TCR Vbeta Research Articles

Novel promiscuous HLA-DQ HIV Nef peptide that induces IFN-gamma-producing memory CD4+ T cells.

We describe the highly conserved sequence 56-68 of the HIV Nef protein as the first promiscuous HLA-DQ HIV-derived peptide. The Nef peptide exhibits an albeit rare capacity to bind 6 different HLA-DQ molecules whereas no binding is observed with the 10 HLA-DR molecules tested. In agreement with these data, after immunization with the Nef peptide, HLA-DQ transgenic Abeta degrees mice display a vigorous cellular and humoral response while the specific immune response of HLA-DR expressing mice is minimal. The promiscuous potentiality of the Nef 56-68 peptide in humans has been confirmed by ex vivo immunization experiments with CD4+ T cells from 14 healthy donors expressing different HLA genotypes. Nef 56-68 specific CD4+ T cells rapidly acquire a memory cell phenotype and are characterized by the preferential usage of the TCR Vbeta 6.1 gene segment and predominant production of IFN-gamma. Taken together, these data indicate that the Nef 56-68 peptide constitutes an attractive component of vaccines aiming at inducing or enhancing HIV-specific T cell immunity.

The ability of variant peptides to reverse the nonresponsiveness of T lymphocytes to the wild-type sequence p53(264-272) epitope.

Recently, we observed that CTL specific for the wild-type (wt) sequence p53(264-272) peptide could only be expanded ex vivo from PBMC of a subset of the HLA-A2.1(+) normal donors or cancer patients tested. Surprisingly, the tumors of the responsive patients expressed normal levels of wt p53 and could be considered unlikely to present this epitope. In contrast, tumors of nonresponsive patients accumulated mutant p53 and were more likely to present this epitope. We sought to increase the responsive rate to the wt p53(264-272) peptide of PBMC obtained from normal donors and patients by identifying more immunogenic variants of this peptide. Two such variants were generated by amino acid exchanges at positions 6 (6T) and 7 (7W) of the peptide. These variants were capable of inducing T cells from PBMC of nonresponsive donors that recognized the parental peptide either pulsed onto target cells or naturally presented by tumors. TCR Vbeta analysis of two T cell lines isolated from bulk populations of effectors reactive against the wt p53(264-272) peptide, using either the parental or the 7W variant peptide, indicated that these T cells were expressing identical TCR Vbeta13.6/complementarity-determining region 3/J region sequences. This finding confirms the heteroclitic nature of at least one of the variant peptides identified in this study. The use of variant peptides of the wt p53(264-272) epitope represents a promising approach to overcoming the nonresponsiveness of certain cancer patients to this self epitope, thereby enhancing its potential use in tumor vaccines for appropriately selected cancer patients.

Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with th2 phenotype acquisition in vivo.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vbeta repertoire, different from the TCR Vbeta profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IA(d)-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.

Cytotoxic HIV-1 p55gag-specific CD4+ T cells produce HIV-inhibitory cytokines and chemokines.

CD4+ T-helper cells appear to be essential in sustaining immune responses in chronic viral infections, as the maintenance of CD8+ cytotoxic T-lymphocyte responses and the control of viremia were demonstrated to depend on CD4+ T cell help. In order to investigate the function of HIV-specific CD4+ T cells in chronic HIV-1-infection, 49 chronically HIV-infected patients were analyzed before and 3 and 6 months after initiation of antiviral treatment. Ten patients showed a substantial, although weak, proliferative response to HIV-1-p55gag protein for which no improvement was observed upon initiation of HAART. From one individual, HIV-1-p55gag-specific CD4-positive T-cell clones were generated that were heterogeneous in their TCR Vbeta gene usage and HLA-DRB1*13 and DRB1*03 restricted, respectively. In addition, some CD4+ TCC produced substantial amounts of IFN-gamma and MIP-1alpha/beta were perforin-positive, and showed cytotoxic activity. These diverse functional features of HIV-specific CD4+ T cells suggest that they may exert direct antiviral activity.

Ex vivo analysis of phenotype and TCR usage in relation to CD45 isoform expression on cytomegalovirus-specific CD8+ T lymphocytes.

Human cytomegalovirus (CMV) is a ubiquitous pathogen which sets up a lifelong persistent infection and which can lead to significant disease in the immunosuppressed. The immunological mechanisms controlling CMV in the long term are not defined completely, but CD8+ T lymphocytes are thought to play an important role. Antiviral CD8+ T lymphocytes may exist in very large pools in healthy individuals. Although the detailed composition of these pools is not completely understood, there is known to be heterogeneity, in particular of CD45 isoform expression. We have therefore investigated the CD8+ T-lymphocyte response against CMV directly ex vivo using Class I tetramers combined with stains for a range of phenotypic markers followed by four-colour flow cytometric analysis. In particular, we examined expression of these phenotypic markers in relation to the expression of CD45 isoforms. We found that a spectrum of phenotypes exists stably, from CD45R0(high)/RA(low) through CD45RA(high)/R0(low), and that expression of other surface markers such as CD28 and CD62L, and also TCR usage, may vary in parallel with CD45 isoform expression. In some individuals, expansions of antigen-specific CD8+ T lymphocytes bearing specific TCR Vbeta chains were restricted to cells of particular CD45 isoforms. Immunity against CMV comprises a large population of CD8+ T lymphocytes with heterogeneous potential, a spectrum in which CD45 isoform expression may play a central role.

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR alpha/beta-mediated recognition.

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8(+) CTL has been shown to lyse certain natural killer (NK)-susceptible target cells via still undefined mechanism(s). These CD8(+) T cells, hereafter referred to as NK-CTL, are frequently composed of cells expressing one single TCR Vbeta expansion (different in different individuals), display a memory phenotype and express HLA class I-specific inhibitory NK receptors. Here we show that cell populations or clones of NK-CTL isolated from three healthy donors homogeneously expressed Vbeta16, Vbeta9 and Vbeta3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vbeta16(+) cells of donor 1 displayed identical TCR Vbeta and Valpha rearrangements, thus suggesting a substantial monoclonality of the NK-CTL subset analyzed. NK-CTL lysed a number of NK-susceptible tumor target cells with the exception of those characterized by beta2-microglobulin (beta2m) deficiency. However, the latter targets became susceptible to lysis upon beta2m transfection. Using monoclonal antibodies specific for the relevant TCR Vbeta or beta2m we provide evidence suggesting that target cell lysis by NK-CTL is mediated by the TCR itself upon recognition of beta2m-associated proteins. The cellular distribution of the potential beta2m-associated proteins in susceptible target cells suggested, as a likely candidate for TCR-mediated recognition, the non-classical HLA-E molecule. The use, as target cells, of the murine TAP2-deficient RMA-S cells, either untransfected or transfected with HLA-E, and loaded with an appropriate HLA-E-binding peptide, provided the direct demonstration that HLA-E represents a ligand recognized by the TCR expressed by NK-CTL. This is the first evidence that human TCR alpha/beta can recognize HLA-E molecules, thus revealing a novel type of TCR-mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.

Expression of inhibitory "killer cell lectin-like receptor G1" identifies unique subpopulations of effector and memory CD8 T cells.

T cell and natural killer (NK) cell functions are regulated by triggering of activating and inhibitory cell surface receptors. Here, we have studied the expression profile and predicted inhibitory function of mouse "killer cell lectin-like receptor G1" (KLRG1) on CD8 T cells. KLRG1 was present on 1 - 3 % of adult splenic CD8 cells that expressed CD8alpha beta heterodimers as well as a polyclonal TCR Vbeta repertoire indicative of conventional CD8 cells. The majority of KLRG1(+) CD8 cells belonged to the memory pool as determined by extensive phenotypic marker analysis. Spontaneous IFN-gamma production by approximately 20 % of KLRG1(+) CD8 cells identified them as pro-inflammatory effector cells. In contrast to NK cells, Ly49 and KLRG1 expression on CD8cells was found to be mutually exclusive. Therefore, distinct programs regulate KLRG1 expression in CD8 and NK cells. Finally, we provide evidence that KLRG1 triggering interferes with TCRalpha beta-mediated Ca(++) mobilization and cytotoxicity, raising the possibility that KLRG1 functionally participates in down-regulation of CD8 T cell responses.

Tissue-specific peptides influence human T cell repertoire to porcine xenoantigens.

Human CD8+ T cells elicit a vigorous response to allo- or xenogeneic MHC class I molecules. However, the influence of a given MHC-bound peptide to the responding allo- or xenoreactive T cell repertoire is not clear. In this study, we analyzed individual T cell responses to unique tissue epitopes presented on syngeneic porcine endothelial and lymphoblastoid cells by limiting dilution analysis and analyzed the responding T cell repertoire by T cell receptor beta (TCR Vbeta) chain spectrotyping. Both porcine endothelial and lymphoblastoid cells were able to elicit swine leukocyte antigen (SLA) class I restricted and peptide-dependent cytotoxic T lymphocyte (CTL) responses. The responding human CD8+ T cells showed a heterogenous but limited TCR Vbeta gene usage. Interestingly, although a large portion of the selected TCR Vbeta gene usage in response to endothelial and lymphoblastoid cells were shared (i.e., Vbeta-1, 2, 6.1, 13), unique Vbeta usage was noted in T cells that respond to either endothelial (Vbeta-5.3) or lymphoblastoid cells (Vbeta-5.1, 11), suggesting that porcine tissue-specific epitopes play a role in modulating the responding T cell repertoire. Limiting dilution cloning analysis revealed that a majority (89%) of the CTL clones stimulated by porcine endothelial cells recognized shared peptides presented by both endothelial cells and syngeneic lymphoblastoid cells. However, a significant portion (11%) of the CTL clones recognized unique peptides presented only in the context of SLA class I molecules on endothelial cells. These results provide evidence for the first time that tissue-specific peptides can directly influence T cell repertoire in response to the xenogeneic stimulus.

Analysis of the in vivo dendritic cell response to the bacterial superantigen staphylococcal enterotoxin B in the mouse spleen.

To investigate the in vivo effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) in the spleen, a single dose of SEB (50 microg/kg) was administered to BALB/c mice by intraperitoneal injection. Afterwards, the mice were sacrificed at 2, 6 and 24 hr, 2, 4, 7 and 15 days, and the spleens were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. The distribution patterns of DCs and their major costimulatory molecules, CD80, CD86 and CD40 in the spleen were identified, and the evidence for maturation of DCs in vivo in response to SEB was obtained. It was found that systemic administration of SEB induced the migration of most of the immature, splenic DCs from the marginal zone to the periarterial lymphatic sheath within 6 hr. This movement paralleled a maturation process, as assessed by upregulation of CD40, CD80 and CD86 expression in the interdigitating dendritic cells (IDCs). The upregulation of costimulatory molecule expression was conspicuous only in DCs in contrast to other antigen-presenting cells (APCs) such as macrophages and B cells which did not show any significant alterations in their costimulatory molecule expression. We also demonstrated the temporal expression pattern of these costimulatory molecules on the activated DCs. The upregulation of costimulatory molecules on DCs reached a peak level 6 hr after SEB injection, while the increase in number of T cells expressing T cell receptor V138 reached a peak level on day 2 after SEB treatment. In conclusion, we demonstrated the in vivo DC response to SEB in the mouse spleen, especially a potent stimulative effect of SEB on DCs in vivo, a temporal distribution pattern of DCs as well as T cells including TCR Vbeta8+ T cells, and a differential expression pattern of costimulatory molecules on the activated DCs. The results of the present study indicate that DCs are the principal type of APCs which mediate T cell activation by SAg in vivo, and that each costimulatory molecule may have different role in the activation of DCs by SAg. Thus, it is plausible to speculate that DCs play a critical role in the T cell clonal expansion by SAgs and other SAg-induced immune responses in vivo.

Competitive inhibition in vivo and skewing of the T cell repertoire of antigen-specific CTL priming by an anti-peptide-MHC monoclonal antibody.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2D(d) and a decamer peptide corresponding to residues 311-320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8(+) CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2D(d). This inhibition is specific for this particular peptide Ag; another H-2D(d)-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2L(d)-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2D(d)/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vbeta usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vbeta7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8(+) T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.

Evidence of T cell clonality in the infectious tolerance pathway: implications toward identification of regulatory T cells.

We have shown that a rare population of regulatory CD4+ T cells plays a key role in the acquisition of infectious tolerance in rat sensitized recipients of cardiac allografts pretreated with nondepleting anti-CD4 mAb. This study was designed to analyze the TCR Vbeta expression patterns in this transplantation model. First, we used Vbeta-specific RT-PCR to show that there was no differential usage of TCR Vbeta genes by T cells mediating rejection or tolerance. Indeed, graft-infiltrating lymphocytes expressed most of the 22 known rat TCR Vbeta genes in both recipient groups, suggesting unrestricted TCR Vbeta repertoire in alloreactive T cells. Then, we applied CDR3 spectrotyping of TCR beta-chain to assess the clonality of T cells at different anatomic sites. CDR3 size restriction, indicative of the presence of T cell clones, was observed in graft-infiltrating lymphocytes but not in draining lymph nodes or spleen of tolerant hosts. Consisent with the clonal expansion, T cells in tolerated grafts exhibited the memory phenotype at a much higher percentage as compared with peripheral lymphoid organs. Moreover, in tolerated graft-infiltrating lymphocytes, the CD3 size restriction occurred in limited Vbeta gene families, with Vbeta8.1 and Vbeta18 most frequently detected. Hence, T cells at the graft site of tolerant recipients contain T cell clones expressing selective Vbeta genes. This phenotypic characteristics of the tolerogenic GILs may potentially be used as a novel marker to identify operational regulatory T cells in organ allograft recipients.

V beta 6+ and V beta 4+ T cells exert cooperative activity in clearance of secondary infection with Histoplasma capsulatum.

We previously studied the lung Vbeta TCR repertoire of C57BL/6 mice during primary infection with the pathogen Histoplasma capsulatum. We observed a consistent oligoclonal expansion of Vbeta4(+) T cells during the peak of infection and early stages of resolution. The Vbeta4(+) family played a role in protective immunity against the fungus. Depletion of this subpopulation of T cells hindered optimal clearance of infection from tissues. In this report we analyze the flux of the Vbeta TCR repertoire in the lungs of C57BL/6 mice with reinfection histoplasmosis. We observed a significant increase in Vbeta6(+) T cells on days 7, 10, and 14, the peak and early resolution phases of infection. This skewing was preceded by an increased number of memory T cells within Vbeta6(+) cells. The VDJ sequences of Vbeta6 chains were oligoclonal during the early stages of the infection, suggesting that the expansion was driven by a small number of Ags. More than 96% of the expanded Vbeta6(+) cells were CD4(+). Depletion of Vbeta6(+) T cells but not Vbeta4(+) T cells induced a modest but significant delay in fungal clearance. Simultaneous depletion of Vbeta4(+) and Vbeta6(+) T cells induced a more pronounced impairment of host resistance. These studies illustrate the dynamic interactions between Vbeta families in the response to microbial challenge.

Differential transcriptional regulation of individual TCR V beta segments before gene rearrangement.

The promoter sequences of individual murine TCR Vbeta segments are dissimilar, but any functional differences between them are masked after productive gene rearrangement by the dominance of the TCRbeta 3' enhancer. However, thymocytes of recombination-activating gene-2 (Rag2)-deficient mice allow the transcriptional activity of Vbeta promoters to be studied before rearrangement. Here we report that many Vbeta segments are detectably transcribed in Rag2(-/-) thymocytes and that there are significant differences in expression among different Vbeta segments. Primer extension and characterization of cDNA clones from SCID thymocytes suggest that these germline Vbeta transcripts generally use the same start sites as those previously determined in mature T cells. The strength of expression before rearrangement does not correlate with proximity to the known enhancer, because members of the most distal Vbeta cluster (Vbeta2.1, Vbeta1.1, Vbeta4.1) are relatively strongly expressed and more proximal Vbeta segments (Vbeta14.1, Vbeta3.1, Vbeta7.1, Vbeta6.1) are only weakly expressed. Different Vbeta segments also show different developmental programs of activation in different thymocyte subsets, with the Vbeta5.1(L)-8.2(V) spliced transcript expressed earliest as well as most strongly overall. Comparison with Rag(+) MHC class I(-/-) and class II(-/-) thymocytes confirms that many of these expression differences are leveled by rearrangement and/or by beta selection, before MHC-dependent selection. However, the expression pattern of Vbeta2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles.

Peripheral T cell expansion in lymphopenic mice results in a restricted T cell repertoire.

In the absence of thymic contribution, the peripheral T cell pool is maintained by division of mature lymphocytes. Recent studies suggest that peripheral T cell expansion may be driven by low-affinity interactions with self ligands. Here we have investigated the consequence of homeostatic proliferation on the T cell repertoire. Following day 3 thymectomy of mice, there is a subsequent 30-fold expansion of the peripheral T cell population. Significantly, expansion of the T cell population results in skewed TCR Vbeta complementarity-determining region (CDR)3 length distributions and, in some cases, a marked bias toward one or two CDR3 lengths. TCR sequence analysis showed that these biases were a consequence of (oligo)clonal T cell expansion. Neonatally thymectomized adult mice have reduced antibody responses to primary challenge with T-dependent antigens. These data demonstrate that peripheral expansion of the T cell pool can result in a limited T cell repertoire, indicating that the array of stimulating ligands that drives homeostatic expansion is restricted.

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo.

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.

Alleviation of renal disease and lymphadenopathy in MRL-Fasp(lrcg)/Fas(lprcg) (MR-lpr(cg)) mice neonatally infected with mouse mammary tumor virus encoding superantigen strongly reactive with TCR Vbeta8.2 element.

Mouse mammary tumor virus transmitted by FM mice (FM-MMTV) encodes a superantigen (SAg) characterized by strong reactivity with TCR Vbeta8.2 element and broad spectrum of Vbeta reactivity. To investigate what effects the expression in vivo of FM-MMTV SAg exhibits on the course of the disease in a lupus-prone model, MRL/MpJ-Fas(lprcg)/Fas(lprcg) (MRL-lpr9cg) mice, neonatally FM-MMTV-infected MRL-lprcg(MMTV) and uninfected MRL-lpr(cg) mice were compared for various disease parameters. In MRL-lprcg(MMTV), survival was significantly prolonged, glomerulonephritis, proteinuria, and lymphadenopathy were clearly ameliorated, and the production of serum immunoglobulin G (IgG), complement-activating IgG2a, and cryogenic IgG3 autoantibodies, which are thought to be pathogenic to kidneys, and circulating immune complexes (IC), and glomerular IC deposition were significantly suppressed. FM-MMTV infection deleted Vbeta8.2+ cells by about 90% and Vbeta14+ cells less efficiently in all of the CD4+, CD8+, and B220+ CD4- CD8- or double-negative (DN) T-cell populations, and Vbeta8.1+ cells in the CD4+ population but not in the others. Similar deletion profiles of CD8+ and DN T cells support that DN T cells are derived from the CD8 lineage. The results imply that the specific regulation of the immune system with viral SAg has a potential for development of an attractive immunomodulatory therapy of autoimmune diseases.

Anti-TCR Vbeta-specific DNA vaccination prolongs heart allograft survival in adult rats.

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-beta chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vbeta13 and Vbeta2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vbeta, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vbeta13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vbeta families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vbeta13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

The role of adaptive immunity in atherosclerosis.

Atherosclerosis is an inflammatory disease induced by a lipid metabolic disturbance at sites of hemodynamic strain in the vasculature. Studies in both man and experimental animal models show an involvement of innate and adaptive immune mechanisms in the disease process. Our recent studies in apoE-knockout mice show that the level of hypercholesterolemia affects the functional properties of the immune response. Modulating immune activity by injections of polyclonal immunoglobulins inhibits disease progression, suggesting that immunomodulation may be useful to treat atherosclerosis. Analysis of T cell receptor (TCR) mRNA in atherosclerotic lesions shows expansions of T cells expressing TCR-V beta 6, a receptor type that is also expressed by T cells recognizing oxidized low density lipoprotein (oxLDL). This suggests that oxLDL is an autoantigen that induces strong, local T cell responses in the plaque. Further characterization of this and other candidate antigens, such as heat shock proteins and macromolecular components of Chlamydia pneumoniae, may provide important information on which specific interference with the disease process could be based.

Human CD4 residue Phe 43 is critical for repertoire development and maturation of MHC class II restricted CD4 single-positive T lineage cells in vivo.

To determine the functional significance of structural alteration of CD4-MHC class II interaction in vivo, two human (h)CD4-transgenic (tg) mice were established on a murine (m)CD4(-/-) H-2(b) background. The MHC class II binding-competent hCD4 (R240AhCD4) rescues the number and helper activity of hCD4(+)CD8(-) single-positive (SP) mature T cells in mCD4(-/-) mice. In contrast, the MHC class II binding-deficient F43I hCD4 mutant cannot facilitate normal differentiation of double-positive thymocytes to CD4(+)CD8(-) SP thymocytes. Hence, only 20 - 25% of CD4(+)CD8(-) SP T cells found in wild-type or R240A hCD4tg mice are generated, with resultant diminished helper responses. Differentiation of F43I hCD4 SP T cells is MHC class II but not class I dependent as demonstrated by crossing F43I hCD4tg mice onto MHC-deficient mice. These cells show a different pattern of TCR Valpha and Vbeta gene usage relative to comparable R240A hCD4 SP T cells from R240 AhCD4tg animals. Expression of activation markers including CD25 and CD69 on F43I hCD4 SP T cells suggests that autoreactive specificites may not have been eliminated intrathymically. Collectively, the results show that CD4-MHC class II interaction significantly influences intrathymic repertoire selection.

Impact of V beta 8+/+ T cells on the development of increased airway reactivity and IgE production in SJL mice.

SJL mice have been extensively characterized as "low-responder" animals in terms of IgE-dependent immediate-type hypersensitivity responses. Since these mice are genetically deficient in certain TCR Vbeta gene segments, we asked whether this might be the reason for the "low-responder" status. Specifically in H-2d mice the TCR-Vbeta8.2 gene element has been shown to play an important role in Th2 immune responses to ovalbumin (OVA). Utilizing a TCR Vbeta8. 2-transgenic SJL (SJL Vbeta8+/+) mouse, we examined whether the H-2s -bearing "low-responder" mouse could be converted into a "high-responder" animal. Remarkably, non-sensitized SJL Vbeta8+/+ mice demonstrated strongly elevated levels of total IgE antibody. Mitogen-stimulated T cells from these mice released high amounts of IL-4 as compared to SJL wild-type (wt) mice. In addition, sensitization to OVA via the airways resulted in the development of increased airway responsiveness in SJL Vbeta8+/+ mice, but not in SJL wt animals. The results indicate that the capacity to produce IgE and IL-4 and to develop increased airway responsiveness can be restored in SJL wt mice by introducing the Vbeta8.2 gene segment into the TCR repertoire.