Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Evidence for in vivo primed and expanded autoreactive T cells as a specific feature of patients with type 1 diabetes

Identifying beta cell autoantigen-reactive T cells that are involved in the pathogenesis of type 1 diabetes has been troublesome for many laboratories. Disease-relevant autoreactive T cells should be in vivo Ag experienced. The aim of this study was to test this hypothesis and then use this principle as a strategy for identifying diabetes-relevant autoreactive T cells. In this study, a CSFE dilution assay was used to detect glutamic acid decarboxylase 65 (GAD65)- and insulin-responsive T cells and HLA-0201*-GAD65(114-122) pentamers were used to detect CD8(+) GAD-responsive T cells in memory CD45RO(+) and naive CD45RO(-) cell populations from patients with type 1 diabetes and healthy control subjects. T cell proliferative history was evaluated by flow cytometry telomere length measurement. CD4(+) and CD8(+) T cells specific for GAD65 and insulin were present in patients with type 1 diabetes and control subjects. Within the naive CD45RO(-) cells, CD4(+) and CD8(+) T cell responses were similar between patients and controls. Within the memory CD45RO(+) cells, CD4(+) T cell responses against whole GAD65 and insulin and HLA-0201*-GAD65(114-122) pentamer-positive CD8(+) T cells were found in patients with type 1 diabetes, but not in control subjects (p < 0.05 for all). Responding cells from the CD45RO(+) T cell population had substantially shorter telomere lengths than responding cells from the CD45RO(-) cell population. Diabetes-specific autoreactive T cells in the circulation have uniquely undergone sustained in vivo proliferation and differentiation into memory T cells. Prior selection of these cells is possible and is a way to identify diabetes-relevant target Ags and epitopes.     

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.     

Immunization of HLA class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells. CD8(+) T cells have recently been assigned a major role in beta cell injury. Consequently, the identification of autoreactive CD8(+) T cells in humans remains essential for development of therapeutic strategies and of assays to identify aggressive cells. However, this identification is laborious and limited by quantities of human blood samples available. We propose a rapid and reliable method to identify autoantigen-derived epitopes recognized by human CD8(+) T lymphocytes in T1D patients. Human histocompatibility leukocyte Ags-A*0201 (HLA-A*0201) transgenic mice were immunized with plasmids encoding the T1D-associated autoantigens: 65 kDa glutamic acid decarboxylase (GAD) or insulinoma-associated protein 2 (IA-2). Candidate epitopes for T1D were selected from peptide libraries by testing the CD8(+) reactivity of vaccinated mice. All of the nine-candidate epitopes (five for GAD and four for IA-2) identified by our experimental approach were specifically recognized by CD8(+) T cells from newly diagnosed T1D patients (n = 19) but not from CD8(+) T cells of healthy controls (n = 20). Among these, GAD(114-123), GAD(536-545) and IA-2(805-813) were recognized by 53%, 25%, and 42% of T1D patients, respectively.     

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.     

Cutting Edge: Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens

Constitutive presentation of tissue Ags by dendritic cells results in tolerance of autoreactive CD8+ T cells; however, the underlying molecular mechanisms are not well understood. In this study we show that programmed death (PD)-1, an inhibitory receptor of the CD28 family, is required for tolerance induction of autoreactive CD8+ T cells. An antagonistic Ab against PD-1 provoked destructive autoimmune diabetes in RIP-mOVA mice expressing chicken OVA in the pancreatic islet cells, which received naive OVA-specific CD8+ OT-I cells. This effect was mediated by the PD ligand (PD-L) PD-L1 but not by PD-L2. An increased number of effector OT-I cells recovered from the pancreatic lymph nodes of anti-PD-L1-treated mice showed down-regulation of PD-1. Furthermore, the blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect OT-I cell division but enhanced its granzyme B, IFN-gamma, and IL-2 production. Thus, during the presentation of tissue Ags to CD8+ T cells, PD-1/PD-L1 interaction crucially controls the effector differentiation of autoreactive T cells to maintain self-tolerance.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

Systemic administration of agonist peptide blocks the progression of spontaneous CD8-mediated autoimmune diabetes in transgenic mice without bystander damage

Insulin-dependent diabetes is an autoimmune disease targeting pancreatic beta-islet cells. Recent data suggest that autoreactive CD8+ T cells are involved in both the early events leading to insulitis and the late destructive phase resulting in diabetes. Although therapeutic injection of protein and synthetic peptides corresponding to CD4+ T cell epitopes has been shown to prevent or block autoimmune disease in several models, down-regulation of an ongoing CD8+ T cell-mediated autoimmune response using this approach has not yet been reported. Using CL4-TCR single transgenic mice, in which most CD8+ T cells express a TCR specific for the influenza virus hemagglutinin HA512-520 peptide:Kd complex, we first show that i.v. injection of soluble HA512-520 peptide induces transient activation followed by apoptosis of Tc1-like CD8+ T cells. We next tested a similar tolerance induction strategy in (CL4-TCR x Ins-HA)F1 double transgenic mice that also express HA in the beta-islet cells and, as a result, spontaneously develop a juvenile onset and lethal diabetes. Soluble HA512-520 peptide treatment, at a time when pathogenic CD8+ T cells have already infiltrated the pancreas, very significantly prolongs survival of the double transgenic pups. In addition, we found that Ag administration eliminates CD8+ T cell infiltrates from the pancreas without histological evidence of bystander damage. Our data indicate that agonist peptide can down-regulate an autoimmune reaction mediated by CD8+ T cells in vivo and block disease progression. Thus, in addition to autoreactive CD4+ T cells, CD8+ T cells may constitute targets for Ag-specific therapy in autoimmune diseases.     

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.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

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.     

Myeloid-derived suppressor cells prevent type 1 diabetes in murine models

Effective immunotherapy for type 1 diabetes (T1D) relies on active induction of peripheral tolerance. Myeloid-derived suppressor cells (MDSCs) play a critical role in suppressing immune responses in various pathologic settings via multiple mechanisms, including expansion of regulatory T cells (Tregs). In this study, we investigated whether MDSCs could act as APCs to induce expansion of Ag-specific Tregs, suppress T cell proliferation, and prevent autoimmune T1D development. We found that MDSC-mediated expansion of Tregs and T cell suppression required MHC-dependent Ag presentation. A murine T1D model was established in INS-HA/RAG(-/-) mice in which animals received CD4-HA-TCR transgenic T cells via adoptive transfer. We found a significant reduction in the incidence of diabetes in recipients receiving MDSC plus HA, but not OVA peptide, leading to 75% diabetes-free mice among the treated animals. To test further whether MDSCs could prevent diabetes onset in NOD mice, nondiabetic NOD/SCID mice were injected with inflammatory T cells from diabetic NOD mice. MDSCs significantly prevented diabetes onset, and 60% of MDSC-treated mice remained diabetes free. The pancreata of treated mice showed significantly lower levels of lymphocyte infiltration in islet and less insulitis compared with that of the control groups. The protective effects of MDSCs might be mediated by inducing anergy in autoreactive T cells and the development of CD4(+)CD25(+)Foxp3(+) Tregs. Thist study demonstrates a remarkable capacity of transferred MDSCs to downregulate Ag-specific autoimmune responses and prevent diabetes onset, suggesting that MDSCs possess great potential as a novel cell-based tolerogenic therapy in the control of T1D and other autoimmune diseases.     

High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 microM to 26 pM). CD8(+) T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (K(D) value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, K(D) 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range-with K(D) values of 450 nM and 4 microM-demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8(+) T cells, introduction of these TCR dramatically increased the reactivity of CD4(+) T cells to tumor cell lines.     

Islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD4+ T cells in human subjects

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4+ T cell responses toward IGRP in human subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4+ T cell epitopes. IGRP(23-35) and IGRP(247-259) were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP(13-25) and IGRP(226-238) were identified as DRA1*0101/DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4+ T cell responses for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both gamma-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP(247-259)-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general population.     

Development of memory-like autoregulatory CD8+ T cells is CD4+ T cell dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8(+) cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8(+) T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8(+) T cell memory is CD4(+) T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti-4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4(+) T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4(+) T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2(-/-) mice, but inhibited it in low-affinity TCR-TG NOD.Rag2(-/-) mice. Thus, in chronic autoimmune responses, CD4(+) Th cells contribute to both promoting and suppressing pathogenic autoimmunity.     

Use of synthetic peptides to establish anti-human acetylcholine receptor CD4+ cell lines from myasthenia gravis patients

Acetylcholine receptor-(AcChR) specific T cell lines were propagated from the PBL of six myasthenia gravis (MG) patients by the use of a pool of synthetic peptides (alpha-pool) corresponding to the complete sequence of the alpha-subunit of the human AcChR. All the lines had CD4+ phenotype and strongly recognized the alpha-pool. Four lines cross-reacted with native Torpedo AcChR. Five lines showed, at certain stages of their propagation, some degree of reactivity to autologous or DR-matched APC. One of the CD4+ T lines was challenged with each one of the peptides present in the alpha-pool. Several peptides, corresponding to the sequence segments 48-67, 101-120, 304-322, 320-337, and 419-437 of the human alpha-subunit were recognized, indicating that different epitopes and multiple T cell clones are involved in the recognition of the autoantigen in MG. Human AcChR-specific CD4+ T cell lines will be useful to identify the repertoire of epitopes recognized by the autoreactive Th cells in MG, to investigate the TCR genes utilized by autoreactive Th cells and to develop specific immunosuppressive treatments using anti-T cell vaccination.     

Cellular and molecular mechanism for Kilham rat virus-induced autoimmune diabetes in DR-BB rats

Kilham rat virus (KRV) causes autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats; however, the mechanism by which KRV induces autoimmune diabetes without the direct infection of beta cells is not well understood. We first asked whether molecular mimicry, such as a common epitope between a KRV-specific peptide and a beta cell autoantigen, is involved in the initiation of KRV-induced autoimmune diabetes in DR-BB rats. We found that KRV peptide-specific T cells generated in DR-BB rats infected with recombinant vaccinia virus expressing KRV-specific structural and nonstructural proteins could not induce diabetes, indicating that molecular mimicry is not the mechanism by which KRV induces autoimmune diabetes. Alternatively, we asked whether KRV infection of DR-BB rats could disrupt the finely tuned immune balance and activate autoreactive T cells that are cytotoxic to beta cells, resulting in T cell-mediated autoimmune diabetes. We found that both Th1-like CD45RC+CD4+ and cytotoxic CD8+ T cells were up-regulated, whereas Th2-like CD45RC-CD4+ T cells were down-regulated, and that isolated and activated CD45RC+CD4+ and CD8+ T cells from KRV-infected DR-BB rats induced autoimmune diabetes in young diabetes-prone BioBreeding (DP-BB) rats. We conclude that KRV-induced autoimmune diabetes in DR-BB rats is not due to molecular mimicry, but is due to a breakdown of the finely tuned immune balance of Th1-like CD45RC+CD4+ and Th2-like CD45RC-CD4+ T cells, resulting in the selective activation of beta cell-cytotoxic effector T cells.     

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.     

Analysis of naïve and memory CD4 and CD8 T cell populations in breast cancer patients receiving a HER2/<Emphasis Type="Italic">neu</Emphasis> peptide (E75) and GM-CSF vaccine

We are conducting clinical trials of the E75 peptide as a vaccine in breast cancer (BrCa) patients. We assessed T cell subpopulations in BrCa patients before and after E75 vaccination and compared them to healthy controls. We obtained 17 samples of blood from ten healthy individuals and samples from 22 BrCa patients prior to vaccination. We also obtained pre- and post-vaccination samples of blood from seven BrCa patients who received the E75/GM-CSF vaccine. CD4, CD8, CD45RA, CD45RO, and CCR7 antibodies were used to analyze the CD4+ and CD8+ T cells by four-color flow cytometry. Compared to healthy individuals, BrCa patients have significantly more memory and less naïve T cells and more effector-memory CD8+ and less effector CD4+ T cells. Phenotypic differences in defined circulating CD4+ and CD8+ T cell subpopulations suggest remnants of an active immune response to tumor distinguished by a predominant memory T cell response and by untapped recruitment of naïve helper and cytotoxic T cells. E75 vaccination induced recruitment of both CD4+ and CD8+ naïve T cells while memory response remained stable. Additionally, vaccination induced global activation of all T cells, with specific enhancement of effector CD4+ T cells. E75 vaccination causes activation of both memory and naïve CD4+ and CD8+ T cells, while recruiting additional naïve CD4+ and CD8+ T cells to the overall immune response.     

An altered self-peptide with superagonist activity blocks a CD8-mediated mouse model of type 1 diabetes

T cell tolerance can be experimentally induced through administration of self-peptides with single amino acid substitution (altered peptide ligands or APLs). However, little is known about the effects of APLs on already differentiated autoreactive CD8+ T cells that play a pivotal role in the pathogenesis of autoimmune diabetes. We generated a panel of APLs derived from an influenza virus hemagglutinin peptide exhibiting in vitro functions ranging from antagonism to superagonism on specific CD8+ T cells. A superagonist APL was further characterized for its therapeutic activity in a transgenic mouse model of type 1 diabetes. When injected i.v. 1 day after the transfer of diabetogenic hemagglutinin-specific CD8+ T cells into insulin promoter-hemagglutinin transgenic mice, the superagonist APL proved more effective than the native hemagglutinin peptide in blocking diabetes. This protective effect was associated with an inhibition of CD8+ T cell cytotoxicity in vivo and with a decreased accumulation of these cells in the pancreas, leading to a marked reduction of intrainsulitis. In conclusion, a superagonist "self-peptide" APL was more effective than the native peptide in treating a CD8+ T cell-mediated diabetes model.