Deletion of antigen-specific immature thymocytes by dendritic cells requires LFA-1/ICAM interactions.

An in vitro assay was used for assessing the participation of various cell surface molecules and the efficacy of various cell types in the deletion of Ag-specific immature thymocytes. Thymocytes from mice expressing a transgenic TCR specific for the male Ag presented by the H-2Db class I MHC molecule were used as a target for deletion. In H-2d transgenic mice, cells bearing the transgenic TCR are not subjected to thymic selection as a consequence of the absence of the restricting H-2Db molecule but, nevertheless, express this TCR on the vast majority of immature CD4+8+ thymocytes. In this report we show that CD4+8+ thymocytes from H-2d TCR-transgenic mice are preferentially killed upon in vitro culture with male APC; DC were particularly effective in mediating in vitro deletion when compared with either B cells or T cells. Deletion of CD4+8+ thymocytes by DC was H-2b restricted and could be inhibited by mAb to either LFA-1 alpha or CD8. Partial inhibition was observed with mAb to ICAM-1, whereas mAb to CD4 and LFA-1 beta were without effect. These results are the first direct evidence of LFA-1 involvement in negative selection and provide further direct support for the participation of CD8/class I MHC interactions in this process. Like the requirements for deletion, activation of mature male-specific CD4-8+ T cells from female H-2b TCR-transgenic mice was also largely dependent on Ag presentation by DC and required both LFA-1/ICAM and CD8/class I MHC interactions; these results support the view that activation and deletion may represent maturation stage-dependent consequences of T cells encountering the same APC. Finally, our results also support the hypothesis that negative selection (deletion) does not require previous positive selection because deletion was observed under conditions where positive selection had not occurred.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

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.     

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 two TCR transgenic mouse lines specific for herpes simplex virus

To better understand the T cell-mediated processes involved in the immune response to herpes simplex virus type 1 (HSV-1)infection, two HSV-specific T cell receptor (TCR) transgenic mouse lines were produced. These mice (gBT-I.1 and gBT-I.3) are MHC class I-restricted and specific for the immunodominant peptide from HSV glycoprotein B (gB), gB498-505. Although derived from the same clone, the mice differ in the chromosomal location of the TCR transgenes and show marked differences in TCR alpha/beta expression on both CD4+ and CD8+ cells in the thymus. Despite this, peripheral CD8+ Tcells from both mice express equally high levels of the transgenic TCR and bind the KbgB498-505 tetramer to the same degree. In concordance with this, both were shown to respond equally well in vitro upon stimulation with the gB498-505 peptide or HSV-infected cells. These data show that selection of broadly equivalent peripheral T-cell subsets can occur in the presence of distinctly different thymic T-cell subsets.     

Thymocyte antigens do not induce tolerance in the CD4+ T cell compartment.

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.     

MHC-mismatched islet allografts are vulnerable to autoimmune recognition in vivo

When transplanted into type 1a diabetic recipients, islet allografts are subject both to conventional allograft immunity and, presumably, to recurrent autoimmune (islet-specific) pathogenesis. Importantly, CD4 T cells play a central role both in islet allograft rejection and in autoimmune disease recurrence leading to the destruction of syngeneic islet transplants in diabetic NOD mice. However, it is unclear how NOD host MHC class II (I-A(g7))-restricted, autoreactive CD4 T cells may also contribute to the recognition of allogeneic islet grafts that express disparate MHC class II molecules. We hypothesized that islet-specific CD4 T cells can target MHC-mismatched islet allografts for destruction via the "indirect" (host APC-dependent) pathway of Ag recognition. To test this hypothesis, we determined whether NOD-derived, islet-specific CD4 T cells (BDC-2.5 TCR transgenic cells) could damage MHC-mismatched islets in vivo independent of conventional allograft immunity. Results demonstrate that BDC-2.5 CD4 T cells can vigorously destroy MHC class II-disparate islet allografts established in NOD.scid recipients. Tissue injury is tissue-specific in that BDC-2.5 T cells destroy donor-type islet, but not thyroid allografts established in the same NOD.scid recipient. Furthermore, BDC-2.5 CD4 T cells acutely destroy MHC class II-deficient islet allografts in vivo, indicating that autoimmune pathogenesis can be completely independent of donor MHC class II expression. Taken together, these findings indicate that MHC-mismatched islet allografts can be vulnerable to autoimmune pathogenesis triggered by autoreactive CD4 T cells, presumably through indirect autoantigen recognition in vivo.     

Cross-priming of diabetogenic T cells dissociated from CTL-induced shedding of beta cell autoantigens

Cross-presentation of self Ags by APCs is key to the initiation of organ-specific autoimmunity. As MHC class I molecules are essential for the initiation of diabetes in nonobese diabetic (NOD) mice, we sought to determine whether the initial insult that allows cross-presentation of beta cell autoantigens in diabetes is caused by cognate interactions between naive CD8(+) T cells and beta cells. Naive splenic CD8(+) T cells from transgenic NOD mice expressing a diabetogenic TCR killed peptide-pulsed targets in the absence of APCs. To ascertain the role of CD8(+) T cell-induced beta cell lysis in the initiation of diabetes, we expressed a rat insulin promoter (RIP)-driven adenovirus E19 transgene in NOD mice. RIP-E19 expression inhibited MHC class I transport exclusively in beta cells and rendered these cells resistant to lysis by CD8(+) (but not CD4(+)) T cells, both in vitro and in vivo. Surprisingly, RIP-E19 expression impaired the accumulation of CD8(+) T cells in islets and delayed the onset of islet inflammation, without affecting the timing or magnitude of T cell cross-priming in the pancreatic lymph nodes, which is the earliest known event in diabetogenesis. These results suggest that access of beta cell autoantigens to the cross-presentation pathway in diabetes is T cell independent, and reveal a previously unrecognized function of MHC class I molecules on target cells in autoimmunity: local retention of disease-initiating clonotypes.     

Functional similarity and differences between selection-independent CD4-CD8- alphabeta T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4-CD8- alphabeta T cells in antigen-expressing mice.

In TCR-alphabeta transgenic mice, CD4-CD8- TCR-alphabeta+ (alphabeta DN) cells arise in the absence of positively selecting MHC molecules and are resistant to clonal deletion in Ag-expressing mice. In this study the activation requirements and functional properties of alphabeta double-negative (DN) cells were compared with those of positively selected CD8+ cells expressing equivalent levels of the same MHC class I-restricted transgenic TCR. We found that positively selected CD8+ cells required a lower density of the antigenic ligand for optimal proliferative responses compared with alphabeta DN cells derived from nonpositively selecting mice. However, when the CD8 coreceptor on CD8+ cells was blocked with an anti-CD8 mAb, both alphabeta DN and CD8+ cells exhibited the same dose-response curve to the antigenic ligand and the same dependence on CD28/B7 costimulation. Positively selected CD8+ cells also differed from alphabeta DN cells in that they differentiated into more efficient killers and IL-2 producers after Ag stimulation, even after CD8 blockade. However, Ag-activated alphabeta DN and CD8+ cells were equally efficient in producing IFN-gamma, suggesting that this functional property is independent of positive selection. We also found that alphabeta DN cells recovered from the lymph nodes of Ag-expressing mice were functionally anergic. This anergic state was associated with defective proliferation and IL-2 production in response to Ag stimulation. These observations indicate that alphabeta DN cells can be anergized in vivo by physiological levels of the antigenic ligand.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Murine B7 antigen provides a sufficient costimulatory signal for antigen-specific and MHC-restricted T cell activation.

We have previously shown that the murine B7 (mB7) molecule, when expressed in Chinese hamster ovary cells in stable fashion, can costimulate with anti-CD3 mAb or Con A to induce T cell activation. We have now derived, by gene transfection, Chinese hamster ovary cell lines that express the I-Ad molecule, either alone or in context with mB7. We have analyzed these transfectants for their capacity to present Ag to murine CD4+ T lymphocytes. I-Ad/mB7-double transfectants were able to stimulate mixed lymphocyte reactions and to present peptide Ag to specific T cells. Chinese hamster ovary cells that expressed only the I-Ad molecule were not able to stimulate T cell proliferation in these systems. Thus, the mB7 protein is a sufficient costimulatory molecule for the physiologic, Ag-dependent/MHC-restricted activation of murine CD4+ T cells. Stimulation of T cell bulk cultures resulted predominantly in the production of IL-2 and not of IL-4. The costimulatory activity of mB7 is not, however, restricted to the IL-2-secreting subset. We have identified one IL-4-secreting T cell clone, CDC35, which is responsive to mB7 triggering. Finally, we present experiments that suggest that mB7 and peptide/MHC complexes need to be expressed on the same cell for optimal induction of T cell activation.     

Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

Nonimmune thyroid destruction results from transgenic overexpression of an allogeneic major histocompatibility complex class I protein.

The overexpression of major histocompatibility complex (MHC) class I molecules in endocrine epithelial cells is an early feature of autoimmune thyroid disease and insulin-dependent diabetes mellitus, which may reflect a cellular response, e.g., to viruses or toxins. Evidence from a transgenic model in pancreatic beta cells suggests that MHC class I overexpression could play an independent role in endocrine cell destruction. We demonstrate in this study that the transgenic overexpression of an allogeneic MHC class I protein (H-2Kb) linked to the rat thyroglobulin promoter, in H-2Kk mice homozygous for the transgene, leads to thyrocyte atrophy, hypothyroidism, growth retardation, and death. Thyrocyte atrophy occurred in the absence of lymphocytic infiltration. Tolerance to allogeneic class I was revealed by the reduced ability of primed lymphocytes from transgenic mice to lyse H-2Kb target cells in vitro. This nonimmune form of thyrocyte destruction and hypothyroidism recapitulates the beta-cell destruction and diabetes that results from transgenic overexpression of MHC class I molecules in pancreatic beta cells. Thus, we conclude that overexpression of MHC class I molecules may be a general mechanism that directly impairs endocrine epithelial cell viability.     

Cutting edge: CD25+ regulatory T cells prevent expansion and induce apoptosis of B cells specific for tissue autoantigens

To study the role of CD25(+) regulatory T cells (T(regs)) in peripheral B cell tolerance, we generated transgenic rat insulin promoter RIP-OVA/HEL mice expressing the model Ags OVA and HEL in pancreatic islet beta cells (where RIP is rat insulin promoter and HEL is hen egg lysozyme). Adoptively transferred transgenic OVA-specific CD4(+) and CD8(+) T cells proliferated only in the autoantigen-draining pancreatic lymph node (PLN), demonstrating pancreas-specific Ag expression. Transferred HEL-specific transgenic B cells (IgHEL cells) disappeared within 3 wk from transgenic but not from nontransgenic mice immunized with autoantigen. Depletion of CD25(+) FoxP3(+) cells completely restored IgHEL cell numbers. T(reg) exerted an analogous suppressive effect on endogenous HEL-specific autoreactive B cells. T(regs) acted by inhibiting the proliferation of IgHEL cells in the spleen and PLN and by systemic induction of their apoptosis. Furthermore, they reduced BCR and MHC II surface expression on IgHEL cells in the PLN. These findings demonstrate that autoreactive B cells specific for a nonlymphoid tissue autoantigen are controlled by T(regs).     

Processing and presentation of self and foreign antigens by the renal proximal tubule.

The renal proximal tubule (PT) in many ways resembles an APC. The PT is one of the few epithelial cells in the body reported to constitutively express the class II MHC molecules required to present Ag to CD4+ T cells. We questioned whether the PT could function as an APC in vitro and in vivo. Fluorescence cytometry demonstrated that the normal CBA/J PT constitutively expressed low levels of class II MHC and that this expression was markedly augmented by either IFN-gamma or systemic Listeria monocytogenes infection. Functionally, the PT from normal CBA/J mice also stimulated T cell hybridomas when cultured in vitro with Ag, and this ability was markedly up-regulated by both IFN-gamma as well as L. monocytogenes infection. To prove that the PT constitutively processed and presented self Ag in vivo, freshly isolated PT from mice transgenic for human alpha 1-antitrypsin were cultured with the appropriate T cell hybridoma in the absence of exogenous Ag. Strong stimulation of the T cell hybridoma occurred. Our data show that the renal proximal tubule processes and presents foreign Ag both in vitro and in vivo, and that it constitutively processes and presents the self Ag hAAT in vivo. These results have important implications for the understanding of renal interstitial autoimmune diseases as well as the interstitial nephritis that occurs in response to foreign Ag.     

Characterization of primary T cell subsets mediating rejection of pancreatic islet grafts

The cellular mechanisms by which pancreatic islet grafts are rejected have not been clearly defined. In order to address the roles of CD4+ and CD8+ T cells in pancreatic islet rejection, we used an adoptive transfer model in which H-2b nude mice were reconstituted with negatively selected H-2b CD4+ or CD8+ T cell subpopulations and engrafted with fully allogeneic pancreatic islet grafts. We found that primary (unprimed) CD4+ T cells mediated the rejection of pancreatic islet grafts, whereas, primary CD8+ T cells failed to do so, even though both T cell subpopulations were competent to reject skin allografts. These data indicate that primary CD4+ T cells are necessary for rejection of allogeneic pancreatic islet grafts, whereas primary CD8+ T lymphocytes are not. Implications concerning the nature of the APC involved in the initiation of the rejection response to islet allografts and the expression of MHC Ag by pancreatic islet cells are discussed.     

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.