A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity.

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

Structural and Mechanistic Implications of Rearrangement Frequencies within Human TCRBV Genes.

The T cell repertoire is a function of thymic V(D)J rearrangement and of peripheral selection. The mature repertoire embodies TCR sequences that are important for survival and can identify important structural aspects of the TCR. Analysis of the circulating TCRBV19 CD8 T cell repertoire showed that a majority of NDN-encoded CDR3 amino acid motifs start at CDR3 position four, well within the V region. Rearrangement at this position indicates that the DNA hairpin loop is not opened at the position adjacent to the recombination signal sequence, but rather is trimmed back three or more bases. In this article, we show that the rearrangement frequency distribution within the V region reveals selection on CDR3 position four. The selection is already established in single-positive CD8 thymocytes. Crystal structures reveal a possible basis for this selection due to the location of this residue in a bend that positions the remaining portion of CDR3 to interact with the peptide and MHC. Examination of other TCRBV families also shows selection for rearrangement within the V region of a number of genes and for CD8 and CD4 cells. The exact profile of rearrangement within the V region appears to be V gene specific. The frequent observation of side chains associated with turn motifs at CDR3 positions three and four fits with the structural need for a bend. The data are discussed in terms of the generation of a structural turn motif, the rearrangement mechanism, and selection of the repertoire on the peptide and MHC.

Narrowing of human influenza A virus-specific T cell receptor α and ß repertoires with increasing age.

UNLABELLED: Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses to this nonpersistent virus, to which recurrent exposure with new strains is common, changed over time with increasing age. Here, we show a direct correlation between increasing age and narrowing of the HLA-A2-restricted IAV Vα and Vß T cell repertoires specific to M1 residues 58 to 66 (M158-66), which simultaneously lead to oligoclonal expansions, including the usage of a single identical VA12-JA29 clonotype in all eight older donors. The Vα repertoire of older individuals also had longer CDR3 regions with increased usage of G/A runs, whose molecular flexibility may enhance T cell receptor (TCR) promiscuity. Collectively, these results suggest that CD8 memory T cell responses to nonpersistent viruses like IAV in humans are dynamic, and with aging there is a reduced diversity but a preferential retention of T cell repertoires with features of enhanced cross-reactivity. IMPORTANCE: With increasing age, the immune system undergoes drastic changes, and older individuals have declined resistance to infections. Vaccinations become less effective, and infection with influenza A virus in older individuals is associated with higher morbidity and mortality. Here, we questioned whether T cell responses directed against the highly conserved HLA-A2-restricted M158-66 peptide of IAV evolves with increasing age. Specifically, we postulated that CD8 T cell repertoires narrow with recurrent exposure and may thus be less efficient in response to new infections with new strains of IAV. Detailed analyses of the VA and VB TCR repertoires simultaneously showed a direct correlation between increasing age and narrowing of the TCR repertoire. Features of the TCRs indicated potentially enhanced cross-reactivity in all older donors. In summary, T cell repertoire analysis in older individuals may be useful as one of the predictors of protection after vaccination.

Emergence of T cells that recognize nonpolymorphic antigens during graft-versus- host disease.

Chronic GVHD is a major cause of morbidity and mortality in allogeneic stem cell transplantation recipients and typically develops from antecedent acute GVHD. In contrast to acute GVHD, chronic GVHD has much broader tissue involvement and clinical manifestations that bear striking similarity to what is observed in autoimmune diseases. How autoimmunity arises out of alloimmunity has been a longstanding unresolved issue. To address this question, in the present study, we performed a comprehensive analysis of the clonotypic T-cell response using complementary murine models that simulate what occurs during the transition from acute to chronic GVHD. These studies revealed repertoire skewing and the presence of high-frequency clonotypes that had undergone significant in vivo expansion, indicating that GVHD-associated autoimmunity was characterized by antigen-driven expansion of a limited number of T-cell clones. Furthermore, we observed that T cells with identical TCRß CDR3 nucleotide sequences were capable of recognizing donor and host antigens, providing evidence that the loss of self-tolerance during acute GVHD leads to the emergence of self-reactive donor T cells that are capable of recognizing nonpolymorphic tissue or commensally derived antigens. These data provide a mechanistic framework for how autoimmunity develops within the context of preexisting GVHD and provide additional insight into the pathophysiology of chronic GVHD.

The TCR repertoires of regulatory and conventional T cells specific for the same foreign antigen are distinct.

The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCRß-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2ß(+/-) TCRα(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of Jα usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.

Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

Corrigendum: Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

[This corrects the article DOI: 10.3389/fimmu.2024.1306490.].

Selective T cell expansion during aging of CD8 memory repertoires to influenza revealed by modeling.

The aging of T cell memory is often considered in terms of senescence, a process viewed as decay and loss of memory T cells. How senescence would affect memory is a function of the initial structure of the memory repertoire and whether the clonotypes that make up the repertoire decay at random. We examine this issue using the T cell memory generated to the conserved influenza A epitope M1(58-66), which induces a strong, focused, but polyclonal CD8 T cell response in HLA-A2 individuals. We analyzed the CD8 T cell memory repertoires in eight healthy middle-aged and eight healthy older blood donors representing an average age difference of ∼ 27 y. Although the repertoires show broadly similar clonotype distributions, the number of observable clonotypes decreases significantly. This decrease disproportionally affects low-frequency clonotypes. Rank frequency analysis shows the same two-component clonotype distribution described earlier for these repertoires. The first component includes lower frequency clonotypes for which distribution can be described by a power law. The slope of this first component is significantly steeper in the older cohort. Generating a representative repertoire for each healthy cohort allowed agent-based modeling of the aging process. Interestingly, simple senescence of middle-aged repertoires is insufficient to describe the older clonotype distribution. Rather, a selective clonotype expansion must be included to achieve the best fit. We propose that responses to periodic virus exposure may drive such expansion, ensuring that the remaining clonotypes are optimized for continued protection.

Naive T cell repertoire skewing in HLA-A2 individuals by a specialized rearrangement mechanism results in public memory clonotypes.

How the naive T cell repertoire arises and forms the memory repertoire is still poorly understood. This relationship was analyzed by taking advantage of the focused TCR usage in HLA-A2-restricted CD8 memory T cell responses to influenza M1(58-66). We analyzed rearranged BV19 genes from CD8 single-positive thymocytes, a surrogate for the naive repertoire, from 10 HLA-A2 individuals. CDR3 amino acid sequences associated with response to influenza were observed at higher frequencies than expected by chance, an indicator of preselection. We propose that a rearrangement mechanism involving long P-nucleotide addition from the J2.7 region explains part of this increase. Special rearrangement mechanisms can result in identical T cells in different individuals, referred to as public responses. Indeed, the rearrangements utilizing long P nucleotide additions were commonly observed in the response to the M1(58-66) epitope in 30 HLA-A2 middle-aged adults. Thus, in addition to negative and positive selection, special rearrangement mechanisms may influence the composition of the naive repertoire, resulting in more robust responses to a pathogen in some individuals.

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.

A clonotype nomenclature for T cell receptors.

T cell receptor (TCR) nucleotide sequences are often generated during analyses of T cell responses to pathogens or autoantigens. The most important region of the TCR is the third complementarity-determining region (CDR3) whose nucleotide sequence is unique to each T cell clone. The CDR3 interacts with the peptide and thus is important for recognizing pathogen or autoantigen epitopes. While conventions exist for identifying the various TCR chains, there is a lack of a concise nomenclature that would identify both the amino acid translation and nucleotide sequence of the CDR3. This deficiency makes the comparison of published TCR genetic and proteomic information difficult. To enhance information sharing among different databases and to facilitate computational assessment of clonotypic T cell repertoires, we propose a clonotype nomenclature. The rules for generating a clonotype identifier are simple and easy to follow, and have a built-in error-checking system. The identifier includes the V and J region, the CDR3 length as well as its human or mouse origin. The framework of this naming system could also be expanded to the B cell receptor.

Age-Based Dynamics of a Stable Circulating Cd8 T Cell Repertoire Component.

T-cell memory to pathogens can be envisioned as a receptor-based imprint of the pathogenic environment on the naïve repertoire of clonotypes. Recurrent exposures to a pathogen inform and reinforce memory, leading to a mature state. The complexity and temporal stability of this system in man is only beginning to be adequately described. We have been using a rank-frequency approach for quantitative analysis of CD8 T cell repertoires. Rank acts as a proxy for previous expansion, and rank-frequency, the number of clonotypes at a particular rank, as a proxy for abundance, with the relation of the two estimating the diversity of the system. Previous analyses of circulating antigen-experienced cytotoxic CD8 T-cell repertoires from adults have shown a complex two-component clonotype distribution. Here we show this is also the case for circulating CD8 T cells expressing the BV19 receptor chain from five adult subjects. When the repertoire characteristic of clonotype stability is added to the analysis, an inverse correlation between clonotype rank frequency and stability is observed. Clonotypes making up the second distributional component are stable; indicating that the circulation can be a depot of selected clonotypes. Temporal repertoire dynamics was further examined for influenza-specific T cells from children, middle-aged, and older adults. Taken together, these analyses describe a dynamic process of system development and aging, with increasing distributional complexity, leading to a stable circulating component, followed by loss of both complexity and stability.

Quantitative measurement of pathogen-specific human memory T cell repertoire diversity using a CDR3 beta-specific microarray.

BACKGROUND: Providing quantitative microarray data that is sensitive to very small differences in target sequence would be a useful tool in any number of venues where a sample can consist of a multiple related sequences present in various abundances. Examples of such applications would include measurement of pseudo species in viral infections and the measurement of species of antibodies or T cell receptors that constitute immune repertoires. Difficulties that must be overcome in such a method would be to account for cross-hybridization and for differences in hybridization efficiencies between the arrayed probes and their corresponding targets. We have used the memory T cell repertoire to an influenza-derived peptide as a test case for developing such a method. RESULTS: The arrayed probes were corresponded to a 17 nucleotide TCR-specific region that distinguished sequences differing by as little as a single nucleotide. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including an equimolar mixture of Cy3-labeled synthetic targets representing all 108 arrayed probes. The same synthetic targets were used to measure the degree of cross hybridization between probes. Reconstitution studies found the system sensitive to input ratios as low as 0.5% and accurate in measuring known input percentages (R2 = 0.81, R = 0.90, p < 0.0001). A data handling protocol was developed to incorporate the differences in hybridization efficiency. To validate the array in T cell repertoire analysis, it was used to analyze human recall responses to influenza in three human subjects and compared to traditional cloning and sequencing. When evaluating the rank order of clonotype abundance determined by each method, the approaches were not found significantly different (Wilcoxon rank-sum test, p > 0.05). CONCLUSION: This novel strategy appears to be robust and can be adapted to any situation where complex mixtures of highly similar sequences need to be quantitatively resolved.

CDR3 motif generation and selection in the BV19-utilizing subset of the human CD8 T cell repertoire.

The amino acids at the V-J rearrangement junction of TCR are encoded by the D region and by N or P nucleotides. Together they comprise the NDN region, the specific pMHC selection surface of the TCR ß-chain. As an extension of our earlier work on the recall response to influenza M158-66 in HLA-A2 individuals, we have been analyzing the circulating BV19 CD8 T cell repertoires. We observed that NDN regions of the CDR3 often start at positions that are V-region encoded. Here we examine NDN encoded amino acid motifs of BV19 rearrangements in circulating CD8 T cells based on the CDR3 length, the CDR3 start position of the NDN, and the motif length. Motifs that start at V region-encoded positions could be expected to be CDR3 length independent as indeed is the case. Motifs that included sequential proline and glycine showed a CDR3 length independent distribution and examining codon usage indicates that a large proportion of these can be explained by P-nucleotide addition from the 5' end of the D region. Other examples of skewed codon usage were observed indicating possible additional rearrangement mechanisms. Another pattern of motif distributions was a shift of position along the CDR3 as a function of the CDR3 length. As these data were collected from an older healthy individual they can be used to model successful repertoire selection and to further define characteristics associated with a positive history of responses to pathogen exposures.

CDR3 clonotype and amino acid motif diversity of BV19 expressing circulating human CD8 T cells.

Generating a detailed description of human T cell repertoire diversity is an important goal in the study of human immunology. The circulation is the source of most T cells used for studies in humans. Here we use high throughput sequencing of TCR BV19 transcripts from CD8 T cells derived from unmanipulated PBMC from an older HLA-A2 individual to provide a quantitative and qualitative description of the clonotypic CDR3 nucleotide and amino acid composition of the TCR ß-chain from this subset of circulating CD8 T cells. Aggregated samples from six time points spanning ∼1.5 years were analyzed to smooth possible temporal fluctuation. BV19 encompasses the well studied RS-encoding clonotypes involved in recognition of the M1(58-66) epitope from influenza A in HLA-A2 individuals. The clonotype distribution was diverse, complex and self-similar. The amino acid composition was generally skewed in favor of glycines and there were specific amino acids observed at higher frequency at the NDN start position. The motif repertoire distribution was also diverse, complex and self-similar with respect to CDR3 length, NDN start and length.

The functional CD8 T cell memory recall repertoire responding to the influenza A M1(58-66) epitope is polyclonal and shows a complex clonotype distribution.

The CD8 memory T cell repertoire to the influenza A derived M1(58-66) epitope shows a restricted V genes and CDR3 sequences usage. The repertoire is highly polyclonal and the clonotype distribution has been described as consisting of two components, one showing a power law-like distribution and the other composed of a few clonotypes with a very high relative frequency. The question is whether the complex repertoire defined by its ability to flourish in a short term recall culture corresponded to functional cells. Here we show that there is a relation between expression of the degranulation marker CD107 and cytotoxicity or IFN-γ production in CD8 T cell lines and clones. We then examine recently degranulated CD8 cells from recall cultures from four middle aged HLA-A2 subjects and show that these functional cells are polyclonal. The clonotype distributions of the CD8(+)CD107(+) repertoires are complex in the same manner as previously reported. The clonotype composition of CD8(+)CD107(+) repertoires is also very similar to CD8 only repertoires, and to CD8(+)HLA-A2-M1(58-66) pentamer positive repertoires. We postulate that multiple exposures during childhood to this conserved influenza A epitope has generated a complex functional repertoire in HLA-A2 individuals.

Developmental dynamics of post-selection thymic DN iNKT.

BACKGROUND: Invariant natural killer T (iNKT) cells develop in the thymus and branch off from the maturation pathway of conventional T cell at the DP stage. While different stages of iNKT cellular development have been defined, the actual time that iNKT cell precursors spend at each stage is still unknown. METHODOLOGY/PRINCIPAL FINDING: Here we report on maturation dynamics of post-selection DN iNKT cells by injecting wild-type DP(dim) thymocytes into the thymus of TCRα(-/-) mice and using the Vα14-Jα18 rearrangements as a molecular marker to follow the maturation dynamics of these cells. CONCLUSION/SIGNIFICANCE: This study shows that the developmental dynamics of DN iNKT cells in DP(dim) are very rapid and that it takes less than 1 day to down-regulate CD4 and CD8 and become DN. These DN cells are precursors of peripheral DN iNKT cells and appear in the spleen in 1-2 days. Thymic DN iNKT residents are predominantly derived from cells that quickly return from the periphery. The expansion of a very small subset of DN iNKT precursors could also play a small role in this process. These data are an example of measuring T cell maturation in the thymus and show that the maturation dynamics of selected DN iNKT cells fall within the same general time frame as conventional αß T cells.

Phospholipase C{gamma}1 is essential for T cell development, activation, and tolerance.

Phospholipase Cgamma1 (PLCgamma1) is an important signaling effector of T cell receptor (TCR). To investigate the role of PLCgamma1 in T cell biology, we generated and examined mice with T cell-specific deletion of PLCgamma1. We demonstrate that PLCgamma1 deficiency affects positive and negative selection, significantly reduces single-positive thymocytes and peripheral T cells, and impairs TCR-induced proliferation and cytokine production, and the activation of ERK, JNK, AP-1, NFAT, and NF-kappaB. Importantly, PLCgamma1 deficiency impairs the development and function of FoxP3(+) regulatory T cells, causing inflammatory/autoimmune symptoms. Therefore, PLCgamma1 is essential for T cell development, activation, and tolerance.