The TCR repertoire of an immunodominant CD8+ T lymphocyte population

The TCR repertoire of an epitope-specific CD8(+) T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8(+) T cell population that recognizes a dominant epitope of the AIDS virus, the CD8(+) T cells recognizing the tetrameric Mamu-A*01/p11C(,CM) complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01(+) rhesus monkeys. This CD8(+) T cell population exhibited selected usage of TCR V beta families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8(+) T cell response was clearly polyclonal, a dominance of selected V beta(+) cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected V beta(+) cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other V beta(+) cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected V beta(+) cell subpopulations and clones and suggests that dominant V beta(+) cell subpopulations and clones can either be stable or evolve during a chronic infection.     

A TCR Affinity Threshold Regulates Memory CD4 T Cell Differentiation following Vaccination

Diverse Ag-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein Ags with TLR agonists are very potent at inducing T cell immune responses, but their capacity to elicit stable and diverse memory CD4 T cell repertoires has not been evaluated. In this study, we examined the evolution of a complex Ag-specific population during the transition from primary effectors to memory T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires, but peptide vaccines skewed the memory CD4 TCR repertoire toward high-affinity clonotypes whereas protein vaccines maintained low-affinity clonotypes in the memory compartment. CD27-mediated signaling was essential for the maintenance of low-affinity clonotypes after protein vaccination but was not sufficient to promote their survival following peptide vaccination. The rapid culling of the TCR repertoire in peptide-immunized mice coincided with a prolonged proliferation phase during which low-affinity clonotypes disappeared despite exhibiting no sign of enhanced apoptosis. Our study reveals a novel affinity threshold for memory CD4 T cell differentiation following vaccination and suggests a role for nonapoptotic cell death in the regulation of CD4 T cell clonal selection.     

Selection and long-term persistence of reactive CTL clones during an EBV chronic response are determined by avidity, CD8 variable contribution compensating for differences in TCR affinities.

Recent studies have suggested that the diversity of TCR repertoire after primary immunization is conserved in memory T cells and that a progressive narrowing of this repertoire may take place during recall infections. It now remains to be investigated which parameters determine the repertoire of the memory response and possibly restrict its diversity after subsequent antigenic challenges. To address this question, we took advantage of a panel of CD8+ T cell clones from the joint of a rheumatoid arthritis patient and selected for their reactivity against a single MHC/peptide complex. Characterization of both TCR chains documented a great diversity among those clones and the persistence of clonotypes over a 2-yr period. Strikingly, despite the observed repertoire heterogeneity, all clones displayed a narrow range of MHC/peptide density requirements in cytotoxicity assays (ED50 between 9 and 36 nM). TCR affinities were then indirectly estimated by blocking CD8 interaction with an anti-CD8 mAb. We found a wide range of TCR affinities among the different clonotypes that segregated with Vbeta usage. We thus propose that during an in vivo chronic response, a narrow range of avidity of the TCR-CD8 complex conditions long-term clonotype persistence, and that the level of CD8 contribution is adjusted to keep clonotypes with variable TCR affinities within this avidity window.     

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates

Antigen-specific CD8⁺ T cells play a key role in the host’s antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8⁺ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8⁺ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8⁺ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8⁺ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.     

GagCM9-specific CD8+ T cells expressing limited public TCR clonotypes do not suppress SIV replication in vivo

Several lines of evidence suggest that HIV/SIV-specific CD8(+) T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag(45-269)) that were subsequently infected with SIVsmE660. These seven Mamu-A*01(+) animals developed CD8(+) T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8(+) T cells could not control virus replication in vivo. GagCM9-specific CD8(+) T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8(+) T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20-250 GagCM9-specific CD8(+) T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8(+) T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8(+) T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8(+) T cell population elicited by vaccination and infection.     

Clonotype and repertoire changes drive the functional improvement of HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation

Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.     

Homogenization of TCR repertoires within secondary CD62Lhigh and CD62Llow virus-specific CD8+ T cell populations

Influenza virus-specific CD8(+) T cell clonotypes generated and maintained in C57BL/6J mice after respiratory challenge were found previously to distribute unequally between the CD62L(low) "effector" (T(EM)) and CD62L(high) "central" (T(CM)) memory subsets. Defined by the CDR3beta sequence, most of the prominent TCRs were represented in both the CD62L(high) and CD62L(low) subsets, but there was also a substantial number of diverse, but generally small, CD62L(high)-only clonotypes. The question asked here is how secondary challenge influences both the diversity and the continuity of TCR representation in the T(CM) and T(EM) subsets generated following primary exposure. The experiments use single-cell RT-PCR to correlate clonotypic composition with CD62L phenotype for secondary influenza-specific CD8(+) T cell responses directed at the prominent D(b)NP(366) and D(b)PA(224) epitopes. In both the acute and long-term memory phases of the recall responses to these epitopes, we found evidence of a convergence of TCR repertoire expression for the CD62L(low) and CD62L(high) populations. In fact, unlike the primary response, there were no significant differences in clonotypic diversity between the CD62L(low) and CD62L(high) subsets. This "TCR homogenization" for the CD62L(high) and CD62L(low) CD8(+) populations recalled after secondary challenge indicates common origin, most likely from the high prevalence populations in the CD62L(high) central memory set. Our study thus provides key insights into the TCR diversity spectrum for CD62L(high) and CD62L(low) T cells generated from a normal, unmanipulated T cell repertoire following secondary challenge. A better understanding of TCR selection and maintenance has implications for improved vaccine and immunotherapy protocols.     

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.     

Rapid Perturbation in Viremia Levels Drives Increases in Functional Avidity of HIV-specific CD8 T Cells

The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype, i.e. co-expressing PD-1/2B4/CD160. Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses.     

Antiretroviral therapy reduces the magnitude and T cell receptor repertoire diversity of HIV-specific T cell responses without changing T cell clonotype dominance

After initiation of antiretroviral therapy (ART), HIV loads and frequencies of HIV epitope-specific immune responses decrease. A diverse virus-specific T cell receptor (TCR) repertoire allows the host to respond to viral epitope diversity, but the effect of antigen reduction as a result of ART on the TCR repertoire of epitope-specific CD8(+) T cell populations has not been well defined. We determined the TCR repertoires of 14 HIV-specific CD8(+) T cell responses from 8 HIV-positive individuals before and after initiation of ART. We used multiparameter flow cytometry to measure the distribution of memory T cell subsets and the surface expression of PD-1 on T cell populations and T cell clonotypes within epitope-specific responses from these individuals. Post-ART, we noted decreases in the frequency of circulating epitope-specific T cells (P = 0.02), decreases in the number of T-cell clonotypes found within epitope-specific T cell receptor repertoires (P = 0.024), and an overall reduction in the amino acid diversity within these responses (P < 0.0001). Despite this narrowing of the T cell response to HIV, the overall hierarchy of dominant T cell receptor clonotypes remained stable compared to that pre-ART. CD8(+) T cells underwent redistributions in memory phenotypes and a reduction in CD38 and PD-1 expression post-ART. Despite extensive remodeling at the structural and phenotypic levels, PD-1 was expressed at higher levels on dominant clonotypes within epitope-specific responses before and after initiation of ART. These data suggest that the antigen burden may maintain TCR diversity and that dominant clonotypes are sensitive to antigen even after dramatic reductions after initiation of ART.     

Quantification of repertoire diversity of influenza-specific epitopes with predominant public or private TCR usage

The H-2Db-restricted CD8 T cell immune response to influenza A is directed at two well-described epitopes, nucleoprotein 366 (NP366) and acid polymerase 224 (PA224). The responses to the two epitopes are very different. The epitope NP366-specific response is dominated by TCR clonotypes that are public (shared by most mice), whereas the epitope PA224-specific response is private (unique within each infected animal). In addition to being public, the NP366-specific response is dominated by a few clonotypes, when T cell clonotypes expressing the Vbeta8.3 element are analyzed. Herein, we show that this response is similarly public when the NP366+Vbeta4+ CD8 T cell response is analyzed. Furthermore, to determine whether these features resulted in differences in total TCR diversity in the NP366+ and PA224+ responses, we quantified the number of different CD8 T clonotypes responding to each epitope. We calculated that 50-550 clonotypes recognized each epitope in individual mice. Thus, although the character of the response to the two epitopes appeared to be different (private and diverse vs public and dominated by a few clonotypes), similar numbers of precursor cells responded to both epitopes and this number was of similar magnitude to that previously reported for other viral CD8 T cell epitopes. Therefore, even in CD8 T cell responses that appear to be oligoclonotypic, the total response is highly diverse.     

Persistence of EBV Antigen-Specific CD8 T Cell Clonotypes during Homeostatic Immune Reconstitution in Cancer Patients

Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an in vivo clinical setting where lymphocyte homeostasis was transiently perturbed, we studied EBV antigen-specific CD8 T cells before and after non-myeloablative lympho-depleting chemotherapy of melanoma patients. Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes (CD27, IL7R, EOMES, CD62L/SELL and CCR5) supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.     

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.     

A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing

The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8(+) T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals.     

Identical beta cell-specific CD8(+) T cell clonotypes typically reside in both peripheral blood lymphocyte and pancreatic islets

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring beta cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8(+) T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3alpha and CDR3beta sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. beta cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Despite biased TRBV gene usage against a dominant HLA B57-restricted epitope, TCR diversity can provide recognition of circulating epitope variants

The role of epitope-specific TCR repertoire diversity in the control of HIV-1 viremia is unknown. Further analysis at the clonotype level is important for understanding the structural aspects of the HIV-1 specific repertoire that directly relate to CTL function and ability to suppress viral replication. In this study, we performed in-depth analysis of T cell clonotypes directed against a dominantly recognized HLA B57-restricted epitope (KAFSPEVIPMF; KF11) and identified common usage of the TCR beta-chain TRBV7 in eight of nine HLA B57 subjects examined, regardless of HLA B57 subtype. Despite this convergent TCR gene usage, structural and functional assays demonstrated no substantial difference in functional or structural avidity between TRBV7 and non-TRBV7 clonotypes and this epitopic peptide. In a subject where TRBV7-usage did not confer cross-reactivity against the dominant autologous sequence variant, another circulating TCR clonotype was able to preferentially recognize the variant peptide. These data demonstrate that despite selective recruitment of TCR for a conserved epitope over the course of chronic HIV-1 infection, TCR repertoire diversity may benefit the host through the ability to recognize circulating epitope variants.