that are secreted by other immunoregulatory T cells can also suppress the immune response. [Reproduced with permission from Bellanti JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012]

    TGFβ & Regulatory Cells Other Than Treg

    • TGF&beta in the presence of IL-6 (e.g. from activated macrophages during infection) and IL-23 can also lead to induction of Th17, which produce IL-17
    • Th17 are associated with antimicrobial immunity as well as autoimmune/inflammatory disorders
    • TGF&beta can therefore either regulate inflammation through Treg, or promote inflammation through Th17
    • CD1-restricted NKT cells have also been implicated in immune regulation, as have CD8+ suppressor T-cells
    • &gamma&deltaCD8+ T-cells that populate MALT are most likely involved in suppression of immune responses initiated by antigen delivered by the mucosal route
    • After inhalation of small quantities of antigen, such CD8+ T-cells are activated in sub-mucosal areas to become suppressors cells and migrate to draining lymph nodes to suppress immune response via production of IL-10 and TGF&beta
    • Ingestion of larger quantities of antigen activates not only such CD8+ suppressors, but also Treg that migrate to areas of inflammation to downregulate T-driven immune responses
    • Th1-type IFN&gamma opposes Th2-type immunity, while Th2-type IL-4 opposes Th1-type immunity
    • Regulatory T-cells and cytokines are also being used as and targeted therapeutically

    B-cell Tolerance

    • During normal B-cell development, a set of processes help induce B-cell central tolerance
    • Education of B-cells and elimination of self-reactive B-cell clones is somewhat different from that of T-cells
    • B-cells are still immature when they relocate from bone marrow to spleen T-cell zones
    • Autoreactive B-cells are not necessarily eliminated during negative selection in the bone marrow
    • B-cells that recognise autoantigens are eliminate via apoptosis or become anergic
    • Autoreactive B-cells that escape negative selection become part of the a maximally-diverse immune repertoire

    B-cell Peripheral Tolerance

    • Peripheral tolerance mechanisms (in secondary lymphoid tissues) exist for various reasons:
    • Imperfect T-cell tolerance: in most autoimmune diseases, B-cells are T-cell dependent, requiring help from pre-activated cognate autoreactive T-cells
    • T-independent B-cells can be activated by autoantigens without T-cell help
    • Microbial antigens structurally similar to autoantigens can lead B-cells to produce cross-reactive antibodies in a phenomenon known as molecular mimicry
    • B-cells hypermutate their receptors on activation, so there is a second chance that they may become self-reactive

    Exploring B-cell Tolerance

    • Mechanisms of B-cell tolerance have been explored using transgenic mice (FIGURE 5)
    • In a classic experiment, monotransgenic mice expressing hen egg lysozyme (after tolerisation via fetal exposure) are bred with monotransgenic mice expressing anti-lysozyme IgM and having B-cell receptors capable of recognising lysozyme
    • The resulting mouse hybrids produce lysozyme, but despite lysozyme-recognising B-cells do not produce anti-lysozyme antibody after immunization with lysozyme
    • This demonstrates that the simultaneous presence of lysozyme and lysozyme-reactive B-cells leads to B-cell anergy
    • B-cells from the hybrid strain transferred into irradiated wild-type mice were indeed able to make anti-lysozyme antibodies
    • This proves that anergy was related to continued stimulation by lysozyme, but could be reversed when B-cells were in a host without the tolerising effects of lysozyme

    Figure 5. Experimental model of B cell anergy. (1) A transgenic mouse expressing hen egg lysozyme (white mouse) was crossed with a mouse transgenic for anti-lysozyme specific B cell production (brown mouse) to produce an F1 hybrid (tan mouse) (2) double transgenic for lysozyme production (3) and anti-lysozymal B cell production these double transgenic hybrids produced lysozyme but did not produce anti-lysozyme antibody. (4) The B cells from these F1 mice, although unable to react to the lysozyme, when transferred to irradiated mice and immunized with lysozyme (5) resulted in the production of anti-lysozyme antibody (Ab) (6). This proves that the B cells in the F1 mice, although capable of producing anti-lysozyme antibody, if transferred to another mouse were functionally anergized in the presence of endogenously produced lysozyme [Reproduced with permission from Bellanti JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012].

    Regulatory B Cells & Tolerance

    • B cells are known to be antibody producers. In fact more specifically, B stimulation occurs in the Germinal Centres of the lymph nodes and develop into antibody secreting plasma cells with the help of T follicular cells.
    • This is a well described pathway and there are many reviews on the topic
    • What is less well known, is the regulatory nature of B cells and how these cells can play a role in regulating immunity, additional to the antibody functions of these cells.

    Regulatory B-cells

    • Regulatory B-cells (Breg) are a B-cell sub-population present in mice and humans.
    • They are important in maintaining immune homeostasis (e.g. they contribute to maintenance of tolerance, and prevention of uncontrolled inflammation).
    • They can also secrete immunoglobulins (especially IgM), however.
    • Breg origins are incompletely understood, but they may develop from marginal zone B-cells, from CD5 + transitional or pre-naïve B-cells, or from the human equivalent of B1 cells.
    • Triggers for Breg development include inflammation, the presence of apoptotic cells, tolerogenic DC (tolDC)-derived IFN&beta, tumor-related TNF&alpha, and interaction with tolDC CD40 or activated helper T-cell surface CD40L. B-cell activating factor (BAFF) and T-cell surface CTLA-4 may also play roles.
    • There is no unequivocal Breg identity, possibly because Breg are a transient phenotype along. The current best definition in humans is CD19 + CD24 hi CD38 hi (equivalent to transitional B-cells), although heterogeneity exists.
    • Various Breg subsets include:
      • Immature transitional (CD19 + CD24 hi CD38 hi ) B-cells &ndash the peripheral B-cell subset producing the highest levels of IL-10.
      • B10 cells (mainly within the CD19 + CD24 hi CD27 + population) &ndash a rare circulating sub-population related to memory B-cells and characterised by IL-10 production as well as a high proliferative capacity on activation, with fully IL-10-dependent regulatory abilities. Some have autoreactive B-cell receptors, and there is interest in elucidating their relationship to the human equivalent of B1 cells.
      • GrB + (CD19 + CD38 + CD1d + IgM + CD147 + ) B-cells &ndash induced by T-cell-derived IL-21, and expressing IL-10, IDO, and granzyme B.
      • Br1 (CD25 hi CD71 hi CD73 lo ) cells &ndash secrete allergen-specific tolerogenic IgG4.
      • Plasmablasts (CD27 int CD38 hi ) &ndash also capable of IL-10 secretion
      • Circulating CD39 + CD73 + Breg &ndash express enzymes which convert pro-inflammatory ATP to adenosine.
      • iBreg (phenotype undefined) &ndash induced by T-cell surface CTLA-4, express TGF&beta and IDO.
      • Subsets down-regulate innate and adaptive responses via diverse molecular mechanisms, including secretion of regulatory cytokines (e.g. IL-10, TGF&beta), expression of regulatory enzymes (e.g. IDO), and direct cell-cell contact (e.g. antigen presentation along with co-stimulation by CD80/86, CD40 or cytotoxicity via granzyme B or diverse TNF and TNF receptor (TNFR) family members, such as Fas/FasL, TRAIL/DR5, and PDL1 and -2).
      • In many cases, suppression is IL-10-dependent, yet IL-10 is not always necessary for Breg suppressive function. The multitude of Breg suppressive mechanisms means that demonstration of suppressive capacity remains the gold standard for Breg
      • Results of Breg suppressive mechanisms include inhibition of T-cell proliferation, suppression of Th1 and Th17 cytokine (IFN&gamma, TNF&alpha, IL-17) production, restoration of the Th1/Th2 balance, promotion of T-cell IL-10 secretion, Treg induction, inhibition of dendritic cell and macrophage function (e.g. phagocytosis, antigen presentation, TNF secretion, NO production), and Breg surface CD1d may facilitate activation of iNKT cells with regulatory functions.
      • Clinically Breg are implicated in inflammation, autoimmunity (e.g. animal models of arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus(SLE)), allergy (e.g. animal models of contact hypersensitivity), cancer, and transplantation. Breg appear to play both tumor-promoting and tumor-inhibiting roles.
      • Most studies on Breg have been done in vitro or in murine models less is known about Breg functions in healthy humans and human autoimmune diseases. Although autoimmune patients display an increased Breg frequency, Breg from SLE patients have impaired suppressive activity due to a defect in IL-10 production

      Islet Graft Alloimmunity

      Islet Replacement Strategies

      The current clinical strategy to replace the lost beta cell function is through whole pancreas or isolated pancreatic islet transplantation from genetically unrelated cadaveric donors. Because donors are limited, currently only T1D patients with hypoglycemia unawareness are considered for transplantation. This has created great interest in cell culture methods to produce large quantities of insulin-producing cells for transplantation. After more than 10 years of development, the Melton laboratory became the first group to develop a reproducible protocol for iPS conversion to insulin-producing beta cells (143), quickly followed by several other groups (144�). However, these methods are not yet suitable for large-scale production of patient-specific iPS-beta cells for transplantation studies since individuals require several hundred thousand individual pancreatic islets. Furthermore, a critical limiting factor of a “universal donor” beta cell line is conventional transplant recognition, described below. In addition, unlike whole pancreas or isolated islet transplantation, iPS-beta cells do not replace the lost alpha cell function. Until these challenges are addressed, transplantation from a cadaveric donor will likely remain the preferred approach in combination with immune suppression (147, 148). A recent phase III clinical trial demonstrated improved glycemic control in islet transplant recipients following multisite standardized processing protocols (149, 150). As less beta cell-toxic immune suppression treatments are developed, we can expect transplant function and long-term survival to continue to improve. In the absence of these treatments, transplanted beta cells in autoimmune recipient patients would be subject to at least two categories of T cell responses: (a) autoimmune (islet-specific) responses by T cells (151, 152), and (b) conventional anti-transplant-reactive T cell responses. However, current immune suppression treatments do not promote immune tolerance as described above, must be continued indefinitely after transplantation, and can render the transplant recipient vulnerable to cancer and infectious agents. Therefore, transplant-specific tolerance-promoting treatments are a highly sought after goal in the islet transplantation field.

      An alternative to replacing the lost beta cell mass would be to stimulate beta cell regeneration. Beta cell regeneration is based on the premise that if autoreactive T cells are removed or inhibited, existing beta cells could proliferate, alpha cells could convert into beta cells, or islet-resident stem cell populations could proliferate and differentiate into beta cells. There is little experimental evidence to support these suppositions to date. Beta cells are exceptionally metabolically active, continuously producing insulin secretory granules. The less beta cell mass is available to produce insulin, the higher the metabolic stress is on each individual islet. Therefore, the ability to regenerate beta cells from existing beta cells could be a significant hurdle. Another theoretical option to replace lost beta cell mass is to promote trans-differentiation of existing alpha cells into beta cells. Recent evidence from Kim’s laboratory at Stanford suggests that alpha cell conversion to beta cells may be feasible (153). However, even if beta cell replacement, alpha cell trans-differentiation, or beta cell regeneration succeed, these strategies do not address the deficiency in alpha cell glucagon production, which precipitates hypoglycemia unawareness, and as such do not represent a complete treatment for this life-threatening diabetic complication on its own. Therefore, whole islet transplantation will remain the clinical standard-of-care over beta cell replacement until these concerns can be fully addressed.

      Concurrent Autoimmune and Alloimmune Pathogenesis

      There are two separate immune recognition pathways leading to the destruction of transplanted beta cells in the autoimmune recipient. As mentioned above, the first is autoimmunity due to antigen-specific memory T cells. Regardless of the source of beta cells transplanted into an individual with T1D, autoimmune T cells would target cells producing insulin and must be inhibited or removed to facilitate long-term transplant function (154). In contrast, autoreactive T cell targeting of a kidney transplant in a diabetic individual would not likely occur, because there would be no pre-existing kidney-specific memory T cells (154). Alloimmunity is the second major concern leading to the destruction of transplanted beta cells. Transplant-reactive or alloreactive T cell responses can target the genetic differences between the transplant donor and recipient (155). This category of immune response occurs against any organ or tissue transplant, in any individual, regardless of autoimmune disease status (156). Importantly, these transplant-specific responses focus primarily on the HLA molecule of the human transplant or MHC in mouse. HLA molecules are the most polymorphic loci in the human genome, and each individual expresses multiple alleles of both class I and class II HLA (154�). All the genetic differences in both alleles are potential antigens and could be targeted by T cells in transplant recipients. The differences in HLA class I are targeted by recipient CD8 T cells, and the differences in HLA class II are targeted by recipient CD4 T cells (156). Ironically, genetic diversity in HLA promotes diverse T cell responses to the same pathogen in different individuals, but unfortunately these genetic differences also promote strong T cell responses against any transplanted organ or tissue. In this section, we describe transplant recognition and alloimmunity separately from autoimmunity.

      Transplant Recognition: Direct and Indirect Pathways

      Donor-derived MHC (or HLA) molecules are the most prevalent transplant-derived antigen seen by the immune system of a transplant recipient. Transplant recipient T cells can interact with donor MHC molecules in two ways termed direct and indirect recognition (157). Direct allorecognition results from T cell interaction with donor MHC (plus some peptide loaded in MHC), whereas indirect allorecognition results from T cell interactions with recipient MHC (plus peptide derived from donor MHC, or any other transplant-derived protein). It is estimated that 1�% of CD8 T cells or CD4 T cells will spontaneously respond to allogeneic MHC I or MHC II, respectively [reviewed in Ref. (158)]. In contrast, we hypothesize that the indirect precursor frequency is even smaller. In support of this hypothesis, recent evidence indicates that only 10% of allograft-reactive CD4 T cells in a mouse model of cardiac allograft rejection are indirect, while the remaining 90% are direct alloreactive CD4 T cells (159). Due to the higher precursor frequency for direct allorecognition than indirect allorecognition [reviewed in Ref. (157)], immune suppression protocols appear to hold direct alloreactivity in check. However, indirect recognition, which leads to antibody formation, CD4 T cell reactivity, and complement activation, is not completely inhibited using current immune suppression treatment regiments, as shown by complement deposition and antibody formation in chronic rejection models (160).

      Importantly, both CD4 and CD8 T cells in the recipient can interact with donor MHC through either the direct or indirect pathway. The frequency and physiologic relevance of direct and indirect allorecognition varies with the nature of the transplanted organ or tissue. For islet allograft recognition, donor MHC class I and direct interaction with recipient CD8 T cells is a high-frequency event, because all cells in the graft express MHC class I. Since beta cells do not express MHC class II at baseline (161), direct recognition via CD4 T cells may not be as high frequency of an event. However, recent evidence suggests that beta cells may express MHC class II following T cell infiltration (161), which suggests that direct alloreactive CD4 T cells may be critical for anti-islet allograft responses. In contrast, indirect allorecognition by CD8 T cells must be therapeutically addressed to prevent islet allograft rejection (see below discussion of CD154 blockade therapy). Table ​ Table2 2 summarizes the roles of direct and indirect CD4 and CD8 T cells in islet allograft rejection in the NOD mouse model.

      Table 2

      Islet allograft recognition pathways and likely players in rejection in autoimmune diabetic recipients.

      Direct or indirectT cellsTargetPrecursor frequency in recipientsFold expansion posttransplantSufficient for rejection?Required for rejection?Reference
      DirectCD4 T cellsDonor MHC II + transplant-derived peptide0.1�% versus individual donor MHC10�YesNo(162�)
      DirectCD8 T cellsDonor MHC I + transplant-derived peptide0.1�% versus individual donor MHC10�YesNo(162�)
      IndirectCD4 T cellsDonor-derived peptide loaded in recipient MHC IILess than 1 in 1,000,000𾄀YesAppears likely(162�)
      IndirectCD8 T cellsDonor-derived peptide loaded in recipient MHC ILess than 1 in 1,000,000𾄀YesAppears not(162�)

      Islet Allograft Tolerance in Non-Autoimmune Diabetic Mice

      Unfortunately, islet transplants are subject to both autoimmune disease recurrence and allograft recognition in T1D mice and humans. To remove autoimmunity as a confounding variable from islet transplant tolerance studies, several labs have made use of the free radical generator streptozotocin (STZ) (165�). STZ induces diabetes due to the relative lack of free radical scavenging enzymes expressed in pancreatic beta cells relative to other cell types (168). Following induction of diabetes with STZ, mice can be transplanted with allogeneic (MHC-disparate) pancreatic islets and treated with candidate transplant tolerance-promoting therapies. In experiments using non-autoimmune diabetic mice, untreated recipients serve as control groups to determine time to normal allograft rejection.

      Multiple different general immune suppressive therapies have been tested in preclinical mouse models and are used clinically (168). These therapies can include anti-CD3, antithymocyte globulin, calcineurin inhibitors, mTOR inhibitors, tacrolimus, or mycophenolate mofetil (169). Interestingly, one of the tolerance-promoting protocols, which reversed diabetes, ECDI-coupled splenocytes, can also promote islet allograft tolerance in non-autoimmune mice (170). Of particular interest, monoclonal antibodies to block T cell co-stimulation (or signal 2) have been tested by several groups (165, 171). For example, short-term monoclonal antibody therapy directed against the T cell-expressed co-stimulation molecule CD154 (CD40L) has been shown by several groups (165, 171) to induce long-term (𾄀 days) islet allograft tolerance across full MHC mismatch donor/recipient pairs (e.g., BALB/c islets transplanted into STZ-treated B6 male mice). This tolerance resides in the CD4 T cell compartment and can be transferred from treated and tolerant mice to naive mice (165). It is controversial whether this therapy induces allo-specific regulatory T cells de novo [suggested by Ferrer et al. (172)] or inhibits reactivity of naive alloreactive CD8 T cells through killing mediated by NK cells (173), or if these effects are simultaneous. In addition, the combination of anti-CD154 antibody with other therapies has been highly efficacious, in particular LFA-1 blockade. LFA-1 (CD11a) is an adhesion molecule expressed on most leukocytes, in particular on neutrophils, macrophages, and activated T cells. LFA-1 inhibition appears to delay and/or prevent islet allograft rejection as a single therapy. Similar to anti-CD154-induced transplant tolerance, uniform (100% of mice), long-term (𾄀�ys) tolerance induced by the combination therapy of LFA-1 blockade and CD154 blockade resided in the CD4 T cell compartment and was serially transferable to multiple islet allograft recipients (165). In summary, STZ-induced diabetes represents a useful, non-autoimmune model system to test candidate islet allograft tolerance-promoting therapies. However, the end goal is to induce islet tolerance in autoimmune recipients, such as the NOD mouse.

      In islet transplantation studies, “indirect” (recipient MHC-restricted) alloreactive CD4 T cells are key perpetrators of islet allograft rejection (174). As such, we hypothesize that co-transfer of islet antigen-specific Tregs at the time of islet transplantation would inhibit alloreactive T cell responses. Indeed, immune tolerance to antigen-presenting cell-depleted islet allografts in non-autoimmune mice requires CD4 T cells in transplant recipient mice (175). An alternative approach is to promote expression of T cell inhibitory receptor ligands on beta cells prior to transplantation (Figure ​ (Figure1). 1 ). One example of this approach is beta cell expression of Fas ligand, which when combined with the immune suppressive drug rapamycin generated Tregs in recipient mice (176). Another example of this approach is a recent report which demonstrated that enforced beta cell-intrinsic PD-L1 and CTLA4 expression significantly delayed islet allograft rejection in NOD mice (177). In conclusion, whether autoimmunity or alloimmunity drives islet transplant rejection, generation, or adoptive transfer of Tregs or pre-arming transplanted beta cells with co-inhibitory molecules represent two distinct strategies to protect beta cells.

      Potential Role for Regulatory CD4 T Cells in the Autoimmune Recipient of an Islet Allograft

      Importantly, regulatory CD4 Foxp3 + T cells engage peptides through the indirect antigen recognition pathway. Therefore, therapies that promote the development of transplant-specific Tregs are highly desirable. One long-term goal of the islet transplantation and autoimmunity field is to either deplete “indirect” autoreactive CD4 T cells or re-educate these CD4 T cells to become Foxp3 + regulatory CD4 T cells, while also generating additional “indirect” Tregs specific for transplant-derived antigens. Based on the above considerations for beta cell MHC II expression in the inflamed transplant recipient, we hypothesize that regulatory CD4 T cells specific for donor MHC II would prolong islet allograft survival. In addition, we hypothesize that conventional self-reactive and “indirect” CD4 T cells, which recognize autoantigens through the transplant recipient’s MHC class II molecule, would prolong graft survival. In combination, we speculate that adoptive transfer of both autoantigen-specific “indirect” Tregs as well as transplant MHC II-specific 𠇍irect” Tregs would synergize to significantly prolong islet allograft survival in autoimmune recipients.

      Failure of Islet Transplant Tolerance in the NOD Mouse

      Laboratories at the Barbara Davis Center (31), Vanderbilt (178), Harvard (179), University of Massachusetts (180), University of North Carolina (181), the University of Miami (182), and the St. Vincent’s Institute in Melbourne (78) have utilized the NOD mouse as a model system to study both autoimmune disease recurrence (rejection of NOD-background islets) or islet allograft rejection (rejection of islet from genetically unrelated donor strains including B6, C3H). Due to its autoimmune disease status, the diabetic NOD female islet transplant recipient is a difficult, but clinically relevant model to test islet transplant tolerance-promoting therapies. Several studies have demonstrated the requirement for both CD4 T cells and CD8 T cells in diabetes recurrence in NOD mice (183, 184). Less data are available in the islet allograft scenario in NOD mice. Due to the sheer number of pancreatic islets required to reverse hyperglycemia and rapid T cell-mediated transplant rejection, diabetic female NOD mice are not frequently used to test transplant tolerance-promoting therapies.

      The NOD mouse is an extremely stringent model to test transplant tolerance-promoting therapies. There are vanishingly few examples of long-term transplant tolerance in NOD mice. In particular, the combination of CD154 and LFA-1 in B6 mice resulted in long-term tolerance (180, 185). It is controversial whether this stringency results from resistance to therapeutic intervention in the autoimmune primed/memory T cell compartment, the alloreactive T cell response in NOD mice, or both. Mouse models and human clinical reports have suggested that autoimmune T cells are less susceptible to conventional immunosuppression (151, 185). In addition and in parallel, data from NOD mice support the existence of an accelerated and therapy-resistant anti-allograft T cell response (162). Additional studies in the Bio Breeder rat further suggested that autoimmune T cells are strongly impervious to tolerance-promoting therapy in this animal model of T1D, whereas the anti-allograft response can be made tolerant (186�). These differences between models, and a lack of peptide-MHC II reagents to separately track both autoreactive and alloreactive CD4 T cells in the same transplant recipient mouse, lead to a lack of consensus in the field and an incomplete understanding of auto- and allo-T cell tolerance, in particular when both immune responses occur simultaneously.

      While global immune suppressive treatments promote survival of transplanted beta cells [with the exception of calcineurin inhibitors, which are toxic to beta cells (190)], it is challenging to interpret effects of immune-modulatory therapies on specific T cell populations. Clinically, in the autoimmune recipient of pancreatic islets, there are at least two concurrent immune responses. As such, a major limiting factor in this analysis is the quality and availability of reagents to reliably and separately track autoreactive and alloreactive T cell responses in human patients. Lack of validated reagents to monitor these responses longitudinally in clinical samples presents a major challenge to interpret therapeutic effects on recurrent autoimmunity versus anti-allograft responses. Lack of reagents to separately assess these two categories of T cell responses in the NOD mouse prevents the development of reagents to preferentially influence either category of T cell response in the preclinical or clinical setting.


      Results

      Mice lacking negative selection dependent on MHC class II expression, but not on MHC class I expression, develop autoimmune GVHD

      To test the hypothesis that impaired thymic negative selection could induce autoimmune disease, we created BM chimeras in which MHC molecules were expressed on the radioresistant thymic epithelium that supports positive selection, but not on the radiosensitive hematopoietic elements responsible for negative selection. It has been shown that negative selection of CD4 + T cells is impaired in [II – / – → wt] chimeras, while [wt → II – / – ] chimeras lack positive selection of CD4 + T cells. 12,27 Similarly, negative selection of CD8 + T cells is impaired in [β2m – / – → wt] chimeras. 27 Flow cytometric analysis of thymic DCs 4 weeks after syngeneic BMT either from II – / – or β2m – / – donors confirmed the replacement of host thymic DCs by donor-derived DCs: thymic DCs from [II – / – → wt] chimeras did not express MHC class II, whereas more than 98% DCs isolated from [β2m – / – → wt] chimeras did not express MHC class I, as previously shown. 27 Analysis of the thymus 10 weeks after BMT showed the emergence of CD4 single-positive (SP) thymocytes and CD8 SP thymocytes in [wt → wt] chimeras comparable with naive wt mice (Table 1), demonstrating preservation of efficient positive selection by the thymus after 13 Gy TBI, as previously shown for 10 Gy TBI. 27 At 4 weeks after BMT, [II – / – → wt] chimeras developed weight loss, scaling of skin, hunched posture, and decreased activity. This constellation of signs was quite similar to acute GVHD seen after allogeneic BMT. 35 Assessment of these mice with a clinical GVHD scoring system 35 showed a rapid and sustained rise in [II – / – → wt] chimeras beginning 4 weeks after BMT (Figure 1A). Scores were slightly elevated in all mice 1 week after BMT due to radiation toxicity, but returned to baseline at 2 weeks after BMT and remained within normal range thereafter in both [wt → wt] and [wt → II – / – ] chimeras. This systemic illness proved lethal in the majority of animals so that only 40% survived at day 130 after BMT (Figure 1B, P < .005).

      Development of multiple organ injury in [II –/– → wt] chimeras

      . Naive wt . [wt → wt] . [II -/- → wt] . [β2m -/- → wt] .
      Pathology scores n = 6 n = 15 n = 14 n = 3
      Liver
      Portal tracts 0.5 ± 0.3 0.3 ± 0.2 4.9 ± 0.6* 0.0 ± 0.0
      Bile ducts 0.0 ± 0.0 0.0 ± 0.0 2.6 ± 0.5* 0.0 ± 0.0
      Vessels 0.2 ± 0.2 0.1 ± 0.1 1.4 ± 0.3† 0.0 ± 0.0
      Hepatocytes 1.2 ± 0.6 1.0 ± 0.2 3.4 ± 0.4* 0.0 ± 0.0
      Total 1.8 ± 0.6 1.3 ± 0.2 12.6 ± 1.5* 0.0 ± 0.0
      Small bowel
      Architecture 1.1 ± 0.5 1.1 ± 0.3 2.6 ± 0.2* 0.0 ± 0.0
      Epithelium 1.3 ± 0.5 1.5 ± 0.4 3.1 ± 0.2† 0.0 ± 0.0
      Total 2.5 ± 1.0 2.5 ± 0.6 5.8 ± 0.3* 0.0 ± 0.0
      Large bowel
      Architecture 1.0 ± 0.0 0.5 ± 0.2 1.7 ± 0.3† 0.0 ± 0.0
      Epithelium 1.5 ± 0.5 0.7 ± 0.2 2.9 ± 0.2* 0.0 ± 0.0
      Total 2.3 ± 0.8 1.2 ± 0.4 4.6 ± 0.4* 0.0 ± 0.0
      Skin 0.0 ± 0.0 0.0 ± 0.0 1.3 ± 0.2* 0.0 ± 0.0
      Immune reconstitution n = 3 n = 4 n = 4 ND
      Thymus, cells × 10 6
      Total thymocytes 82 ± 5 62 ± 13 1 ± 2 ND
      CD4 SP cells 11 ± 1 8 ± 2 0 ± 0 ND
      CD8 SP cells 2 ± 1 2 ± 1 0 ± 0 ND
      Spleen, cells × 10 6
      CD4 + T cells 21 ± 2 18 ± 2 8 ± 4† ND
      CD8 + T cells 9 ± 1 6 ± 1 3 ± 1† ND
      B220 + cells 43 ± 3 55 ± 5.8 22 ± 8.6† ND
      . Naive wt . [wt → wt] . [II -/- → wt] . [β2m -/- → wt] .
      Pathology scores n = 6 n = 15 n = 14 n = 3
      Liver
      Portal tracts 0.5 ± 0.3 0.3 ± 0.2 4.9 ± 0.6* 0.0 ± 0.0
      Bile ducts 0.0 ± 0.0 0.0 ± 0.0 2.6 ± 0.5* 0.0 ± 0.0
      Vessels 0.2 ± 0.2 0.1 ± 0.1 1.4 ± 0.3† 0.0 ± 0.0
      Hepatocytes 1.2 ± 0.6 1.0 ± 0.2 3.4 ± 0.4* 0.0 ± 0.0
      Total 1.8 ± 0.6 1.3 ± 0.2 12.6 ± 1.5* 0.0 ± 0.0
      Small bowel
      Architecture 1.1 ± 0.5 1.1 ± 0.3 2.6 ± 0.2* 0.0 ± 0.0
      Epithelium 1.3 ± 0.5 1.5 ± 0.4 3.1 ± 0.2† 0.0 ± 0.0
      Total 2.5 ± 1.0 2.5 ± 0.6 5.8 ± 0.3* 0.0 ± 0.0
      Large bowel
      Architecture 1.0 ± 0.0 0.5 ± 0.2 1.7 ± 0.3† 0.0 ± 0.0
      Epithelium 1.5 ± 0.5 0.7 ± 0.2 2.9 ± 0.2* 0.0 ± 0.0
      Total 2.3 ± 0.8 1.2 ± 0.4 4.6 ± 0.4* 0.0 ± 0.0
      Skin 0.0 ± 0.0 0.0 ± 0.0 1.3 ± 0.2* 0.0 ± 0.0
      Immune reconstitution n = 3 n = 4 n = 4 ND
      Thymus, cells × 10 6
      Total thymocytes 82 ± 5 62 ± 13 1 ± 2 ND
      CD4 SP cells 11 ± 1 8 ± 2 0 ± 0 ND
      CD8 SP cells 2 ± 1 2 ± 1 0 ± 0 ND
      Spleen, cells × 10 6
      CD4 + T cells 21 ± 2 18 ± 2 8 ± 4† ND
      CD8 + T cells 9 ± 1 6 ± 1 3 ± 1† ND
      B220 + cells 43 ± 3 55 ± 5.8 22 ± 8.6† ND

      The extent of GVHD was assessed 10 weeks after BMT. Liver, small intestine, large intestine, and skin were analyzed using the histopathologic scoring system described in “Materials and methods.” Thymus and spleens were phenotyped by a flow cytometric analysis. II -/- mice have normal counts of DP cells in the thymus and of CD8 + and B220 + cells in spleen. 43 Data are expressed as mean ± SE. SP indicates single positive ND, not done and DP, double positive.

      [II – / – → wt] chimeric mice develop systemic and lethal autoimmune disease. Wt or II – /– mice were lethally irradiated and received transplants from either wt or II –/– B6 donors. (A) Clinical GVHD scores 35 and (B) survivals after BMT were monitored. [wt → wt], n = 38 [wt → II – /– ], n = 29 and [II – /– → wt], n = 35. Results from 3 similar experiments are combined and mean ± SE of clinical scores is shown. *P < .001, ** P < .005.

      [II – / – → wt] chimeric mice develop systemic and lethal autoimmune disease. Wt or II – /– mice were lethally irradiated and received transplants from either wt or II –/– B6 donors. (A) Clinical GVHD scores 35 and (B) survivals after BMT were monitored. [wt → wt], n = 38 [wt → II – /– ], n = 29 and [II – /– → wt], n = 35. Results from 3 similar experiments are combined and mean ± SE of clinical scores is shown. *P < .001, ** P < .005.

      Histologic examination of the liver, skin, and small and large intestine 10 weeks after BMT showed standard pathologic features of acute GVHD in [II – / – → wt] chimeras (Table 1). GVHD was severe in the liver, showing characteristic damage of acute GVHD in portal tracts, bile ducts, vasculature, and parenchyma. 44 The portal tract was expanded with dense mononuclear cell infiltrates and necrotic hepatocytes (Figure 2A), producing a hepatitis-like picture characterized by acidophilic bodies. Mononuclear cells infiltrated the bile duct epithelium, causing nuclear pleomorphism, multilayering, pyknosis, and endothelialitis (Figure 2B). GVHD was also in the skin with mononuclear cell infiltration of the dermis (Figure 2C), and in the intestine with villous atrophy, crypt cell apoptosis, and lymphocytic infiltrates (Figure 2D). Immunodefi-ciency, another cardinal feature of GVHD after allogeneic BMT, 45 was present with profound reductions in the number of double-positive (DP) thymocytes, splenic CD4 + T, CD8 + T, and B cells (Table 1).

      Histologic analysis of [II – / – → wt] chimeric mice reveals systemic GVHD. Histologic analysis of [II – /– → wt] chimeras 10 weeks after BMT showed periportal mononuclear infiltrates (A) and endothelialitis (B, arrow) in liver, dermal mononuclear infiltrates in the skin (C), and crypt cell apoptosis in the small intestine (D, arrow). Original magnification × 200 (A, C, D) and × 400 (B).

      Histologic analysis of [II – / – → wt] chimeric mice reveals systemic GVHD. Histologic analysis of [II – /– → wt] chimeras 10 weeks after BMT showed periportal mononuclear infiltrates (A) and endothelialitis (B, arrow) in liver, dermal mononuclear infiltrates in the skin (C), and crypt cell apoptosis in the small intestine (D, arrow). Original magnification × 200 (A, C, D) and × 400 (B).

      We then investigated whether the similar absence of MHC class I expression on thymic antigen-presenting cells (APCs) could also cause GVHD. In contrast to [II – / – → wt] chimeras, [β2m – / – → wt] chimeras did not show any clinical signs of GVHD and 95% of these mice survived at day 140 after BMT (Figure 3). Analysis of the liver, intestine, and skin 10 weeks after BMT showed no histologic evidence of GVHD (Table 1).

      [β2m – / – → wt] chimeric mice do not develop autoimmune diseases. Wt mice were lethally irradiated and received transplants from wt or β2m – / – B6 donors. Clinical GVHD scores (A) and survival (B) after BMT were monitored in [wt → wt], n = 32 and [β2m – /– → wt], n = 56. Results from 3 similar experiments are combined and mean ± SE of clinical scores is shown.

      [β2m – / – → wt] chimeric mice do not develop autoimmune diseases. Wt mice were lethally irradiated and received transplants from wt or β2m – / – B6 donors. Clinical GVHD scores (A) and survival (B) after BMT were monitored in [wt → wt], n = 32 and [β2m – /– → wt], n = 56. Results from 3 similar experiments are combined and mean ± SE of clinical scores is shown.

      Intact thymus is required for the development of autoimmune GVHD

      To confirm the causative role of thymus in the development of GVHD, wt mice were thymectomized prior to BMT and underwent transplantation as above. Control [II – / – → wt] chimeras developed severe, lethal GVHD with only 40% survival at day 60 after BMT, whereas all thymectomized [II – / – → wt] chimeras survived this period (Figure 4A) without any evidence of GVHD as judged by clinical GVHD scores (Figure 4B) and histology scores of the liver and intestine (Figure 4C). Thus, we confirmed the causal association of the thymus with the development of autoimmune GVHD.

      Thymectomy of BM transplant recipients prevents the development of GVHD. (A-C) Wt mice were thymectomized (Tx) and received transplants of TCD BM from II – / – donors following 13 Gy TBI. Survival (A) and clinical GVHD scores (B) are shown after BMT (n = 5-9/group). (C) Pathology scores of the liver and intestine at 7 weeks after BMT (n = 3/group). Mean ± SE is shown. *P < .01, **P < .05.

      Thymectomy of BM transplant recipients prevents the development of GVHD. (A-C) Wt mice were thymectomized (Tx) and received transplants of TCD BM from II – / – donors following 13 Gy TBI. Survival (A) and clinical GVHD scores (B) are shown after BMT (n = 5-9/group). (C) Pathology scores of the liver and intestine at 7 weeks after BMT (n = 3/group). Mean ± SE is shown. *P < .01, **P < .05.

      Autoreactive CD4 + T cells confer GVHD to the secondary hosts when peripheral regulatory mechanisms are eliminated

      We next investigated the pathogenic role of CD4 + T cells in this disease process. CD4 + T cells that were isolated 8 weeks after BMT from spleens of [II – / – → wt] chimeras proliferated and produced IL-2 and IFN-γ in response to B6 (self) stimulators in vitro, while CD4 + T cells isolated from [wt → wt] chimeras did not (Table 2). [II – / – → wt] CD4 + T cells showed an activation/memory phenotype with the reduction of CD62L and CD45RB expression compared with [wt → wt] CD4 + T cells (CD62L + , 11% vs 66% CD45RB + , 60% vs 90%), respectively, confirming the emergence of activated, autoreactive CD4 + Th1 cells in the absence of thymic negative selection.

      Development of autoreactive CD4 + T cells in the [II –/– → wt] chimeras

      Mice . Stimulation with B6-PC . Proliferation, cpm . IFN-γ, pg/mL . IL-2, pg/mL .
      wt B6
      - 538 ± 34 UD UD
      + 860 ± 164 UD UD
      [wt → wt]
      - 916 ± 24 UD UD
      + 780 ± 157 UD UD
      [wt → II -/- ]
      - 733 ± 237 UD UD
      + 535 ± 181 UD UD
      [II -/- → wt]
      - 949 ± 54 UD UD
      + 5640 ± 459 698 ± 22 64.1 ± 9.5
      Mice . Stimulation with B6-PC . Proliferation, cpm . IFN-γ, pg/mL . IL-2, pg/mL .
      wt B6
      - 538 ± 34 UD UD
      + 860 ± 164 UD UD
      [wt → wt]
      - 916 ± 24 UD UD
      + 780 ± 157 UD UD
      [wt → II -/- ]
      - 733 ± 237 UD UD
      + 535 ± 181 UD UD
      [II -/- → wt]
      - 949 ± 54 UD UD
      + 5640 ± 459 698 ± 22 64.1 ± 9.5

      CD4 + T cells were isolated from spleens (3 spleens/group) 8 weeks after BMT and were stimulated at 2 × 10 5 cells/well with irradiated (20 Gy) peritoneal cells (1 × 10 5 /well) from B6 mice (B6-PC). After 3 days of culture, supernatants were harvested for cytokine measurements and cell proliferation was determined after pulsing with [ 3 H]thymidine for an additional 16 hours. Data represent mean ± SD. UD indicates undetectable.

      To determine whether [II – / – → wt] CD4 + T cells transmit the disease to secondary hosts, CD4 + T cells isolated from spleens of [II – / – → wt] chimeras were adoptively transferred to secondary syngeneic wt recipients. Transfer of 1 × 10 7 [II – / – → wt] CD4 + T cells into unirradiated wt B6 mice produced no clinical disease (Figure 5A). We therefore hypothesized that unirradiated recipients did not develop disease after transfer of autoreactive CD4 + T cells because (a) the CD4 + T cells were suppressed by peripheral regulatory cells and/or (b) the absence of proinflammatory milieu in unirradiated secondary hosts did not sustain activation of the CD4 + T cells. To test this hypothesis, wt B6 mice were sublethally irradiated (6.5 Gy) and then injected with 1 × 10 7 CD4 + T cells isolated either from [wt → wt] or [II – / – → wt] chimeras. Recipients of [II – / – → wt] CD4 + T cells developed lethal GVHD with only 17% surviving 5 weeks after transfer (Figure 5A). These mice exihibited significantly elevated clinical GVHD scores (4.5 ± 1.0, P < .01) and showed hepatic and intestinal pathology similar to the original [II – / – → wt] chimeras (Figure 5B). Flow cytometric analysis of the spleen 6 weeks after adoptive transfer of [II – / – → wt] CD4 + T cells (CD45.2 + ) into irradiated congeneic B6.Ly-5a (CD45.1 + ) mice confirmed the expansion of donor-derived CD45.2 + CD4 + T cells (79 ± 3%).

      Autoreactive CD4 + T cells that escape thymic negative selection cause GVHD only when peripheral regulatory mechanisms by T cells are eliminated. At 10 weeks after BMT, splenic CD4 + T cells (1 × 10 7 ) isolated from [wt → wt] and [II – /– → wt] chimeras were injected into either irradiated (6.5 Gy) or unirradiated wt mice (n = 5 or 6 per group). Survival after transfer (A) and pathology scores of the liver and small and large intestine at 6 weeks after transfer (B) (n = 3 per group) are shown. Results from 2 similar experiments are combined and represent mean ± SE. Survival (C) and clinical score (D) after adoptive transfer of CD4 + T cells from [II – /– → wt] chimeras into lethally irradiated wt mice with or without cotransfer of splenic CD4 – CD8 – cells (non-T cells, 2 × 10 7 ), CD4 + T cells (1 × 10 7 ), 1 × 10 7 CD8 + T cells (1 × 10 7 ), or splenic CD4 + plus CD8 + T cells (2 × 10 7 ) isolated from naive wt mice. (E) DP thymocytes were enumerated 5 weeks after transfer. UD indicates undetectable. *P < .05.

      Autoreactive CD4 + T cells that escape thymic negative selection cause GVHD only when peripheral regulatory mechanisms by T cells are eliminated. At 10 weeks after BMT, splenic CD4 + T cells (1 × 10 7 ) isolated from [wt → wt] and [II – /– → wt] chimeras were injected into either irradiated (6.5 Gy) or unirradiated wt mice (n = 5 or 6 per group). Survival after transfer (A) and pathology scores of the liver and small and large intestine at 6 weeks after transfer (B) (n = 3 per group) are shown. Results from 2 similar experiments are combined and represent mean ± SE. Survival (C) and clinical score (D) after adoptive transfer of CD4 + T cells from [II – /– → wt] chimeras into lethally irradiated wt mice with or without cotransfer of splenic CD4 – CD8 – cells (non-T cells, 2 × 10 7 ), CD4 + T cells (1 × 10 7 ), 1 × 10 7 CD8 + T cells (1 × 10 7 ), or splenic CD4 + plus CD8 + T cells (2 × 10 7 ) isolated from naive wt mice. (E) DP thymocytes were enumerated 5 weeks after transfer. UD indicates undetectable. *P < .05.

      We then tested whether normal, resting T cells could suppress the pathology of autoreactive CD4 + T cells. Transfer of [II – / – → wt] CD4 + T cells (1 × 10 7 ) with TCD BM (5 × 10 6 ) from wt mice into lethally irradiated wt mice conferred GVHD. However, cotransfer of both CD4 + and CD8 + T cells from wt mice completely prevented GVHD development, while cotransfer of non-T cells or either CD4 + or CD8 + T cells alone did not prevent it (Figure 5C-D). Cotransfer of CD4 + and CD8 + T cells completely prevented GVHD-associated thymic damage measured by the number of DP thymocytes (Figure 5E). These results demonstrate that autoreactive CD4 + T cells that escape negative selection in the thymus become pathogenic when peripheral T cells are eliminated by irradiation.

      CD4 + effector T cells lyse target cells primarily by Fas ligand and cytokines. 46 Serum levels of TNF-α (46 ± 11 pg/mL) and IL-1 (20 ± 3 pg/mL) were elevated in [II – / – → wt] chimeras but not in controls. Therefore, in order to determine the functional relevance of these proinflammatory cytokines, we neutralized both cytokines by intraperitoneal injections of TNFR/Fc and αIL-1R, as described in “Materials and methods.” This neutralization significantly delayed GVHD compared with control-treated animals (median survival time 22 days versus 12 days, P < .05). These results demonstrate an important role for inflammatory cytokines in autoimmune GVHD caused by autoreactive CD4 + T cells that escaped negative thymic selection, as has been shown in other autoimmune diseases. 47


      Multiple Choice

      A. a protein that is highly efficient at stimulating a single type of productive and specific T cell response
      B. a protein produced by antigen-presenting cells to enhance their presentation capabilities
      C. a protein produced by T cells as a way of increasing the antigen activation they receive from antigen-presenting cells
      D. a protein that activates T cells in a nonspecific and uncontrolled manner

      To what does the TCR of a helper T cell bind?

      A. antigens presented with MHC I molecules
      B. antigens presented with MHC II molecules
      C. free antigen in a soluble form
      D. haptens only

      Cytotoxic T cells will bind with their TCR to which of the following?

      A. antigens presented with MHC I molecules
      B. antigens presented with MHC II molecules
      C. free antigen in a soluble form
      D. haptens only

      A ________ molecule is a glycoprotein used to identify and distinguish white blood cells.

      A. T-cell receptor
      B. B-cell receptor
      C. MHC I
      D. cluster of differentiation

      Name the T helper cell subset involved in antibody production.


      Immunologic Tolerance and Autoimmunity: Self–Nonself Discrimination in the Immune System and Its Failure

      One of the remarkable properties of the normal immune system is that it can react to an enormous variety of microbes but does not react against the individual’s own (self) antigens. This unresponsiveness to self antigens, also called immunologic tolerance , is maintained despite the fact that the molecular mechanisms by which lymphocyte receptor specificities are generated are not biased to exclude receptors for self antigens. In other words, lymphocytes with the ability to recognize self antigens are constantly being generated during the normal process of lymphocyte maturation. Furthermore, many self antigens have ready access to the immune system, so unresponsiveness to these antigens cannot be maintained simply by concealing them from lymphocytes. The process by which antigen-presenting cells (APCs) display antigens to T cells does not distinguish between foreign and self proteins, so self antigens are normally seen by lymphocytes. It follows that there must exist mechanisms that prevent immune responses to self antigens. These mechanisms are responsible for one of the cardinal features of the immune system—namely, its ability to discriminate between self and nonself (usually microbial) antigens. If these mechanisms fail, the immune system may attack the individual’s own cells and tissues. Such reactions are called autoimmunity , and the diseases they cause are called autoimmune diseases. In addition to tolerating the presence of self antigens, the immune system has to coexist with many commensal microbes that live on the epithelial barriers of their human hosts, often in a state of symbiosis, and the immune system of a pregnant female has to accept the presence of a fetus that expresses antigens derived from the father. Unresponsiveness to commensal microbes and the fetus is maintained by many of the same mechanisms involved in unresponsiveness to self.

      In this chapter we address the following questions:

      How does the immune system maintain unresponsiveness to self antigens?

      What are the factors that may contribute to the loss of self-tolerance and the development of autoimmunity?

      How does the immune system maintain unresponsiveness to commensal microbes and the fetus?

      This chapter begins with a discussion of the important principles and features of self-tolerance. Then we discuss the different mechanisms that maintain tolerance to self antigens, as well as commensal microbes and the fetus, and how tolerance may fail, resulting in autoimmunity.

      Immunologic Tolerance: General Priniciples and Significance

      Immunologic tolerance is a lack of response to antigens that is induced by exposure of lymphocytes to these antigens . When lymphocytes with receptors for a particular antigen encounter this antigen, any of several outcomes is possible. The lymphocytes may be activated to proliferate and to differentiate into effector and memory cells, leading to a productive immune response antigens that elicit such a response are said to be immunogenic . The lymphocytes may be functionally inactivated or killed, resulting in tolerance antigens that induce tolerance are said to be tolerogenic . In some situations, the antigen-specific lymphocytes may not react in any way this phenomenon has been called immunologic ignorance, implying that the lymphocytes simply ignore the presence of the antigen. Normally, microbes are immunogenic and self antigens are tolerogenic.

      The choice between lymphocyte activation and tolerance is determined largely by the nature of the antigen and the additional signals present when the antigen is displayed to the immune system. In fact, the same antigen may be administered in different ways to induce an immune response or tolerance. This experimental observation has been exploited to analyze what factors determine whether activation or tolerance develops as a consequence of encounter with an antigen.

      The phenomenon of immunologic tolerance is important for several reasons. First, as we stated at the outset, self antigens normally induce tolerance, and failure of self-tolerance is the underlying cause of autoimmune diseases. Second, if we learn how to induce tolerance in lymphocytes specific for a particular antigen, we may be able to use this knowledge to prevent or control unwanted immune reactions. Strategies for inducing tolerance are being tested to treat allergic and autoimmune diseases and to prevent the rejection of organ transplants. The same strategies may be valuable in gene therapy to prevent immune responses against the products of newly expressed genes or vectors and even for stem cell transplantation if the stem cell donor is genetically different from the recipient.

      Immunologic tolerance to different self antigens may be induced when developing lymphocytes encounter these antigens in the generative (central) lymphoid organs, a process called central tolerance, or when mature lymphocytes encounter self-antigens in peripheral (secondary) lymphoid organs or peripheral tissues, called peripheral tolerance ( Fig. 9.1 ). Central tolerance is a mechanism of tolerance only to self antigens that are present in the generative lymphoid organs—namely, the bone marrow and thymus. Tolerance to self antigens that are not present in these organs must be induced and maintained by peripheral mechanisms. We have only limited knowledge of which self antigens induce central or peripheral tolerance or are ignored by the immune system.

      With this brief background, we proceed to a discussion of the mechanisms of immunologic tolerance and how the failure of each mechanism may result in autoimmunity. Tolerance in T cells, particularly CD4 + helper T lymphocytes, is discussed first because many of the mechanisms of self-tolerance were defined by studies of these cells. In addition, CD4 + helper T cells orchestrate virtually all immune responses to protein antigens, so tolerance in these cells may be enough to prevent both cell-mediated and humoral immune responses against self proteins. Conversely, failure of tolerance in helper T cells may result in autoimmunity manifested by T cell–mediated attack against tissue self antigens or by the production of autoantibodies against self proteins.

      Central T Lymphocyte Tolerance

      The principal mechanisms of central tolerance in T cells are death of immature T cells and the generation of CD4 + regulatory T cells ( Fig. 9.2 ). The lymphocytes that develop in the thymus consist of cells with receptors capable of recognizing many antigens, both self and foreign. If a lymphocyte that has not completed its maturation interacts strongly with a self antigen, displayed as a peptide bound to a self major histocompatibility complex (MHC) molecule, that lymphocyte receives signals that trigger apoptosis. Thus, the self-reactive cell dies before it can become functionally competent. This process, called negative selection (see Chapter 4 ), is a major mechanism of central tolerance. The process of negative selection affects self-reactive CD4 + T cells and CD8 + T cells, which recognize self peptides displayed by class II MHC and class I MHC molecules, respectively. Why immature lymphocytes die upon receiving strong T cell receptor (TCR) signals in the thymus, while mature lymphocytes that get strong TCR signals in the periphery are activated, is not fully understood.

      Some immature CD4 + T cells that recognize self antigens in the thymus with high affinity do not die but develop into regulatory T cells and enter peripheral tissues (see Fig. 9.2 ). The functions of regulatory T cells are described later in the chapter. What determines whether a thymic CD4 + T cell that recognizes a self antigen will die or become a regulatory T cell is also not established.

      Immature lymphocytes may interact strongly with an antigen if the antigen is present at high concentrations in the thymus and if the lymphocytes express receptors that recognize the antigen with high affinity. Antigens that induce negative selection may include proteins that are abundant throughout the body, such as plasma proteins and common cellular proteins.

      Surprisingly, many self proteins that are normally present only in certain peripheral tissues, called tissue-restricted antigens, are also expressed in some of the epithelial cells of the thymus. A protein called AIRE (autoimmune regulator) is responsible for the thymic expression of these peripheral tissue antigens. Mutations in the AIRE gene are the cause of a rare disorder called autoimmune polyendocrine syndrome. In this disorder, several tissue antigens are not expressed in the thymus because of a lack of functional AIRE protein, so immature T cells specific for these antigens are not eliminated and do not develop into regulatory cells. These cells mature into functionally competent T cells that enter the peripheral immune system and are capable of reacting harmfully against the tissue-restricted antigens, which are expressed normally in the appropriate peripheral tissues even in the absence of AIRE. Therefore, T cells specific for these antigens emerge from the thymus, encounter the antigens in the peripheral tissues, and attack the tissues and cause disease. It is not clear why endocrine organs are the most frequent targets of this autoimmune attack. Although this rare syndrome illustrates the importance of negative selection in the thymus for maintaining self-tolerance, it is not known if defects in negative selection contribute to common autoimmune diseases.

      Central tolerance is imperfect, and some self-reactive lymphocytes mature and are present in healthy individuals. As discussed next, peripheral mechanisms may prevent the activation of these lymphocytes.

      Peripheral T Lymphocyte Tolerance

      Peripheral tolerance is induced when mature T cells recognize self antigens in peripheral tissues, leading to functional inactivation (anergy) or death, or when the self-reactive lymphocytes are suppressed by regulatory T cells ( Fig. 9.3 ). Each of these mechanisms of peripheral T cell tolerance is described in this section. Peripheral tolerance is clearly important for preventing T cell responses to self antigens that are not present in the thymus, and it also may provide backup mechanisms for preventing autoimmunity in situations where central tolerance to antigens that are expressed in the thymus is incomplete.

      Antigen recognition without adequate costimulation results in T cell anergy or death or makes T cells sensitive to suppression by regulatory T cells. As noted in previous chapters, naive T lymphocytes need at least two signals to induce their proliferation and differentiation into effector and memory cells: Signal 1 is always antigen, and signal 2 is provided by costimulators that are expressed on APCs, typically as part of the innate immune response to microbes (or to damaged host cells) (see Chapter 5 , Fig. 5.6 ). It is believed that dendritic cells in normal uninfected tissues and peripheral lymphoid organs are in a resting (or immature) state, in which they express little or no costimulators, such as B7 proteins (see Chapter 5 ). These dendritic cells constantly process and display the self antigens that are present in the tissues. T lymphocytes with receptors for the self antigens are able to recognize the antigens and thus receive signals from their antigen receptors (signal 1), but the T cells do not receive strong costimulation because there is no accompanying innate immune response. Thus, the presence or absence of costimulation is a major factor determining whether T cells are activated or tolerized.

      Anergy

      Anergy in T cells refers to long-lived functional unresponsiveness that is induced when these cells recognize self antigens ( Fig. 9.4 ). Self antigens are normally displayed with low levels of costimulators, as discussed earlier. Antigen recognition without adequate costimulation is thought to be the basis of anergy induction, by mechanisms that are described later. Anergic cells survive but are incapable of responding to the antigen.

      The two best-defined mechanisms responsible for the induction of anergy are abnormal signaling by the TCR complex and the delivery of inhibitory signals from receptors other than the TCR complex.

      When T cells recognize antigens without costimulation, the TCR complex may lose its ability to transmit activating signals. In some cases, this is related to the activation of enzymes (ubiquitin ligases) that modify signaling proteins and target them for intracellular destruction by proteases.

      On recognition of self antigens, T cells also may preferentially use one of the inhibitory receptors of the CD28 family, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4, or CD152) or programmed cell death protein 1 (PD-1, CD279), which were introduced in Chapter 5 . Anergic T cells may express higher levels of these inhibitory receptors, which will inhibit responses to subsequent antigen recognition. The functions and mechanisms of action of these receptors are described in more detail below.

      Regulation of T Cell Responses by Inhibitory Receptors

      Immune responses are influenced by a balance between engagement of activating and inhibitory receptors . This idea is established for B and T lymphocytes and natural killer (NK) cells. In T cells, the main activating receptors are the TCR complex and costimulatory receptors such as CD28 (see Chapter 5 ), and the best-defined inhibitory receptors, also called coinhibitors, are CTLA-4 and PD-1. The functions and mechanisms of action of these inhibitors are complementary ( Fig. 9.5 ).

      CTLA-4. CTLA-4 is expressed transiently on activated CD4 + T cells and constitutively on regulatory T cells (described later). It functions to suppress the activation of responding T cells. CTLA-4 works by blocking and removing B7 molecules from the surface of APCs, thus reducing costimulation by CD28 and preventing the activation of T cells (see Fig. 9-5A ). The choice between engagement of CTLA-4 or CD28 is determined by the affinity of these receptors for B7 and the level of B7 expression. CTLA-4 has a higher affinity for B7 molecules than does CD28, so it binds B7 tightly and prevents the binding of CD28. This competition is especially effective when B7 levels are low (as would be expected normally when APCs are displaying self and probably tumor antigens) in these situations, the receptor that is preferentially engaged is the high-affinity blocking receptor CTLA-4. However, when B7 levels are high (as in infections), not all the ligands will be occupied by CTLA-4 and some B7 will be available to bind to the low-affinity activating receptor CD28, leading to T cell costimulation.

      PD-1 . PD-1 is expressed on CD8 + and CD4 + T cells after antigen stimulation. Its cytoplasmic tail has inhibitory signaling motifs with tyrosine residues that are phosphorylated upon recognition of its ligands PD-L1 or PD-L2. Once phosphorylated, these tyrosines bind a tyrosine phosphatase that inhibits kinase-dependent activating signals from CD28 and the TCR complex (see Fig. 9-5B ). Because the expression of PD-1 on T cells is increased upon chronic T cell activation and expression of the ligands is increased by cytokines produced during prolonged inflammation, this pathway is most active in situations of chronic or repeated antigenic stimulation. This may happen in responses to chronic infections, tumors, and self antigens, when PD-1–expressing T cells encounter the ligand on infected cells, tumor cells, or APCs.


      Acknowledgement

      This work was supported by funding from the Australian National Health and Medical Research Council, Australia.

      A model of thymic and peripheral CD25 + T regulatory (Treg) cell selection. In the thymus, differentiation of immature thymocytes into the CD25 + Treg lineage is dependent on TCR interaction with self-peptide/MHC class II complexes within a moderate-to-high range of avidity. In the periphery, TCR interaction with self-antigen is required for survival of CD25 + Treg cells therefore, the final repertoire is also likely to be influenced by the set of peripheral self-antigens. For conventional naive CD4 + T cells, however, contact with self-antigen in the periphery is also required for survival, but will result in clonal deletion if the avidity of TCR interaction is high.