Cross-reactive memory T cells for Epstein-Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by T cells and its role in alloreactivity

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.     

The clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.     

Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity

BACKGROUND & AIMS: High rate of chronicity after acute hepatitis C virus (HCV) infection cannot be explained in the presence of a multispecific cytotoxic T lymphocyte (CTL) response. The aim of this study was to investigate the effect of virus variants on CTL activity in patients in whom chronicity developed. METHODS: CTL clones specific to a decapeptide epitope derived from hypervariable region 1 were generated from 5 HLA-A2-positive patients with acute hepatitis C by in vitro stimulation with synthetic peptides. The sequential change of this CTL epitope and its influence on the CTL recognition were examined. RESULTS: Virus variants did not appear in 3 patients with recovery, whereas variants with altered peptide ligands capable of antagonizing CTL activity emerged rapidly in the remaining 2 patients in whom chronicity developed. Importantly, these HLA-A2-restricted, hypervariable region 1-specific CTL clones shared the use of T-cell receptor (TCR) genes AV6 and BV17. CONCLUSIONS: These data suggest that there is only a narrow T-cell repertoire responding to a single viral peptide/HLA ligand. The emergence of HCV variants with altered peptide ligands as TCR antagonists accompanied by a limited TCR repertoire may provide a mechanism for HCV chronicity.     

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.     

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.     

Accessing Epstein-Barr virus-specific T-cell memory with peptide-loaded dendritic cells

The conventional means of studying Epstein-Barr virus (EBV)-induced cytotoxic T-lymphocyte (CTL) memory, by in vitro stimulation with the latently infected autologous lymphoblastoid cell line (LCL), has important limitations. First, it gives no information on memory to lytic cycle antigens; second, it preferentially amplifies the dominant components of latent antigen-specific memory at the expense of key subdominant reactivities. Here we describe an alternative approach, based on in vitro stimulation with epitope peptide-loaded dendritic cells (DCs), which allows one to probe the CTL repertoire for any individual reactivity of choice; this method proved significantly more efficient than stimulation with peptide alone. Using this approach we first show that reactivities to the immunodominant and subdominant lytic cycle epitopes identified by T cells during primary EBV infection are regularly detectable in the CTL memory of virus carriers; this implies that in such carriers chronic virus replication remains under direct T-cell control. We further show that subdominant latent cycle reactivities to epitopes in the latent membrane protein LMP2, though rarely undetectable in LCL-stimulated populations, can be reactivated by DC stimulation and selectively expanded as polyclonal CTL lines; the adoptive transfer of such preparations may be of value in targeting certain EBV-positive malignancies.     

CD4+ type 1 and CD8+ type 2 T cell subsets in human leishmaniasis have distinct T cell receptor repertoires

The mechanism of protective immunity and immunologic resistance against intracellular pathogens is believed to involve the activation of Ag-specific T cells. The T cells involved in protection/resistance to Leishmania can be studied using localized American cutaneous leishmaniasis (LCL) as a model, because the disease is often self-healing. Our study was undertaken to identify specific T cell populations that had accumulated in LCL lesions on the basis of TCR V beta gene usage. RNA was derived from skin lesions and blood of eight LCL patients, as well as from purified CD4+ and CD8+ subsets from the lesions and blood of three patients. After synthesis of cDNA, V beta gene usage was assessed by polymerase chain reaction. In all eight patients, several V beta gene families were overrepresented in lesions compared to blood. More importantly, the TCR V beta repertoires of both lesional CD4+ and CD8+ subsets were skewed compared to the repertoire of the respective subsets in the blood of the same donor. The overrepresented V beta s in the CD4+ and CD8+ subsets from lesions were in most instances disparate, particularly with the V beta 6 TCR skewed in the lesional CD8+ subset. Not only were the TCR repertoires of the overrepresented V beta in the lesional CD4+ and CD8+ subsets generally distinct, but the cytokine mRNA expressed by these subsets were also discrete. Strikingly, the CD4+ subset was characterized by IFN-gamma mRNA expression and the CD8+ subset by IL-4 and IL-10 mRNA expression. These data indicate that the pathogenesis of human leishmaniasis may be explained by the balance of CD4+ type 1 and CD8+ type 2 T cells, which probably recognize distinct sets of Ag.     

A class I MHC-restricted recall response to a viral peptide is highly polyclonal despite stringent CDR3 selection: implications for establishing memory T cell repertoires in "real-world" conditions

In this study, we analyze the recall response to influenza A matrix peptide M1(58-66) restricted by HLA-A2 in one individual and find a strict CDR3 selection as well as a high degree of polyclonality. The TCR beta-chain repertoire of memory T cells specific for this Ag system has been shown previously to be constrained by the use of the BV17 family and the I/sRS(A)/S amino acid motif in the CDR3 region. Our sequence analysis of BV17 TCR from a CTL line showed the repertoire to be highly polyclonal, as 95 distinct CDR3 sequences (clonotypes) were identified expressing this CDR3 motif. The clonotype frequencies showed a power law distribution with an extensive low-frequency tail. The clonotypes present in the high-frequency component of the distribution could be measured directly in the PBMC. This measurement showed that the relative frequencies of these clonotypes before stimulation were similar to their frequencies after culturing. Analysis of short-term cultures showed that the responding clonotypes have a similar ability to proliferate, which is independent of TCR beta-chain CDR3 sequence or precursor frequency. These data indicate that the memory T cell repertoire is composed of a surprisingly diverse set of T cell clonotypes with a limited potential for expansion. We propose that the high-frequency component represents T cells that have existed the longest. In keeping with this hypothesis, these clonotypes were measured over a 2-year period, during which their precursor frequency did not change.     

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.     

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.     

Influence of HLA genes on T cell receptor V segment frequencies and expression levels in peripheral blood lymphocytes

The effect of the HLA complex on the TCR repertoire in human peripheral blood was assessed by using nine V beta- and V alpha-specific mAb and the quantitative polymerase chain reaction specific for 22 V beta segments. Studies in randomly selected and unrelated individuals failed to show any influence of the HLA complex on the TCR repertoire. In contrast, studies in large families with multiple siblings showed a strong influence on the TCR repertoire by the HLA complex. In pairwise comparisons, HLA-identical sibs had more similar patterns of V segment frequencies, as measured with the nine V segment-specific mAb, as well as more similar expression levels of V beta-specific RNA, as measured by quantitative polymerase chain reaction, than totally mismatched or haplo-identical sibs. When the amount of V beta-specific RNA expressed in CD4+ and CD8+ T cells was compared, it was found that V beta 2, 5.1, 9, and 20 were skewed toward CD4+ T cells; on the other hand, V beta 7 and 14 showed a bias in expression for CD8+ T cells, suggesting that the former were positively selected predominantly by HLA class II gene products whereas the latter V beta segments were positively selected predominantly by HLA class I gene products. These studies unequivocally document the effects of HLA genes on TCR V segment frequencies and expression levels in peripheral blood T lymphocytes.     

Swedish gynecologists'' and general practitioners'' views on the climacteric period: knowledge, attitudes and management strategies

The cytolytic T lymphocyte (CTL) response has often been used to assess the reconstitution of T cell function after allogeneic or autologous bone marrow transplantation (BMT). Less is known, however, about the reconstitution of the CTL response after peripheral blood stem cell transplantation (PBSCT). Therefore, we investigated the CTL response against Epstein-Barr virus (EBV) of patients undergoing autologous PBSCT. CTLs of six patients with relapsed non-Hodgkin's lymphoma and multiple myeloma were established before and at different times after PBSCT by in vitro stimulation of peripheral blood lymphocytes with autologous EBV-transformed lymphoblastoid cell lines (LCLs). The efficiency of T cell priming by LCLs was assessed at the time of initiation of CTL lines; the proliferative response was strongly reduced during the first 4 months and increased 5 months or more following PBSCT. Cytolytic activity was measured after three or four restimulations of CTLs. All patients investigated had a detectable EBV-specific CTL response which was poor during the first weeks after transplantation, accompanied by a strong non-MHC-restricted cytotoxic activity and a high proportion of CD56-positive T cells. Five or more months after PBSCT, a specific CTL response against EBV was seen which was similar to the situation prior to PBSCT, while the unspecific cytotoxic response decreased. Blocking experiments with monoclonal anti-CD3, anti-CD8 or anti-MHC I antibodies resulted in substantial inhibition of autologous LCL lysis, whereas anti-CD4 or anti-MHC II antibodies had no effect. Finally, autologous PHA blasts of a patient with the HLA haplotype A1/9+, B5/8+, Cw4/7+, were loaded with various EBNA-derived nonapeptides known to be presented by HLA B8 or A11, and exposed to autologous, EBV-directed CTLs. Specific lysis by CTLs only occurred with HLA B8-, but not with HLA A11-restricted nonapeptides. This demonstrated the existence of an MHC I-restricted anti-EBV CTL response after PBSCT. Taken together, the results show that the anlaysis of the EBV-directed CTL activity may serve as a surrogate marker to assess the reconstitution of the cellular immune response in patients undergoing autologous PBSCT.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Hemoglobin autooxidation/oxidation mechanisms and methemoglobin prevention or reduction processes in the bloodstream. Literature review and outline of autooxidation reaction

The human carcinoembryonic antigen (CEA), which is expressed in several cancer types is a potential target for antigen-specific immunotherapy. In this study, we show that dendritic cells (DC) pulsed with an HLA class I restricted CEA cytotoxic T lymphocyte (CTL) peptide epitope can stimulate T cells to kill CEA peptide loaded T2 target cells as well as CEA expressing tumor lines in the presence of interleukin-7 (IL-7) in an HLA-restricted manner. This has been demonstrated for carcinoma patients as well as healthy donors. The DC-CEA + IL-7 stimulated cultures contained predominantly CD3+CD8+CD56- cells indicative of MHC class I restricted CTL. In addition, DC-CEA + IL-7 stimulated cells showed higher levels of CD69 expression compared with cells stimulated with IL-7 alone, implying an activated phenotype. When the T-cell receptor (TCR) from CTL cultures stimulated with DC-CEA + IL-7 was analyzed, an oligoclonal pattern of expression was found for certain V beta subfamilies compared with the polyclonal patterns shown by IL-7 or phytohemagglutinin stimulated T cells from the same donors. This TCR restriction appeared to be maintained and enhanced after additional rounds of restimulation with DC-CEA + IL-7. The association between cytotoxicity and TCR restriction suggests that TCR analysis may be useful as an in vitro indicator to monitor alterations in the T-cell population in response to antigen-specific immunotherapies.     

Dominant clonotypes in the repertoire of peripheral CD4+ T cells in rheumatoid arthritis

Clonal expansion of T cell specificities in the synovial fluid of patients has been taken as evidence for a local stimulation of T cells. By studying the T cell receptor (TCR) repertoire of CD4+ T cells in the synovial and peripheral blood compartments of patients with early rheumatoid arthritis (RA), we have identified clonally expanded CD4+ populations. Expanded clonotypes were present in the peripheral blood and the synovial fluid but were not preferentially accumulated in the joint. Dominant single clonotypes could not be isolated from CD4+ cells of HLA-DRB1*04+ normal individuals. Clonal expansion involved several distinct clonotypes with a preference for V beta 3+, V beta 14+, and V beta 17+CD4+ T cells. A fraction of clonally related T cells expressed IL-2 receptors, indicating recent activation. The frequencies of clonally expanded V beta 17+CD4+ T cells fluctuated widely over a period of one year. Independent variations in the frequencies of two distinct clonotypes in the same patient indicated that different mechanisms, and not stimulation by a single arthritogenic antigen, were involved in clonal proliferation. These data support the concept that RA patients have a grossly imbalanced TCR repertoire. Clonal expansion may result from intrinsic defects in T cell generation and regulation. The dominance of expanded clonotypes in the periphery emphasizes the systemic nature of RA and suggests that T cell proliferation occurs outside of the joint.     

Evolutionary dynamics of genetic variation in Epstein-Barr virus isolates of diverse geographical origins: evidence for immune pressure-independent genetic drift

The question whether immune pressure exerted by cytotoxic T lymphocytes (CTLs) can influence the long-term evolution of genetically stable viruses such as Epstein-Barr virus (EBV) has generated considerable scientific interest, primarily due to its important implications for the overall biology of the virus. While arguing for a role of CTLs in the evolution of viruses, it is important to differentiate between genetic variation in virus and immune recognition of these variant virus by CTLs. To assess the role of genetic selection in the long-term evolution of EBV, we have analyzed a large panel of type 1 EBV isolates from African, Southeast Asian, Papua-New Guinean (PNG), and Australian Caucasian individuals. Seven different regions of the EBV genome, which include nine CTL epitopes restricted through a range of HLA class I alleles, were sequenced and compared. Although numerous nucleotide changes were identified within these isolates, comparison of synonymous and nonsynonymous substitutions in the CTL epitope indicated that the genetic variation was generated mostly independently of immune selection pressure. Surprisingly, an inverse correlation between genetic variation within certain CTL epitopes and the frequency distribution of HLA alleles that present the CTL epitopes was seen, suggesting that the evolutionary pressures on the CTL epitopes of the virus may be toward their conservation rather than their inactivation. Furthermore, molecular evolutionary genetic analysis of nucleotide sequences revealed that viral isolates from PNG are evolving as a lineage distinct from isolates from African, Southeast Asian, and Australian Caucasian individuals.     

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection

In HIV-1 infection, circulating HIV-1-specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV-1-specific CTL is capable of clonal expansion upon culture with IL-2 without exogenous antigen. The IL-2-expandable HIV-1-specific CTL precursor frequency was reduced in patients with advancing infection, although HIV-1-specific memory CTL could still be detected by stimulation in vitro with allele-specific HIV-1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL-2-expandable HIV-1-specific CTL precursor (CTLp) frequency without a decline in Epstein-Barr virus (EBV)-specific or allo-specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL-2-expandable HIV-specific CTL response, the requirements for in vitro generation of HIV-1-specific and EBV-specific effector CTL were examined. In the absence of exogenous IL-2 in limiting dilution, generation of EBV-specific CD8+ effector CTL was dependent upon help from CD4+ cells. CD4+ help for EBV-specific CD8+ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL-2, CD4+ cells could also provide help for the optimal generation of HIV-1 peptide-specific CD8+ CTL, because in vitro depletion of CD4+ cells prior to culture using stimulation with an MHC class I-restricted HIV-1 peptide reduced the peptide-specific CD8+ CTL response. We conclude that there is a decline in the IL-2-expandable HIV-1-specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4+ T cell help for in vivo antigen-activated CD8+ T cells.     

Unusually high frequency of Epstein-Barr virus genetic variants in Papua New Guinea that can escape cytotoxic T-cell recognition: implications for virus evolution

Cytotoxic T lymphocytes (CTLs) which recognize viral antigens in association with human leukocyte antigens (HLAs) play an important role in controlling persistent virus infections. These viruses use several mechanisms to evade the immune response, including mutations that affect either T-cell receptor recognition or binding of viral epitopes to the HLA. It has recently been proposed that the distribution of HLA frequencies and the specific CTL response may influence the long-term evolution of Epstein-Barr virus (EBV) by selecting variants which lack immunodominant CTL epitopes. To test this hypothesis, we have studied EBV isolates from two genetically distinct Papua New Guinea (PNG) populations, residing in coastal and highland regions, for polymorphism within seven viral CTL epitope sequences restricted through several class I HLAs. Surprisingly, all EBV isolates analyzed displayed identical amino acid substitutions within HLA A11-, B35- and B8-restricted CTL epitope sequences which completely abrogated CTL recognition and binding of synthetic peptides to HLA molecules. Furthermore, these substitutions revealed no correlation with the contemporary distribution of HLAs in the different PNG populations, which argues for a minimal influence of immune pressure. The sequence homology between EBV isolates from coastal and highland PNG suggests that the virus may have had a single origin and, more importantly, that these isolates are genetically distinct from those present in a Caucasian population.     

TCR alpha-chain usage can determine antigen-selected TCR beta-chain repertoire diversity

There is considerable variation in the TCR repertoire diversity selected by different peptide Ags. Certain responses show limited V region bias with minimal restrictions in the remainder of the sequence while others can be dominated by a single TCR clonotype repeatedly isolated from different individuals. CTL specific for a Kb-restricted determinant from the herpes simplex virus glycoprotein B (gB) preferentially express a dominant TCRBV10 beta-chain subset with extensive conservation located at the V-D junction. However, unlike some biased responses, no single beta-chain V-D-J combination appears to dominate these CTL. Different animals respond with a large array of unique or "private" beta-chain sequences with little J region preference. Here we examine the contribution of the TCR alpha-chain to the gB-specific CTL diversity. The TCR alpha-chains from different TCRBV10-positive gB-specific CTL clones were found to exhibit extensive sequence variation. However, when T cells were forced to use a single alpha-chain in TCR alpha-chain transgenic mice, gB-specific CTL showed limited variation in their beta-chain selection. These T cells retained the TCRBV10 bias but were now dominated by a single beta-chain sequence that could be repeatedly isolated from different transgenic animals. This "public" TCR consisted of the transgenic alpha-chain and a common TCRBV10D2J2S6 beta-chain. These results suggest that preferential use of one TCR subunit can restrict the level of diversity in the other chain due to interchain interactions involving J-derived sequences.     

Alterations in TCR-MHC contacts subsequent to cross-recognition of class I MHC and singly substituted peptide variants

Vesicular stomatitis virus (VSV) elicits H-2Kb-restricted CTLs specific for the immunodominant VSV octapeptide RGYVYQGL. To study the structural features important for interaction between the TCR beta-chain and the peptide/MHC complex, we immunized TCR alpha-chain transgenic mice with the VSV peptide and raised a panel of anti-VSV CTL clones with identical TCR alpha-chains. Consistent with our previous analysis of uncloned populations of primary CTLs, the anti-VSV CTL clones were all Vbeta13+ and expressed TCR beta-chains with highly homologous complementarity-determining region 3 (CDR3) loops. Although the clones expressed similar TCRs, they differed in their ability to cross-react with VSV peptide variants singly substituted at TCR contact positions 4 and 6. These findings allowed us to identify short stretches of amino acids in the C-terminal region of the CDR3beta loop that, when altered, modify the cross-reaction capability of the TCR to position 4 and position 6 variant peptides. To further probe the structural correlates of biologic cross-reactivity, we used cross-reactive CTL clones and cell lines expressing point mutations in H-2Kb to investigate the effect of single amino acid changes in the peptide on the pattern of recognition of the TCR for the peptide/MHC complex. Single conservative substitutions in the peptide were sufficient to alter the recognition contacts between a cross-reactive TCR and the MHC molecule, supporting the idea that the TCR can make overall structural adjustments in MHC contacts to accommodate single amino acid changes in the peptide.