Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

The adaptive phenotype of cortical thymic epithelial cells

Epithelial cells in the thymus are required for positive and negative selection of developing thymocytes. Although medullary epithelial cells play a major role in negative selection owing to their facility of expressing peripheral self‐antigens, the adaptive features of cortical epithelial cells are largely unknown. A paper in this issue of the European Journal of Immunology shows that the putative serine protease Prss16 affects positive selection of a subset of CD4⁺ T cells. A survey of chordate genomes indicates that the Prss16 gene emerged in vertebrates as a paralogue of evolutionarily older members of the serine carboxypeptidase 28 family; thus, Prss16 is not associated with the appearance of the adaptive immune system and the thymus in jawed vertebrates. Nevertheless, it appears that Prss16 has later evolved as an essential contributor to the MHC class II peptide/ligand repertoire.     

Activation of CD4+ CD25+ regulatory T cell suppressor function by analogs of the selecting peptide

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells can undergo both thymic selection and peripheral expansion in response to self peptides that are agonists for their T cell receptors (TCR). However, the specificity by which these TCR must recognize peptide:MHC complexes to activate Treg cell function is not known. We show that CD4⁺CD25⁺Foxp3⁺ Treg cells can mediate suppression in response to peptides that are only weakly cross‐reactive with the self peptide that induced their formation in vivo. Moreover, suppression could be efficiently activated by peptide analogs that were inefficient at inducing CD69 up‐regulation, and that also induced little or no proliferation of naïve CD4⁺CD25^–Foxp3^– T cells expressing the same TCR. These findings provide evidence that self peptide‐specific CD4⁺CD25⁺Foxp3⁺ Treg cells can exert regulatory function in response to self‐ and/or pathogen‐derived peptides with which they are only weakly cross‐reactive.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

On integrity in immunity during ontogeny or how thymic regulatory T cells work

The Standard model of T cell recognition asserts that T cell receptor (TCR) specificities are positively and negatively selected during ontogeny in the thymus and that peripheral T cell repertoire has mild self‐major histocompatibility complex (MHC) reactivity, known as MHC restriction of foreign antigen. Thus, the TCR must bind both a restrictive molecule (MHC allele) and a peptide reclining in its groove (pMHC ligand) in order to transmit signal into a T cell. The Standard and Cohn's Tritope models suggest contradictory roles for complementarity‐determining regions (CDRs) of the TCRs. Here, I discuss both concepts and propose a different solution to ontogenetic mechanism for TCR‐MHC–conserved interaction. I suggest that double (CD4⁺CD8⁺)‐positive (DP) developing thymocytes compete with their αβTCRs for binding to self‐pMHC on cortical thymic epithelial cells (cTECs) that present a selected set of tissue‐restricted antigens. The competition between DPs involves TCR editing and secondary rearrangements, similar to germinal‐centre B cell somatic hypermutation. These processes would generate cells with higher TCR affinity for self‐pMHC, facilitating sufficiently long binding to cTECs to become thymic T regulatory cells (tTregs). Furthermore, CD4⁺ Foxp3⁺ tTregs can be generated by mTECs via Aire‐dependent and Aire‐independent pathways, and additionally on thymic bone marrow–derived APCs including thymic Aire‐expressing B cells. Thymic Tregs differ from the induced peripheral Tregs, which comprise the negative feedback loop to restrain immune responses. The implication of thymocytes’ competition for the highest binding to self‐pMHC is the co‐evolution of species‐specific αβTCR V regions with MHC alleles.     

H2‐M3‐restricted CD8+ T cells augment CD4+ T‐cell responses by promoting DC maturation

Infection with Listeria monocytogenes triggers the activation and expansion of nonconventional CD8⁺ T cells restricted by the MHC class Ib molecule, H2‐M3. H2‐M3‐restricted CD8⁺ T cells exhibit a memory phenotype, rapidly produce cytokines, and reach peak frequencies sooner than conventional MHC class Ia‐restricted CD8⁺ T cells. In this study, we found that simultaneous in vivo priming of H2‐M3‐restricted T cells and adoptively transferred OT‐II CD4⁺ T cells on the same DC enhances the survival of OT‐II cells. Stimulation of H2‐M3‐restricted T cells were found to induce DC maturation resulting in costimulatory molecule upregulation and production of T_H1‐type cytokines, which was dependent on both cell‐to‐cell contact and soluble factors, particularly TNF‐α, produced by activated H2‐M3‐restricted T cells. Interestingly, H2‐M3‐restricted T cells were more efficient than activated NK cells in inducing DC maturation. Furthermore, we found that OVA_323–339‐coated DC matured by coculturing with peptide‐stimulated H2‐M3‐restricted T cells were more efficient in stimulating the proliferation of Ag‐activated OT‐II cells. This study indicates that H2‐M3‐restricted T cells promote immune responses by CD4⁺ T cells by inducing DC maturation and suggests novel mechanisms for vaccine development.     

Evidence for a selective and multi-step model of T cell differentiation: CD4+CD8low thymocytes selected by a transgenic T cell receptor on major histocompatibility complex class I molecules

We have characterized a prominent (15-20 %) thymocyte population expressing CD4 at a high and CD8 at a low level “CD4⁺8^lo” in mice transgenic for a T cell receptor “TCR” restricted by major histocompatibility complex “MHC” class I molecules. The results demonstrate that the CD4⁺8^lo population is an intermediate stage between immature CD4⁺8⁺ and end-stage CD4⁺8^- thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4⁺8^lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi-step model of T cell differentiation in the thymus.     

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4⁺ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4⁺ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4⁺ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4⁺ T cells were characterized as CD45RA^? CD62L⁺ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4⁺ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.     

Role of TCR specificity in CD4+CD25+ regulatory T-cell selection

Summary: CD4⁺CD25⁺ regulatory T cells play a crucial role in preventing autoimmune disease and can also modulate immune responses in settings such as transplantation and infection. We have developed a transgenic mouse system in which the role that T‐cell receptor (TCR) specificity for self‐peptides plays in the formation of CD4⁺CD25⁺ regulatory T cells can be examined. We have shown that interactions with a single self‐peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4⁺CD25⁺ regulatory T cells and that thymocytes bearing TCRs with low affinity for the selecting peptide do not appear to undergo selection into this pathway. In addition, thymocytes with identical specificity for the selecting self‐peptide can undergo overt deletion versus abundant selection to become CD4⁺CD25⁺ regulatory T cells in response to variations in expression of the selecting peptide in different lineages of transgenic mice. Finally, we have shown that CD4⁺CD25⁺ T cells proliferate in response to their selecting self‐peptide in the periphery, but these cells do not proliferate in response to lymphopenia in the absence of the selecting self‐peptide. These studies are determining how the specificity of the TCR for self‐peptides directs the thymic selection and peripheral expansion of CD4⁺CD25⁺ regulatory T cells.     

CD4+ T‐cell development in a mouse expressing a transgenic TCR derived from a Treg

CD4⁺Foxp3⁺ Treg maintain peripheral tolerance and influence immune responses to foreign antigens. The thymus is an important source of Treg, but controversy exists as to whether T cells are selected into the Treg lineage based on signals received through TCR specific for self‐peptides. To examine the specificity of TCR expressed by Treg and its effect on CD4⁺ T‐cell development, we generated Treg‐TCR transgenic mice. Deletion of >90% of CD4⁺ T cells in RAG‐sufficient mice, and nearly 100% deletion in RAG^?/? mice expressing this TCR indicate that the TCR is specific for an unknown, naturally expressed peptide in the thymus. Deletion occurs late in development, suggesting this peptide is presented by APC in the thymic medulla. These studies are the first to describe the effects of expressing a Treg‐TCR on CD4⁺ T‐cell development. The implications of our data for models of Treg selection are discussed.     

Quantitative Analysis of the Efficiency of Clonal Deletion in the Thymus

One of the major mechanisms for establishing self-tolerance is the clonal deletion of self-reactive T cells during their development in the thymus. Using a TCR transgenic mouse model, we have established a quantitative ex vivo assay for examining the sensitivity and specificity of negative selection. Thymic organ cultures established from mice of varying MHC haplotypes were incubated with antigen, and the efficiency of clonal deletion assessed. We show here that clonal deletion of CD4⁺8⁺ thymocytes is sensitive to both the gene dosage and the allelic variation of MHC class II molecules expressed on thymic antigen-presenting cells. We also find that when epithelial cells in the thymic cortex are the only antigen-presenting cells expressing the appropriate MHC class II molecules, negative selection of CD4⁺8⁺ cells is as efficient as when antigen is presented on all thymic antigen-presenting cells. These studies demonstrate that the induction of self-tolerance via clonal deletion in the thymus is a function not only of antigen concentration, but also of MHC class II cell-surface density. In addition, together with the reports of others, these results confirm that cortical epithelial cells can mediate negative selection, and demonstrate that they do so in the intact thymic microenvironment.     

CD8 T cells from major histocompatibility complex class II-deficient mice respond vigorously to class II molecules in a primary mixed lymphocyte reaction

Mature CD4⁺ and CD8⁺ T cells are restricted by major histocompatibility complex (MHC) class II and class I molecules, respectively. In a primary mixed lymphocyte reaction (MLR), CD8⁺ T cells from C57BL/6 (B6) mice can respond to allo-class I molecules, but not allo-class II molecules. However, a significant fraction of CD8⁺ T cells from C57BL/6 class II-deficient (B6Aα^?) mice violate this rule by responding vigorously in a MLR to class II molecules. The frequency of responding cells is ～ 50 % of that of B6 CD8⁺ T cells responding to B6bm1 allo-class I molecules. This response requires neither appropriate co-receptor, i.e. CD4, nor exogenous lymphokines, indicating that interactions between the T cell receptors (TCR) and class II molecules are remarkably efficient. Since these CD8⁺ T cells are positively selected by class I molecules in the thymus of class II-deficient mice, these CD8⁺ T cells should interact with both classes of MHC molecules. The absence of thymic negative selection by class II molecules may result in the production of these CD8⁺ T cells. The data imply that a substantial fraction of CD4⁺CD8⁺ double-positive thymocytes in wild-type mice interacts with both classes of MHC molecules prior to thymic selection.     

Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8⁺ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP^?/? mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP^?/? Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8⁺ T cell development and repertoire selection. In comparing SLAP^?/? OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP^?/? Vβ5 mice. We have found that SLAP^?/? OT-1 mice have fewer CD8⁺ thymocytes but have increased CD5 expression. SLAP^?/? OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8⁺ splenocytes upon tetramer staining. Our data demonstrate that SLAP^?/? Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8⁺ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8⁺ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8⁺ T cell development influences repertoire selection.     

Composition of the HLA‐DR‐associated human thymus peptidome

Major histocompatibility complex class II (MHC‐II) molecules bind to and display antigenic peptides on the surface of antigen‐presenting cells (APCs). In the absence of infection, MHC‐II molecules on APCs present self‐peptides and interact with CD4⁺ T cells to maintain tolerance and homeostasis. In the thymus, self‐peptides bind to MHC‐II molecules expressed by defined populations of APCs specialised for the different steps of T‐cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC‐II‐associated peptides from five fresh human thymus samples. The data show a diverse self‐peptide repertoire, mostly consisting of predicted MHC‐II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA‐II‐bound peptidome and provide insight into how this peptidome is generated and how it drives T‐cell repertoire formation.