Deriving quantitative constraints on T cell selection from data on the mature T cell repertoire

The T cell repertoire is shaped in the thymus through positive and negative selection. Thus, data about the mature repertoire may be used to infer information on how TCR generation and selection operate. Assuming that T cell selection is affinity driven, we derive the quantitative constraints that the parameters driving these processes must fulfill to account for the experimentally observed levels of alloreactivity, self MHC restriction and the frequency of cells recognizing a given foreign Ag. We find that affinity-driven selection is compatible with experimental estimates of these latter quantities only if 1) TCRs see more peptide residues than MHC polymorphic residues, 2) the majority of positively selected clones are deleted by negative selection, 3) between 1 and 3.6 clonal divisions occur on average in the thymus after completion of TCR rearrangement, and 4) selection is driven by 103-105 self peptides.     

A novel role for autophagy in T cell education

During T cell development in the thymus, scanning of peptide/major histocompatibility (MHC) molecule complexes on the surface of thymic epithelial cells ensures that only useful (self-MHC restricted) and harmless (self-tolerant) thymocytes survive. In recent years, a number of distinct cell-biological features of thymic epithelial cells have been unraveled that may have evolved to render these cells particularly suited for T cell selection, e.g., cortical epithelial cells use unique proteolytic enzymes for the generation of MHC/peptide complexes, whereas medullary epithelial cells "promiscuously" express otherwise tissue-restricted self-antigens. We recently showed that macroautophagy in thymic epithelial cells contributes to CD4 T cell selection and is essential for the generation of a self-tolerant T cell repertoire. We propose that the unusually high constitutive levels of autophagy in thymic epithelial cells deliver endogenous proteins to MHC class II molecules for both positive and negative selection of developing thymocytes.     

Peptide specificity of thymic selection of CD4+CD25+ T cells.

The CD4(+)CD25(+) regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4(+)CD25(+) cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4(+)CD25(+) cells in the thymus. By comparing the development of CD4(+)CD25(-) and CD4(+)CD25(+) cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4(+)CD25(+) cells appear during natural selection of CD4(+) T cells. The proportion of CD4(+)CD25(+) cells in the population of CD4(+) thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4(+)CD25(+) cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4(+) T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4(+)CD25(+) thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.     

Mature CD4+ T cells perceive a positively selecting class II MHC/peptide complex in the periphery

A repertoire of TCRs is selected in the thymus by interactions with MHC bound to self-derived peptides. Whether self peptides bound to MHC influence the survival of mature T cells in the periphery remains enigmatic. In this study, we show that the number of naive CD4+ T cells that developed in mice with class II MHC bound with endogenous peptides (Abwt) diminished when transferred into mice with Ab covalently bound with a single peptide (AbEp). Moreover, transfer of a mixture of naive CD4+ T cells derived from Abwt and from AbEp mice into AbEp mice resulted in the expansion of the latter and decline of the former. In contrast, when wild-type activated CD4+ T cells were transferred into AbEp or Abwt mice, these cells survived in both recipients for more than 4 wk, but further expanded in the Abwt host. We conclude that to survive, naive CD4+ T cells favor peripheral expression of the class II MHC/peptide complex(es) involved in their thymic selection, whereas some of activated CD4+ T cells may require them only for expansion.     

The role of polymorphic amino acids of the MHC molecule in the selection of the T cell repertoire

Allelic variants of MHC molecules expressed on cells of the thymus affect the selection and the specificity of the T cell repertoire. The selection is based on either the direct recognition by the TCR of the MHC molecules, or the recognition of a complex determinant formed by self-peptides bound to MHC molecules. In an analysis of the T cell repertoire in bone marrow chimeras that express allelic forms of MHC class II molecules in the thymus epithelium, we find that amino acid substitutions that are predicted to affect peptide binding influence the selection of the T cell repertoire during thymic selection.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.     

Editing autoreactive TCR enables efficient positive selection

Allelic exclusion is inefficient at the TCRalpha locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4(+)CD8(-) thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, approximately 100% more CD4(+)CD8(-) thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4(+)CD8(-) thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.     

Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

The role of CD8 alpha' in the CD4 versus CD8 lineage choice.

During thymic development the recognition of MHC proteins by developing thymocytes influences their lineage commitment, such that recognition of class I MHC leads to CD8 T cell development, whereas recognition of class II MHC leads to CD4 T cell development. The coreceptors CD8 and CD4 may contribute to these different outcomes through interactions with class I and class II MHC, respectively, and through interactions with the tyrosine kinase p56lck (Lck) via their cytoplasmic domains. In this paper we provide evidence that an alternatively spliced form of CD8 that cannot interact with Lck (CD8 alpha') can influence the CD4 vs CD8 lineage decision. Constitutive expression of a CD8 minigene transgene that encodes both CD8 alpha and CD8 alpha' restores CD8 T cell development in CD8 alpha mutant mice, but fails to permit the development of mismatched CD4 T cells bearing class I-specific TCRs. These results indicate that CD8 alpha' favors the development of CD8-lineage T cells, perhaps by reducing Lck activity upon class I MHC recognition in the thymus.     

Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides

Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.     

Survival and homeostatic proliferation of naive peripheral CD4+ T cells in the absence of self peptide:MHC complexes

TCR-self peptide:MHC interactions play a critical role in thymic positive selection, yet relatively little is known of their function in the periphery. It has been suggested that continued contact with selecting MHC molecules is necessary for long-term peripheral maintenance of naive T cells. More recent studies have also demonstrated a role for specific self peptide:MHC complexes in the homeostatic expansion of naive T cells in lymphopenic mice. Our examination of these processes revealed that, whereas self class II MHC molecules do have a modest effect on long-term survival of individual CD4+ T cells, interactions with specific TCR ligands are not required for peripheral naive CD4+ T cell maintenance. In contrast, selective engagement of TCRs by self-peptide:MHC complexes does promote proliferation of CD4+ T cells under severe lymphopenic conditions, and this division is associated with an activation marker phenotype that is different from that induced by antigenic stimulation. Importantly, however, the ability of naive T cells to divide in response to homeostatic stimuli does not appear to be stringently dependent on TCR-self peptide:MHC interactions. Therefore, these results show that the factors regulating survival and homeostatic expansion of naive T cells in the periphery are not identical. In addition, we provide evidence for a novel form of T cell proliferation that can occur independently of TCR signaling and suggest that this reflects another mechanism regulating homeostatic T cell expansion.     

Alpha beta TCRs differ in the degree of their specificity for the positively selecting MHC/peptide ligand

We have tested the peptide specificity of positive selection using three transgenic alphabetaTCRs, originally selected on class II MHC (A(b)) covalently bound with one peptide Ealpha (52-68) (Ep). The transgenic TCR specific for the cytochrome c-derived (43-58) peptide was selected on A(b) bound with different arrays of endogenous peptides or the analogue of Ep covalently bound to A(b), but not on the original A(b)Ep complex. In contrast, transgenic TCRs specific for two different analogues of the Ep peptide and A(b) did not mature as CD4(+) T cells in various thymic environments, including the A(b)EpIi(-) mice. These results show that TCRs can be promiscuous or specific for the selecting MHC/peptide complex, and suggest that in mice described in this study transgenic expression of the TCR changes the original requirements for the positively selecting MHC/peptide complex. Future studies will determine whether the latter phenomenon is general or specific for this system.     

Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors

The MHC determines susceptibility and resistance to type 1 diabetes in humans and nonobese diabetic (NOD) mice. To investigate how a disease-associated MHC molecule shapes the T cell repertoire in NOD mice, we generated a series of tetramers from I-A(g7)/class II-associated invariant chain peptide precursors by peptide exchange. No CD4 T cell populations could be identified for two glutamic acid decarboxylase 65 peptides, but tetramers with a peptide mimetic recognized by the BDC-2.5 and other islet-specific T cell clones labeled a distinct population in the thymus of young NOD mice. Tetramer-positive cells were identified in the immature CD4(+)CD8(low) population that arises during positive selection, and in larger numbers in the more mature CD4(+)CD8(-) population. Tetramer labeling was specific based on the use of multiple control tetramers, including one with a single amino acid analog peptide in which a critical TCR contact residue was substituted. The T cell population was already present in the thymus of 2-wk-old NOD mice before the typical onset of insulitis and was detected in B10 mice congenic for the NOD MHC locus, but not B10 control mice. These results demonstrate that a T cell population can expand in the thymus of NOD mice to levels that are at least two to three orders of magnitude higher than estimated for a given specificity in the naive T cell pool. Based on these data, we propose a model in which I-A(g7) confers susceptibility to type 1 diabetes by biasing positive selection in the thymus and later presenting peptides from islet autoantigens to such T cells in the periphery.     

The effect of thymus environment on T cell development and tolerance

During development in the thymus, T cells are deleted if their receptors are able to recognize self major histocompatibility complex (MHC) proteins. We show that such clonal deletion can occur because of interaction between receptors on T cells and MHC expressed on bone marrow-derived cells. In addition, development in the thymus picks out T cells to mature if their receptors will be restricted for antigen recognition in association with self MHC alleles expressed on thymus epithelial cells. This process is usually thought to involve positive selection of T cells bearing receptors with high and low affinity for MHC on thymus epithelium, and subsequent deletion of high affinity cells by interaction with bone marrow-derived cells. Our data do not fit such a model, but rather suggest that MHC molecules on thymus epithelium and bone marrow-derived cells may not be seen identically by T cell receptors.     

The level of CD8 expression can determine the outcome of thymic selection.

During thymic development, thymocytes that can recognize major histocompatability complex (MHC) molecules on thymic epithelial cells are selected to survive and mature (positive selection), whereas thymocytes that recognize MHC on hematopoietic cells are destroyed (negative selection). It is not known how MHC recognition can mediate both death and survival. One model to explain this paradox proposes that thymocytes whose T cell antigen receptors (TCRs) recognize MHC with high affinity are eliminated by negative selection, whereas low affinity TCR-MHC interactions are sufficient to mediate positive selection. Here we report that, while the expression of a 2C TCR transgene leads to positive selection of thymocytes in H-2b mice, expression of both a CD8 transgene and a 2C TCR transgene causes negative selection. This observation indicates that quantitative differences in TCR-MHC recognition are a critical determinant of T cell fate, a finding predicted by the affinity model for thymic selection.     

The study of high-affinity TCRs reveals duality in T cell recognition of antigen: specificity and degeneracy

TCRs exhibit a high degree of Ag specificity, even though their affinity for the peptide/MHC ligand is in the micromolar range. To explore how Ag specificity is achieved, we studied murine T cells expressing high-affinity TCRs engineered by in vitro evolution for binding to hemoglobin peptide/class II complex (Hb/I-Ek). These TCRs were shown previously to maintain Ag specificity, despite having up to 800-fold higher affinity. We compared the response of the high-affinity TCRs and the low-affinity 3.L2 TCR toward a comprehensive set of peptides containing single substitutions at each TCR contact residue. This specificity analysis revealed that the increase in affinity resulted in a dramatic increase in the number of stimulatory peptides. The apparent discrepancy between observed degeneracy in the recognition of single amino acid-substituted Hb peptides and overall Ag specificity of the high-affinity TCRs was examined by generating chimeric peptides between the stimulatory Hb and nonstimulatory moth cytochrome c peptides. These experiments showed that MHC anchor residues significantly affected TCR recognition of peptide. The high-affinity TCRs allowed us to estimate the affinity, in the millimolar range, of immunologically relevant interactions of the TCR with peptide/MHC ligands that were previously unmeasurable because of their weak nature. Thus, through the study of high-affinity TCRs, we demonstrated that a TCR is more tolerant of single TCR contact residue substitutions than other peptide changes, revealing that recognition of Ag by T cells can exhibit both specificity and degeneracy.     

T-cell recognition of glycolipids presented by CD1 proteins

The most well-known molecular paradigm of antigen recognition by T cells involves partial digestion of proteins to generate small peptides, which bind to major histocompatibility complex (MHC) proteins. Recent studies of CD1, an MHC class I homolog encoded outside the MHC, have revealed that it presents diverse glycolipids to T cells. The molecular mechanism for lipid antigen recognition involves insertion of the lipid portion of antigens into a hydrophobic groove to form CD1-lipid complexes, which contact T-cell receptors (TCRs). Here, we examine the known antigen structures presented by CD1, the majority of which have sugar moieties that are capable of interacting with TCRs. Recognition of carbohydrate epitopes is precise, and lipid-reactive T cells alter systemic immune responses in models of infectious and autoimmune disease. These findings provide a previously unrecognized mechanism by which the cellular immune system can recognize alterations in many types of carbohydrate structures.     

Competition for specific intrathymic ligands limits positive selection in a TCR transgenic model of CD4+ T cell development

Efficient positive selection of a broad repertoire of T cells is dependent on the presentation of a diverse array of endogenous peptides on MHC molecules in the thymus. It is unclear, however, whether the development of individual TCR specificities is influenced by the abundance of their selecting ligands. To examine this, we analyzed positive selection in a transgenic mouse carrying a TCR specific for the human CLIP:I-Ab class II complex. We found that these mice exhibit significantly reduced CD4+ T cell development compared with two other transgenic mice carrying TCRs selected on I-Ab. Moreover, many of the selected cells in these mice express endogenous and transgenic receptors as a consequence of dual TCRalpha expression. Dramatic enhancement of the selection efficiency is observed, however, when fewer transgenic cells populate the thymus in mixed bone marrow chimeras. These results suggest that positive selection is limited by the availability of selecting peptides in the thymus. This becomes apparent when large numbers of thymocytes compete for such peptides in TCR transgenic animals. Under such conditions, thymocytes appear to undergo further TCRalpha gene rearrangement to produce a receptor that may be selected more efficiently by other thymic self-peptides.     

Opposite effects of endogenous peptide-MHC class I on T cell activity in the presence and absence of CD8

Nonstimulatory or endogenous peptide-MHC (pepMHC) presented on the surfaces of APCs, either alone or alongside agonist pepMHC, plays various roles in T cell selection and activation. To examine these properties in more detail, we explored several model systems of TCR and pepMHC ligands with sufficient affinity to be activated in the absence of CD8. The TCRs had a range of affinities for agonist and nonstimulatory ligands and were restricted by MHC class I alleles with different properties. We observed CD8-independent antagonism from TCR-pepMHC interactions with very low affinities (e.g., K(D) = 300 μM). In addition, endogenous peptide-L(d) complexes on APCs antagonized activation of coreceptor (CD8)-negative 2C T cells even by the strong agonist QL9-L(d). In contrast, TCRs m33 and 3D-PYY, restricted by K(b) and D(b), respectively, did not show signs of antagonism by endogenous pepMHC in the absence of CD8. This did not appear to be an inherent difference in the ability of the TCRs to be antagonized, as altered peptide ligands could antagonize each TCR. In the presence of CD8, endogenous pepMHC ligands acted in some cases as coagonists. These results show that endogenous pepMHC molecules exhibit complex behavior in T cells, leading to either reduced activity (e.g., in cases of low coreceptor levels) or enhanced activity (e.g., in presence of coreceptor). The behavior may be influenced by the ability of different TCRs to recognize endogenous pepMHC but also perhaps by the inherent properties of the presenting MHC allele.