Repertoire enhancement with adoptively transferred female lymphocytes controls the growth of pre-implanted murine prostate cancer

Background

In prostate cancer, genes encoding androgen-regulated, Y-chromosome-encoded, and tissue-specific antigens may all be overexpressed. In the adult male host, however, most high affinity T cells targeting these potential tumor rejection antigens will be removed during negative selection. In contrast, the female mature T-cell repertoire should contain abundant precursors capable of recognizing these classes of prostate cancer antigens and mediating effective anti-tumor immune responses.

Methodology/Principal Findings

We find that syngeneic TRAMP-C2 prostatic adenocarcinoma cells are spontaneously rejected in female hosts. Adoptive transfer of naïve female lymphocytes to irradiated male hosts bearing pre-implanted TRAMP-C2 tumor cells slows tumor growth and mediates tumor rejection in some animals. The success of this adoptive transfer was dependent on the transfer of female CD4 T cells and independent of the presence of CD25-expressing regulatory T cells in the transferred lymphocytes. We identify in female CD4 T cells stimulated with TRAMP-C2 a dominant MHC II-restricted response to the Y-chromosome antigen DBY. Furthermore, CD8 T cell responses in female lymphocytes to the immunodominant MHC I-restricted antigen SPAS-1 are markedly increased compared to male mice. Finally, we find no exacerbation of graft-versus-host disease in either syngeneic or minor-antigen mismatched allogeneic lymphocyte adoptive transfer models by using female into male versus male into male cells.

Conclusions/Significance

This study shows that adoptively transferred female lymphocytes, particularly CD4 T cells, can control the outgrowth of pre-implanted prostatic adenocarcinoma cells. This approach does not significantly worsen graft-versus-host responses suggesting it may be viable in the clinic. Further, enhancing the available immune repertoire with female-derived T cells may provide an excellent pool of prostate cancer reactive T cells for further augmentation by combination with either vaccination or immune regulatory blockade strategies.     

Chronic modulation of the TCR repertoire in the lymphoid periphery

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

Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28(-), CD57(+), CCR7(-)). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.     

Comprehensive analysis of the functional TCR repertoire at the single-cell level

A Vβ TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the Vα-Vβ pair is difficult to analyze because no suitable analytical method is available. Here, we have applied the single-cell 5′-RACE method for amplifying TCR cDNAs from single T-cells and analyzed the repertoire of Vα-Vβ pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR Vβ profile of the SEB-stimulated CD4⁺ T-cells was in accordance with the previous reports, that the TCR Vα profile also exhibited a prominent difference, and that the TCR Vα-Vβ pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCRα expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of Vα-Vβ pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics.     

A single-amino-acid variant of the H60 CD8 epitope generates specific immunity with diverse TCR recruitment

TCR of CD8 T cells recognizes peptides of 8–9 amino acids in length (epitope) complexed with MHC class I. Peptide ligands differing from an epitope by one or two amino acids are thought to modulate the immune response specific to that epitope. H60 is a minor histocompatibility antigen for which the specific CD8 T-cell response dominates during alloresponse after MHC-matched allogeneic transplantation. In the present study, we developed a transgenic mouse (designated H60H Tg) expressing a variant of H60, designated H60H, in which the arginine residue at position 4 of the H60 epitope sequence (LTFNYRNL) is replaced by a histidine residue (LTFHYRNL). Immunization of female C57BL/6 mice with splenocytes from male H60H Tg induced a CD8 T cell primary response and memory response after re-challenge. The response was CD4 help-dependent, demonstrating the potency of H60H as a cellular antigen. The response induced by the H60H cellular antigen was comparable to that induced by H60 in its peak magnitude and overall immune kinetics. H60H challenge recruited broadly diverse TCRs to the specific response, shaping a TCR repertoire different from that of the natural H60 epitope. However, some of the TCRs did overlap between the H60H- and H60-specific CD8 T cells, suggesting that H60H might modulate the H60-specific response. These results may provide a basis for the modulation of the H60-specific CD8 T-cell response.     

The impact of a large and frequent deletion in the human TCR β locus on antiviral immunity

The TCR plays a critical role in recognizing intracellular pathogens and initiating pathways leading to the destruction of infected cells by the immune system. Although genetic variability is known to greatly impact on the human immune system and the outcome of infection, the influence of sequence variation leading to the inactivation or deletion of TCR gene segments is unknown. To investigate this issue, we examined the CD8(+) T cell response to an HLA-B7-restricted epitope ((265)RPHERNGFTVL(275)) from the pp65 Ag of human CMV that was highly biased and frequently dominated by a public TCR β-chain encoded by the variable gene segment TRBV4-3. Approximately 40% of humans lack T cells expressing TRBV4-3 because of a 21.5-kb insertion/deletion polymorphism, but these individuals remain responsive to this epitope, using a diverse T cell repertoire characterized by private TCR usage. Although most residues within the bulged 11-mer peptide were accessible for TCR contact, the public and private TCRs showed distinct patterns of sensitivity to amino acid substitution at different positions within the peptide, thereby suggesting that the repertoire diversity generated in the absence of the dominant public TRBV4-3(+) TCR could lead to better protection from viral escape mutation. Thus, variation in the size of the TRBV repertoire clearly contributes toward interindividual variability in immune responses and is presumably maintained in many ethnic groups to enhance the diversity of Ag-specific T cell responses.     

Regulatory T cells constrain the TCR repertoire of antigen‐stimulated conventional CD4 T cells

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non‐self‐antigen, a TCRβ transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T‐cell repertoire was used. The response of EF4.1 mice to an I‐Ab‐associated epitope of the F‐MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg‐depleted EF4.1 mice were immunized, and the extent of the Vα2‐bearing, antigen‐specific TCR repertoire was characterized by high‐throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre‐immune repertoire. Injection of anti‐CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non‐self‐antigens.     

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

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

Differential role of lipid rafts in the functions of CD4+ and CD8+ human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4+ and CD8+ T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4+ and CD8+ T cells. The defect in IL-2 production by CD4+ T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56(Lck) and LAT and their association/recruitment with the lipid raft fractions of CD4+ and CD8+ T cells. p56Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4+ T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4+ and CD8+ T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4+ and CD8+ T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.     

Determinants of public T cell responses

Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.     

Antigen-specific T cell repertoire modification of CD4+CD25+ regulatory T cells

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).     

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαβ+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRβ repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.     

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

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

Characterization of MHC class-I restricted TCRαβ<Superscript>+</Superscript> CD4<Superscript>−</Superscript> CD8<Superscript>−</Superscript> double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination

The immune attack against malignant tumors require the concerted action of CD8⁺ cytotoxic T lymphocytes (CTL) as well as CD4⁺ T helper cells. The contribution of T cell receptor (TCR) αβ⁺ CD4⁻ CD8⁻ double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2⁺ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo. A. Mackensen and K. Fischer contributed equally to this work and should be considered joint senior authors. This work was supported by the Deutsche Forschungsgemeinschaft (MA 1351/5-1, KFO 146) and NIH grants CA90873, CA102280, 104947 (MIN). Companion paper: “Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition” by Tamson V. Moore et al. doi: .     

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

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

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.     

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.     

Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy

Negative selection is designed to purge the immune system of high-avidity, self-reactive T cells and thereby protect the host from overt autoimmunity. In this in vivo viral infection model, we show that there is a previously unappreciated dichotomy involved in negative selection in which high-avidity CD8(+) T cells specific for a dominant epitope are eliminated, whereas T cells specific for a subdominant epitope on the same protein preferentially escape deletion. Although this resulted in significant skewing of immunodominance and a substantial depletion of the most promiscuous T cells, thymic and/or peripheral deletion of high-avidity CD8(+) T cells was not accompanied by any major change in the TCR V beta gene family usage or an absolute deletion of a single preferred complementarity-determining region 3 length polymorphism. This suggests that negative selection allows high-avidity CD8(+) T cells specific for subdominant or cryptic epitopes to persist while effectively deleting high-avidity T cells specific for dominant epitopes. By allowing the escape of subdominant T cells, this process still preserves a relatively broad peripheral TCR repertoire that can actively participate in antiviral and/or autoreactive immune responses.     

Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.