Shaping of the autoreactive regulatory T cell repertoire by thymic cortical positive selection

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.     

Multiple glycines in TCR alpha-chains determine clonally diverse nature of human T cell memory to influenza A virus

Detailed assessment of how the structural properties of T cell receptors affect clonal repertoires of Ag-specific cells is a prerequisite for a better understanding of human antiviral immunity. Herein we examine the alpha TCR repertoires of CD8 T cells reactive against the influenza A viral epitope M1(58-66), restricted by HLA-A2.1. Using molecular cloning, we systematically studied the impact of alpha-chain usage in the formation of T cell memory and revealed that M1(58-66)-specific, clonally diverse VB19 T cells express alpha-chains encoded by multiple AV genes with different CDR3 sizes. A unique feature of these alpha TCRs was the presence of CDR3 fitting to an AGA(G(n))GG-like amino acid motif. This pattern was consistent over time and among different individuals. Further molecular assessment of human CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes led to the conclusion that the poly-Gly/Ala runs in CDR3alpha were a property of immune, but not naive, repertoires and could be attributed to influenza exposure. Repertoires of T cell memory are discussed in the context of clonal diversity, where poly-Gly/Ala runs in the CDR3 of alpha- and beta-chains might provide high levels of TCR flexibility during Ag recognition while gene-encoded CDR1 and CDR2 contribute to the fine specificity of the TCR-peptide MHC interaction.     

Cutting edge: size and diversity of CD4+CD25high Foxp3+ regulatory T cell repertoire in humans: evidence for similarities and partial overlapping with CD4+CD25- T cells

Both differentiation and function of CD4+CD25(high) naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the alphabetaTCR repertoire sizes of human peripheral blood Treg and CD4+CD25- T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted alphabeta TCR repertoire, the size and diversity of which are closely similar to those of CD4+CD25- T cells, with a mean estimated size of 3.5 x 10(6) distinct alphabeta TCR vs 4.7 x 10(6) distinct alphabetaTCR for CD4+CD25- T cells. In addition, a 24% overlap between the repertoires of these two CD4+ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.     

Public T cell receptor beta-chains are not advantaged during positive selection

Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.     

Immunodominance in the TCR repertoire of a [corrected] TCR peptide-specific CD4+ Treg population that controls experimental autoimmune encephalomyelitis

Immunodominance in self-Ag-reactive pathogenic CD4(+) T cells has been well established in several experimental models. Although it is clear that regulatory lymphocytes (Treg) play a crucial role in the control of autoreactive cells, it is still not clear whether immunodominant CD4(+) Treg clones are also involved in control of autoreactivity. We have shown that TCR-peptide-reactive CD4(+) and CD8(+) Treg play an important role in the spontaneous recovery and resistance from reinduction of experimental autoimmune encephalomyelitis in B10.PL mice. We report, by sequencing of the TCR alpha- and beta-chain associated with CD4(+) Treg, that the TCR repertoire is limited and the majority of CD4(+) Treg use the TCR Vbeta14 and Valpha4 gene segments. Interestingly, sequencing and spectratyping data of cloned and polyclonal Treg populations revealed that a dominant public CD4(+) Treg clonotype expressing Vbeta14-Jbeta1.2 with a CDR3 length of 7 aa exists in the naive peripheral repertoire and is expanded during the course of recovery from experimental autoimmune encephalomyelitis. Furthermore, a higher frequency of CD4(+) Treg clones in the naive repertoire correlates with less severity and more rapid spontaneous recovery from disease in parental B10.PL or PL/J and (B10.PL x PL/J)F(1) mice. These findings suggest that unlike the Ag-nonspecific, diverse TCR repertoire among the CD25(+)CD4(+) Treg population, TCR-peptide-reactive CD4(+) Treg involved in negative feedback regulation of autoimmunity use a highly limited TCR V-gene repertoire. Thus, a selective set of immunodominant Treg as well as pathogenic T cell clones can be targeted for potential intervention in autoimmune disease conditions.     

Reciprocal expression in CD4 or CD8 subsets of different members of the V alpha 11 gene family correlates with sequence polymorphism

Previous staining studies with TCR V alpha 11-specific mAbs showed that V alpha 11.1/11.2 (AV11S1 and S2) expression was selectively favored in the CD4+ peripheral T cell population. As this phenomenon was essentially independent of the MHC haplotype, it was suggested that AV11S1 and S2 TCRs exert a preference for recognition of class II MHC molecules. The V alpha segment of the TCR alpha-chain is suggested to have a primary role in shaping the T cell repertoire due to selection for class I or II molecules acting through the complementarity determining regions (CDR) 1 alpha and CDR2 alpha residues. We have analyzed the repertoire of V alpha 11 family members expressed in C57BL/6 mice and have identified a new member of this family; AV11S8. We show that, whereas AV11S1 and S2 are more frequent in CD4+ cells, AV11S3 and S8 are more frequent in CD8+ cells. The sequences in the CDR1 alpha and CDR2 alpha correlate with differential expression in CD4+ or CD8+ cells, a phenomenon that is also observed in BALB/c mice. With no apparent restriction in TCR J alpha usage or CDR3 alpha length in C57BL/6, these findings support the idea of V alpha-dependent T cell repertoire selection through preferential recognition of MHC class I or class II molecules.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.     

High clonality of virus-specific T lymphocytes defined by TCR usage in the brains of mice infected with West Nile virus

It has been reported that brain-infiltrating T lymphocytes play critical roles in the clearance of West Nile virus (WNV) from the brains of mice. We characterized brain-infiltrating T lymphocytes by analyzing the TCR α- and β-chain repertoires, T cell clonality, and CDR3 sequences. CD3(+)CD8(+) T cells were localized in the WNV-infected brains. The expression of CD3, CD8, CD25, CD69, perforin, and granzymes positively correlated with viral RNA levels, and high levels of expression of IFN-γ, TNF-α, and IL-2 were detected in the brains, suggesting that Th1-like cytotoxic CD8(+) T cells are expanded in the brains in response to WNV infection. The brain-infiltrating T lymphocytes dominantly used TCR genes, VA1-1, VA2-1, VB5-2, and VB8-2, and exhibited a highly oligoclonal TCR repertoire. Interestingly, the brain-infiltrating T lymphocytes had different patterns of TCR repertoire usages among WNV-, Japanese encephalitis virus-, and tick-borne encephalitis virus-infected mice. Moreover, CD8(+) T cells isolated from the brains of WNV-infected mice produced IFN-γ and TNF-α after in vitro stimulation with peritoneal cells infected with WNV, but not with Japanese encephalitis virus. The results suggest that the infiltrating CD8(+) T cells were WNV-specific, but not cross-reactive among flaviviruses. T cells from the WNV-infected brains exhibited identical or similar CDR3 sequences in TCRα among tested mice, but somewhat diverse sequences in TCRβ. The results indicate that WNV-specific CD3(+)CD8(+) T cells expanding in the infected brains are highly oligoclonal, and they suggest that TCR α-chains play a dominant and critical role in Ag specificity of WNV-specific T cells.     

V beta T cell repertoire of CD8+ splenocytes selected on nonpolymorphic MHC class I molecules

In this work, we have studied the role of the MHC class Ib molecules in the selection and maintenance of CD8(+) T splenocytes. We have compared the CD8(+) T cell repertoires of wild-type, H-2K-deficient, H-2D-deficient, or double knockout C57BL/6 mice. We show that the different CD8(+) repertoires, selected either by class Ia and class Ib or by class Ib molecules only, use the various V alpha (AV) and V beta (BV) rearrangements in the same proportion and without biases in the CDR3 size distribution. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice. Interestingly, we have found that the BV repertoire size is proportional to the overall number of CD8(+) splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8(+) cells, even when the number of CD8(+) splenocytes is dramatically reduced (90% in the double knockout mice).     

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.     

Distinct and non-overlapping T cell receptor repertoires expanded by DNA vaccination in wild-type and HER-2 transgenic BALB/c mice

Central tolerance to tumor-associated Ags is an immune-escape mechanism that significantly limits the TCR repertoires available for tumor eradication. The repertoires expanded in wild-type BALB/c and rat-HER-2/neu (rHER-2) transgenic BALB-neuT mice following DNA immunization against rHER-2 were compared by spectratyping the variable (V)beta and the joining (J)beta CDR 3. Following immunization, BALB/c mice raised a strong response. Every mouse used one or more CD8+ T cell rearrangements of the Vbeta9-Jbeta1.2 segments characterized by distinct length of the CDR3 and specific for 63-71 or 1206-1214 rHER-2 peptides. In addition, two CD4+ T cell rearrangements recurred in >50% of mice. Instead, BALB-neuT mice displayed a limited response to rHER-2. Their repertoire is smaller and uses different rearrangements confined to CD4+ T cells. Thus, central tolerance in BALB-neuT mice acts by silencing the BALB/c mice self-reactive repertoire and reducing the size of the CD8+ T cell component. CD8+ and CD4+ T cells from both wild-type and transgenic mice home to tumors. This definition of the T cell repertoires available is critical to the designing of immunological maneuvers able to elicit an effective immune reaction against HER-2-driven carcinogenesis.     

T cell development in mice expressing CD1d directed by a classical MHC class II promoter

CD1d and nonclassical MHC molecules differ markedly from classical MHC ligands in their ability to promote the selection and differentiation of developing T cells. Whereas classical MHC-restricted T cells have a predominantly naive phenotype and a broad TCR repertoire, most other T cells have a memory and/or NKT phenotype with a restricted repertoire. Because the nonclassical ligands selecting these memory-type cells are expressed by bone marrow-derived cells, it has been suggested that the development of large repertoires of naive-type cells was dependent on the classical MHC expression pattern in the thymus cortex, high on epithelial cells and low on cortical thymocytes. We redirected CD1d expression using the classical MHC II Ealpha promoter. pEalpha-CD1d mice lacked memory-type NKT cells, but, surprisingly, they did not acquire the reciprocal ability to select a diverse population of naive CD1d-restricted cells. These findings suggest that, whereas the development of NKT cells is dependent on the pattern of CD1d expression, the absence of a broad, naive CD1d-restricted T cell repertoire may reflect intrinsic limitations of the pool of TCR genes or lipid Ags.     

Chronic modulation of the TCR repertoire in the lymphoid periphery

Using TCR V beta 5 transgenic mice as a model system, we demonstrate that the induction of peripheral tolerance can mold the TCR repertoire throughout adult life. In these mice, three distinct populations of peripheral T cells are affected by chronic selective events in the lymphoid periphery. First, CD4+V beta 5+ T cells are deleted in the lymphoid periphery by superantigens encoded by mouse mammary tumor viruses-8 and -9 in an MHC class II-dependent manner. Second, mature CD8+V beta 5+ T cells transit through a CD8lowV beta 5low deletional intermediate during tolerance induction by a process that depends upon neither mouse mammary tumor virus-encoded superantigens nor MHC class II expression. Third, a population of CD4-CD8-V beta 5+ T cells arises in the lymphoid periphery in an age-dependent manner. We analyzed the TCR V alpha repertoire of each of these cellular compartments in both V beta 5 transgenic and nontransgenic C57BL/6 mice as a function of age. This analysis revealed age-related changes in the expression of V alpha families among different cellular compartments, highlighting the dynamic state of the peripheral immune repertoire. Our work indicates that the chronic processes maintaining peripheral T cell tolerance can dramatically shape the available TCR repertoire.     

The structure of H-2K(b) and K(bm8) complexed to a herpes simplex virus determinant: evidence for a conformational switch that governs T cell repertoire selection and viral resistance

Polymorphism within the MHC not only affects peptide specificity but also has a critical influence on the T cell repertoire; for example, the CD8 T cell response toward an immunodominant HSV glycoprotein B peptide is more diverse and of higher avidity in H-2(bm8) compared with H-2(b) mice. We have examined the basis for the selection of these distinct antiviral T cell repertoires by comparing the high-resolution structures of K(b) and K(bm8), in complex with cognate peptide Ag. Although K(b) and K(bm8) differ by four residues within the Ag-binding cleft, the most striking difference in the two structures was the disparate conformation adopted by the shared residue, Arg(62). The altered dynamics of Arg(62), coupled with a small rigid-body movement in the alpha(1) helix encompassing this residue, correlated with biased Valpha usage in the B6 mice. Moreover, an analysis of all known TCR/MHC complexes reveals that Arg(62) invariably interacts with the TCR CDR1alpha loop. Accordingly, Arg(62) appears to function as a conformational switch that may govern T cell selection and protective immunity.     

The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical alpha beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors alpha beta T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either gamma delta T cells or NK cells. In contrast to NK cells, not all NKR(+) T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on alpha beta T cells of one donor. Overall, alpha beta T cells exhibited a distinct pattern of NKR expression different from that of gamma delta T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR(+) T cells sharing identical TCR alpha- and beta-chains. Remarkably, among 55 T cell clones sharing the same TCR alpha beta rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.     

Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.