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Immunosuppression by UV light contributes significantly to the induction of skin cancer by suppressing the cell-mediated immune responses which control the development of carcinogenesis. The B7/CD28-CTLA-4 signaling pathway provides costimulatory signals essential for Ag-specific T cell activation. To investigate the role of this pathway in photocarcinogenesis, we utilized transgenic (Tg) mice which constitutively express CTLA-4Ig, a high-affinity CD28/CTLA-4 antagonist that binds to both B7-1 and B7-2. The transgene is driven by a skin-specific promoter yielding high levels of CTLA-4Ig in the skin and serum. Chronic UV exposure of CTLA-4Ig Tg mice resulted in significantly reduced numbers of skin tumors, when compared to control mice. In addition, Tg mice were resistant to UV-induced suppression of delayed-type hypersensitivity responses to alloantigens. Most importantly, upon stimulation with mitogens and alloantigens, T cells isolated from CTLA-4Ig Tg mice produced significantly less IL-4 but more IFN-γ compared to control T cells, suggesting an impaired Th2 response and a relative increase of Th1-type immunity. Together, these data show that overall B7 engagement directs immune responses toward the Th2 pathway. Moreover, they point out the crucial role of Th1 immune reactions in the protection against photocarcinogenesis.

Ag-specific T cell activation requires a CD28-mediated costimulatory interaction. This observation has suggested novel approaches to suppress donor-specific immunity, including the use of soluble CD28 antagonists, such as CTLA4Ig, which suppresses transplant rejection in small animal models. In this study, CTLA4Ig therapy was examined in a non-human primate model of allogeneic pancreatic islet transplantation. Two of five CTLA4Ig-treated monkeys showed prolonged graft survival, which correlated with donor-specific hyporesponsiveness in vitro. Humoral responses to the transplanted tissue were suppressed in all treated animals. These results suggest that CTLA4Ig is effective in suppressing both humoral and cellular immune responses in a non-human primate model of allogeneic transplantation.

Previous studies have shown that TCR-gammadelta cells expressing Vgamma2 region elements are selectively expanded in vivo in C57BL/6 (B6), but not DBA/2, mice. Genetic analysis demonstrated that the expansion of Vgamma2+ was linked to the TCR alphadelta loci, suggesting that a particular Vgamma-Vdelta pair may be necessary for the expansion. In the studies presented here, we find that the expanding TCR gammadelta cells in B6 mice express a Vgamma2+/Vdelta7+ TCR. The Vgamma2-Jgamma and Vdelta7-Ddelta-Jdelta junctional amino acid sequences of these cells display wide variation in length, suggesting that expansion is based on variable region usage and not junctional diversity. The kinetics and dynamics of Vgamma2+/Vdelta7+ T cell expression were studied to determine the biological basis of clonal expansion. Although expression of the Vgamma2+ cells in B6 and DBA/2 neonates was similar, Vgamma2+ cells in the B6 mice expanded fourfold by 4 wk of age, while the expression in DBA/2 mice remained constant. In addition, expansion of the Vgamma2+ cells occurred in athymic nude mice, suggesting that expansion was driven by extrathymic stimuli. Finally, B6 mice housed under germfree conditions expressed expanded levels of Vgamma2+ gammadelta T cells similar to their normally housed counterparts. Thus, expansion and diversification of Vgamma2+/Vdelta7+ T cells are postnatal extrathymic events that do not require microbial antigenic exposure.

The CD28/CTLA-4 and B7 families of cell surface molecules regulate a complex pathway of T cell signals that profoundly affect peripheral T cell responses. On the one hand, CD28/B7-mediated T cell costimulation is essential for the development and maintenance of T cell responses. In contrast, the prevailing data suggest that the engagement of CTLA-4 with the same B7 ligands down-regulates the initiation and progression of immune responses. This commentary examines the role of CTLA-4-mediated signaling in the regulation of the immune response, leading to the hypothesis that the CTLA-4/B7 interaction plays an essential role in the establishment of peripheral self-tolerance as well as the regulation of normal T cell immunity.

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.

We have previously shown evidence for the early recruitment of gamma delta T cells during the disease course of primary infections with Listeria monocytogenes or Salmonella choleraesuis in mice. Since gamma delta T cells at this stage of the disease do not produce IL-2, the growth factor for the gamma delta T cells remains unknown. IL-15 is a novel cytokine that uses beta- and gamma-chain of IL-2R for signal transduction, and is produced by activated monocytes/macrophages. In this study, we investigated the proliferative activity of IL-15 for gamma delta T cells appearing after primary infection with S. choleraesuis 31N-1. The gamma delta T cells, which expressed beta- and gamma-chains of IL-2R, proliferated in the presence of rIL-15 and produced appreciable levels of gamma-IFN and IL-4. Addition of anti-IL-2R beta mAb significantly inhibited the IL-15-induced proliferation of the gamma delta T cells. Furthermore, the gamma delta T cells produced gamma-IFN in response to monocyte/macrophage cell line, J774A.1 infected with S. choleraesuis, which expressed an abundant level of IL-15 mRNA. This cytokine production was inhibited significantly by anti-IL-15 Ab. Taken together, these results suggest that IL-15 derived from infected macrophages may contribute to the early activation of gamma delta T cells during salmonellosis.

Studies of classical and nonclassical MHC class I molecules have shown that unique peptides are associated and functionally recognized by alloreactive T cells. We have recently shown that an alloreactive TCR-gamma delta cell recognizes a nonclassical MHC molecule, T10b. However, T cell recognition of this glycoprotein did not appear to require typical peptide recognition based on studies using transporter-defective mutant cell lines. In the current study, we have analyzed in detail, the role of peptide in T10b expression and recognition. The findings reveal that the recognition of the nonclassical MHC molecule by TCR-gamma delta cells is independent of species, tissue type, both the class I and class II Ag processing and presentation pathways, or the presence of peptides. In fact, biochemical analysis of the T10b chimeric molecule, T10b/Ld, transfected into CHO cells using radiolabeled [3H]leucine, HPLC, and mass spectrometry suggest that peptides are not associated with this nonclassical class I molecule. Therefore, some class I molecules, e.g., T10b, do not associate with polymorphic peptides typical of classical MHC class I molecules and can be expressed in the absence of peptides on the cell surface in a functionally active form.

145-2C11, a hamster mAb directed against the mouse CD3 complex, is a potent immunosuppressive agent. Upon initial treatment, 145-2C11 triggers a systemic release of multiple cytokines that is responsible for the acute toxicity of the mAb. This cellular activation is a consequence of the cross-linking between T lymphocytes and Fc gamma R-bearing cells, mediated by the high affinity of the hamster mAb for murine Fc gamma Rs. Repeated mAb injections result in the onset of a neutralizing humoral response. Therefore, there has been an increased interest in developing nonmitogenic forms of anti-CD3 mAbs, although it is not clear whether these Abs will retain immunosuppressive properties. To determine whether the initial cytokine production is necessary for the immunosuppressive properties and the immunogenicity of anti-CD3 mAbs in vivo, we have generated chimeric (hamster 145-2C11 F(ab')2 region/mouse Fc gamma portion) mAbs using murine isotypes with different affinities for Fc gamma Rs. The 145-2C11 and a chimeric IgG2a isotype, both of which bind murine Fc gamma Rs avidly, had similar activating, immunogenic, and immunosuppressive properties in mice. The administration of a chimeric IgG3 isotype with a very low affinity for murine Fc gamma Rs did not result in cytokine production, a humoral response against the mAb, or TCR desensitization. Nevertheless, prolongation of skin graft survival was similar in the IgG3, IgG2a, and 145-2C11-treated mice, indicating that Fc gamma R nonbinding anti-CD3 mAbs retain potent immunosuppressive properties in vivo while not being immunogenic. This enhanced therapeutic to toxic profile may be beneficial in clinical transplantation.

Isolated granulomas provide a unique model to study T cells in the site of inflammation. Schistosoma mansoni-infected mice develop liver granulomas in response to schistosome egg deposition and the granulomas contain gamma delta T cells that appear to be activated (Pgp-1high and L-selectin low). Analysis of kinetics and TCR gene usage of granuloma gamma delta T cells revealed a limited TCR repertoire restricted to V gamma 1.1, V delta 4, and V delta 6 genes, suggesting that the occurrence of gamma delta T cells in the granuloma is influenced by TCR V gene usage. The V delta 4+, but not the V delta 6+, gamma delta T cells expressed CD69, a marker of recent activation. To determine if there was a preferred order of accumulation of the gamma delta T cells in granulomas, s.c. sponge grafts were implanted into schistosome-infected mice, schistosome eggs were injected into the grafts, and accumulated T cells were sequentially analyzed. The earliest gamma delta T cell immigrants expressed V delta 6 and later immigrants expressed V delta 4 V genes. Additional evidence for a role of TCR specificity in the accumulation of gamma delta T cells in granulomas is their absence from schistosome granulomas in TCR transgenic mice that express only a single MHC-specific gamma delta TCR. Finally, gamma delta T cell recruitment into the granulomas did not require beta 2-microglobulin, since gamma delta T cells were present in liver granulomas of beta 2-microglobulin gene-disrupted mice. The analysis of the influx of gamma delta T cells into schistosome-induced, liver granulomas and schistosome egg-containing sponges provides a model system to investigate the role, if any, of gamma delta T cells in schistosome infections.

We have previously shown that depletion of TCR-V beta 8+ T cells by treatment with mAb reduces the curative effectiveness of low dose melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large MOPC-315 tumor and extensive metastases. Here we show that V beta 8+/CD8+ T cell lines derived from mice that are in the process of immune-mediated eradication of a large MOPC-315 tumor as a consequence of low dose L-PAM therapy (L-PAM TuB mice) are capable of mediating tumor eradication in vivo upon adoptive transfer. Analysis of the possible mechanisms through which these cell lines bring about tumor eradication revealed that the V beta 8+/CD8+ cells can exert in vitro a potent lytic activity and secrete large amounts of IFN-gamma. Both of these activities can be triggered by the MOPC-315 but not the MOPC-104E plasmacytoma and are restricted by the MHC class I H-2Kd molecule. In vivo neutralization of IFN-gamma by treatment with mAb was found to cause a noticeable delay in tumor rejection in mice subjected to adoptive chemoimmunotherapy with low dose L-PAM and V beta 8+/CD8+ cells; however, all tumors did regress after initial growth. Thus, the V beta 8+/CD8+ cells use an IFN-gamma-dependent mechanism for the realization of their in vivo tumor-eradicating immunity. However, an IFN-gamma-independent mechanism, most likely involving direct V beta 8+/CD8+ CTL activity, is apparently also used.

Previous studies have shown that human hemopoietic cells can be adoptively transferred into immunodeficient C.B-17 scid/scid (SCID) mice that lack autologous T and B lymphocytes, to generate chimeric animals. The future development of novel immunomodulatory drugs in transplantation will depend increasingly on experimental animal models to investigate the properties of the agents on human cells before starting clinical trials. However, in previous models of SCID mice engrafted with human PBLs, human T cells have been found either to be in an unresponsive state, unable to respond to mitogenic stimulations in vitro, or to mediate skin graft rejection only when HLA-primed in vivo before their adoptive transfer into SCID mice. In addition, T cells and other leukocyte subsets engraft quite poorly in the lymphoid tissues of the animals. In an attempt to develop a useful model for transplantation research, we have inoculated SCID mice with fresh human splenocytes from cadaveric organ donors (hu-Spl-SCID mice). In this model, various leukocyte subsets engraft effectively in different lymphoid compartments. In addition, human T cells retain their proliferative responses to mitogens and to alloantigens when tested 3 wk after engraftment into SCID mice. Finally, mice engrafted with unprimed human spleen cells acutely reject human foreskin allografts. Treatment of hu-Spl-SCID mice with OKT3, an immunosuppressive mAb directed against the human CD3 complex associated with the TCR, prevents the rejection of most human skin allografts, indicating a major role for human T cells in this phenomenon. Thus, this hu-Spl-SCID model may be useful for the study of immunosuppressive therapies in a preclinical in vivo setting.

A long term goal of investigators working in our laboratory has been to develop new mAbs for use as immunosuppressive agents. As a means toward achieving this goal, several new mAbs (hybridomas) have been developed by screening fusions for supernatants that possess T cell-inhibitory properties. Of these new mAbs, one mAb, designated 5H7, has been shown to possess both a unique combination of specificity for a monomorphic determinant of the alpha 3 domain of human class I MHC heavy chains and highly potent T cell inhibitory properties. mAb 5H7 profoundly inhibited T cell proliferation in response to anti-CD3 mAb in primary or secondary allogenic MLR or in primary human anti-mouse xenogenic MLR. mAb 5H7 inhibited expression of an early T cell activation marker, Leu23 (CD69); expression of IL-2Rs; and IL-2 production by both CD4+ and CD8+ T cells. mAb 5H7 inhibited IL-2 release by the Jurkat (E6-1) human T cell leukemia line in response to immobilized anti-CD3 mAb, thus, providing further evidence that 5H7 can inhibit activation directly at the level of the T cell. 5H7 profoundly blocked CD3-dependent (anti-CD3, anti-CD3 plus PMA, or anti-CD3 plus anti-CD28) pathways, but only partially blocked a CD3-independent (anti-CD28 plus PMA) pathway of T cell activation. In conclusion, class I MHC molecules that are expressed on the T cell may regulate early TCR/CD3-dependent signaling events. In addition, 5H7 mAb may provide a reagent for suppression of cellular immunity in vivo.

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching. In this study, we demonstrate that incubation with IL-4 enhances the ability of small splenic murine B cells, normally poor stimulators of murine Th1 clones, to stimulate lymphokine production and proliferation by Th1 clones. Moreover, small resting B cells induce anergy, whereas IL-4-treated B cells do not. IL-4-treated B cells were found to express both B7 (B7-1) and a second ligand for CTLA4Ig (B7-2). Although IL-4 induces both B7-1 and B7-2, the kinetics of expression of these molecules are different: B7-2 is detected by 6 h, whereas B7-1 is not detectable until 48 h. In addition, only CTLA4Ig fully blocks IL-4 induced costimulatory activity; a mAb to B7-1 does not. Thus, these results suggest that IL-4 may function indirectly as a costimulatory factor by inducing costimulatory molecules on resting B cells. Additionally, these findings support our previous findings that an alternative ligand for CD28 and CTLA4 is important in providing costimulation.

We have recently demonstrated the importance of V beta 8+/CD8+ cells for the curative effectiveness of a suboptimal low dose (0.75 mg/kg) of melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large s.c. MOPC-315 tumor and extensive metastases. Here we show that staphylococcal enterotoxin B (SEB), which is known to selectively stimulate T cells expressing members of the TCR-V beta 8 gene family, substantially improved the curative effectiveness of the suboptimal dose of L-PAM for mice bearing a large MOPC-315 tumor. Moreover, treatment of mice with mAb F23.1 (anti-V beta 8) abrogated the in vivo therapeutic effect of SEB for low dose L-PAM-treated MOPC-315 tumor bearers (L-PAM TuB mice). Analysis of the effect of SEB on the tumor-infiltrating lymphocytes (TILs) demonstrated that the SEB-mediated therapeutic effect was associated with a significant increase in: 1) the percentage of V beta 8+ cells among the CD8+ T cells that accumulated in the s.c. tumor nodules, 2) the total number of V beta 8+/CD8+ cells present per tumor on day 4 after low dose L-PAM therapy, and 3) the ability of the TILs to lyse MOPC-315 tumor cells in vitro in a short term assay. Furthermore, treatment of mice with mAb F23.1 abolished the ability of SEB to render the TIL population of L-PAM TuB mice more cytotoxic in vitro for MOPC-315 tumor cells. Thus, the SEB-mediated improvement in the therapeutic outcome of low dose L-PAM therapy for mice bearing a large MOPC-315 tumor may be due in part to SEB-mediated increase in the contribution of the V beta 8+ T cells to tumor eradication through enhancement in the magnitude of the anti-MOPC-315 lytic activity exhibited by the TIL population.

The role of costimulation in the activation of TCR-gamma delta cells in normal mice and mice transgenic (tg) for a TCR-gamma delta receptor was investigated. Activation of TCR-gamma delta cells required two signals. One signal was mediated by TCR occupancy, whereas a second signal was provided by accessory cells. The importance of the CD28/B7 interaction in the delivery of the second signal was demonstrated in multiple ways. First, addition of a soluble fusion protein homolog of CD28, CTLA4Ig, significantly inhibited the activation of G8 tg splenic TCR-gamma delta lymphocytes and intestinal epithelial TCR-gamma delta lymphocytes by Ag-bearing lymphocytes during primary stimulation. Similarly, both proliferation and IFN-gamma production were inhibited by addition of CTLA4Ig to secondary antigenic stimulation of G8 tg TCR-gamma delta cells. Second, an Ag-bearing thymoma, EL-4, was only able to stimulate expanded G8 tg TCR-gamma delta cells when the thymoma expressed B7. This stimulation was blocked by both CTLA4Ig and anti-B7 antibody. Third, antibodies to CD28 were able to mimic the costimulatory affect of APC. TCR-gamma delta cells cultured with either Ag-bearing fixed stimulator cells or submitogenic concentrations of immobilized anti-pan TCR-gamma delta mAb proliferated only in the presence of anti-CD28 mAb. Finally, G8 tg cells produced IL-2 only in the presence of APC costimulation or anti-CD28 antibodies, and the addition of exogenous rIL-2 overcame the need for costimulation. Thus, autocrine IL-2 production is one of the major consequences of TCR-gamma delta cell costimulation. Together these data demonstrate that costimulation is necessary for the activation of TCR-gamma delta cells and can occur through CD28 interaction.

Murine collagen-induced arthritis (CIA) is a T cell-mediated disease which is induced by injection of type II collagen. Previous studies have shown that CD4+ cells which express particular V beta TCR genes are involved in the induction of arthritis in this model. In the present report we demonstrate that CD4-, CD8-, TCR gamma delta cells are present in arthritic joints, expanded in peripheral lymphoid tissue of DBA/1 lac J mice with CIA, and respond in vitro to the anti-TCR gamma delta mAb UC7-13D5 (13D5). In order to directly investigate the role of the gamma delta TCR in murine CIA, DBA/1 lac J mice were injected with 13D5 before or 40 days after injection of type II collagen. Our results demonstrate that i.p. injections of 13D5 initiated 1 day before injection of type II collagen significantly delays both the onset and severity of CIA compared with treatment with type II collagen alone. In contrast, anti-TCR gamma delta mAb injection of arthritic mice 40 days after collagen injection resulted in the rapid onset of severe arthritis which was accompanied by increased bone erosion and cell infiltration into inflamed joints compared with arthritic mice injected with either control hamster IgG or F(ab')2 fragments of 13D5. Arthritic mice injected with intact 13D5 rapidly lost weight, suggesting that 13D5 may induce a cytokine-mediated syndrome similar to that observed in mice and humans after the injection of anti-CD3. Flow cytometry analysis of joint cells isolated after collagenase digestion from arthritic mice demonstrated that 13D5 injection induces the accumulation of CD4-, CD8-, PgP-1 (CD44)+ cells within arthritic joints, whereas arthritic joints from mice injected with control hamster IgG contained cells with a CD4+, CD8- phenotype. CD3+ T cell lines which express the gamma delta TCR from inflamed joints of arthritic mice were established and examined for V gamma usage by the polymerase chain reaction. V gamma 2 rearrangements were predominant in both T cell lines established from inflamed synovium as well as freshly isolated synovial cells from arthritic mice, whereas synovial cells from nonarthritic mice did not demonstrate V gamma 2 rearrangements. Taken together, the results described in this report suggest a direct role for gamma delta TCR T cells in the pathogenesis of CIA in DBA/1 lac J mice.

We have previously shown that the curative efficacy of low dose melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large s.c. MOPC-315 tumor requires the participation of CD8+ (but not CD4+) T cell-dependent antitumor immunity. Here we show that CD8+ T cells obtained from regressing tumors on day 4 or 5 after low dose L-PAM therapy of MOPC-315 tumor bearers (L-PAM TuB mice) display a preferential enhancement in the utilization of the TCR-V beta 8.3 gene segment as compared to CD8+ T cells from normal lymph nodes. Treatment of L-PAM TuB mice with mAb F23.1, which leads to the depletion of V beta 8.3+ cells, as well as V beta 8.1 and 8.2+ cells, led to a significant reduction in the ability of their tumor-infiltrating lymphocytes as well as their spleen cells to lyse MOPC-315 tumor cells in vitro in a short term assay. In addition, the mAb F23.1 treatment almost completely abrogated the lytic activity of the tumor-infiltrating lymphocytes against another syngeneic, antigenically related plasmacytoma (the MOPC-104E). Moreover, the mAb F23.1 treatment significantly reduced the curative effectiveness of low dose L-PAM for mice bearing a large MOPC-315 tumor. In contrast, mAb KJ16 treatment, which leads to the depletion of V beta 8.1 and 8.2+ cells (but not V beta 8.3+ cells), did not reduce significantly the curative effectiveness of low dose L-PAM for such MOPC-315 tumor bearers. Thus, V beta 8.3+ T cells are important for the curative effectiveness of low dose L-PAM therapy for MOPC-315 tumor bearers, and it is conceivable that the V beta 8.3+ cells mediate their effect (at least in part) by contributing to the acquisition of CTL activity against plasmacytoma-shared Ag.

The development of TCR-gamma delta cells during thymic ontogeny has been studied using fetal thymic organ cultures of normal and transgenic (Tg) mice. The expression of the cell-surface markers--heat stable Ag (HSA), MEL-14, CD5, CD25 (IL-2R), and CD44 (Pgp-1)--correlated with TCR-gamma delta maturation. As the fetal thymus developed, there was an increase in HSA-, CD5dull, and CD44+ cells for each TCR-gamma delta cell subset. Moreover, the expression of recombination activating genes-1 and -2 (RAG-1 and RAG-2) also correlated with TCR-gamma delta maturation as only HSA+ TCR-gamma delta cells transcribed these genes. Cyclosporin A inhibited the development of the TCR-gamma delta thymocytes if it was introduced early during thymic ontogeny by arresting the differentiation of TCR-gamma delta thymocytes at the HSA+ stage. Immature HSA+ TCR-gamma delta thymocytes isolated from both TCR-gamma delta Tg and normal mice did not respond to nominal Ag or anti-TCR mAb unless exogenous IL-2 was added to the cultures. In contrast, HSA- TCR-gamma delta cells from Tg and normal mice responded to TCR/ligand interactions in the absence of additional IL-2. Finally, the development of functionally mature TCR-gamma delta cells could be induced in vitro. Interaction of the HSA+ Tg+ TCR-gamma delta cells with anti-TCR-gamma delta mAb or Ag-bearing thymic stromal cells resulted in RAG-1 and RAG-2 down-regulation. These data strongly suggest that TCR-gamma delta HSA+, RAG+ thymocytes differentiate into a more mature stage under the pressure of positive selection and that TCR-gamma delta cell development is regulated in a manner similar to TCR-alpha beta cells. In addition, the ability of Cyclosporin A to inhibit TCR-gamma delta cell development combined with the findings that Ag-bearing stromal cells can induce Tg TCR-gamma delta cell development suggests that maturation and selection of TCR-gamma delta cells depends on receptor-mediated physiologic stimuli delivered during thymic development.

We have investigated the in vivo administration of nonmitogenic anti-CD3F(ab')2 fragments for the prevention of lethal graft-vs-host disease (GVHD) in irradiated recipients of fully allogeneic bone marrow cells plus splenocyte (BMS) inocula. Recipients of anti-CD3F(ab')2 fragments administered for 1 mo post-bone marrow transplantation (BMT) had 100% survival without clinical or histopathological evidence of GVHD. Controls given saline injections succumbed by 39 days post-BMT. Similar results were obtained in groups of recipient mice given BMS in which T cells were depleted by in vitro anti-Thy-1.2 plus C' treatment. Further studies were undertaken to define mechanistic differences in the two approaches. Using Ly-5 congenic sources of donor bone marrow and spleen, we determined that anti-CD3F(ab')2 fragments induced TCR modulation and T cell depletion. Mature splenic-derived CD4+ cells were depleted to a greater extent than CD8+ cells. Early post-BMT, recipients receiving injections with control saline had the highest number of CD4+ and CD8+ cells (which may cause GVHD) followed by recipients of anti-CD3F(ab')2 fragments, with the fewest CD8+ cells observed in the anti-Thy-1.2 + C' treated group. CD3+CD4-CD8- cells (which may suppress GVHD generation) were present in higher numbers early post-BMT in recipients given anti-CD3F(ab')2 fragments as compared to recipients given anti-Thy-1.2 + C'-treated BMS. In long term survivors, a mononuclear T cell containing infiltrate without evidence of destruction was observed in sites of GVHD (lung and liver), consistent with a "Quilty" effect, which was not observed in either of the other two groups. Although survivors were tolerant of donor skin grafts and rejected third party grafts, recipients given anti-CD3F(ab')2 fragments but not anti-Thy-1.2 + C'-treated BMS had vigorous anti-host proliferative responses. These results demonstrate that although in vitro anti-Thy-1.2 + C' treatment of BMS (which is highly depletionary) and in vivo administration of anti-CD3F(ab')2 fragments (which is modulatory and less depletionary) are both effective strategies for GVHD, the cellular events involved in achieving GVHD prevention are indeed different.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.

The binding specificity of the murine OKT3 has been transferred into a human antibody framework to reduce its immunogenicity. This "humanized" anti-CD3 mAb (gOKT3-5) was previously shown to retain, in vitro, all the properties of native OKT3, including T cell activation, which has been correlated, in vivo, with the severe side effects observed in transplant recipients after the first administration of the mAb. T cell activation is thought to be triggered by the cross-linking mediated by the antibodies between T cells and Fc receptor-bearing cells. In this study, we introduced a single amino acid mutation from a leucine to a glutamic acid at position 235 in the Fc receptor binding segment of the gOKT3-5 mAb to produce Glu-235 mAb. This mutation generated a 100-fold decrease in the affinity of the antibody for the Fc receptor on U937 cells, without affecting Ag binding. In parallel, we observed a marked reduction in the T cell activation triggered by the mAb (proliferation, cell surface expression of early activation markers including Leu 23 and IL-2R, and release of TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF). In contrast, the mutated mAb retained suppressive properties similar to the gOKT3-5 mAb, as assessed by significant modulation of the T cell receptor complex and suppression of Ag-specific CTL activity. We conclude that this anti-CD3 mAb bearing a single amino acid mutation in its Fc portion retains important immunosuppressive properties, while exhibiting significantly less T cell activation than OKT3 in vitro. This drug might achieve potent immunosuppression while minimizing acute toxicity in vivo and thus be useful in transplantation as well as in autoimmune diseases.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

The role of TCR-gamma delta T lymphocytes in immune responses is currently not well understood. TCR-gamma delta cells have a limited repertoire suggesting that TCR-gamma delta T a limited number of evolutionarily conserved Ag such as nonpolymorphic MHC and heat shock proteins. TCR-gamma delta T lymphocytes appear in enhanced numbers in skin lesions produced by Mycobacterium leprae and in the synovial fluid of joints affected by rheumatoid arthritis, raising the possibility that this subset of T lymphocytes may play a role in control of infectious processes and in autoimmune diseases. We report the identification of a TCR-gamma delta T cell clone isolated from a HSV-infected mouse that recognizes glycoprotein I of HSV type 1. Clone recognition of glycoprotein I does not appear to require the expression of MHC class I or class II gene products. These data suggest that TCR-gamma delta lymphocytes may play an important role in the immune response to viral infections.

Intestinal intraepithelial lymphocytes (IEL) from mice are greater than 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.

T lymphocytes bearing the gamma/delta T cell receptor (TCR-gamma/delta) express a limited number of germline variable gene segments, generating receptor sequence diversity primarily through junctional mechanisms. To examine the role of V(D)J junctional sequences in antigen recognition by TCR-gamma/delta, we derived an alloreactive murine TCR-gamma/delta+ T cell line, LKD1, specific for the I-Ad class II major histocompatibility complex (MHC) molecule, and compared its receptor with that expressed by a previously characterized class II MHC alloreactive T cell line, LBK5, specific for I-Ek,b,s Ia molecules. Both LKD1 and LBK5 express receptors encoded by rearranged V gamma 1.2J gamma 2 and V delta 5D delta 2J delta 1 gene elements, differing in sequence only in the V(D)J junctional regions of the gamma and delta genes. These results demonstrate that junctionally encoded sequences corresponding to the putative third complementarity determining region can influence the antigen specificity of TCR-gamma/delta.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.

C57BL/6 mice are protected from a lethal pneumonia caused by Sendai virus when treated with low doses of mAb directed to the CD3 Ag. The protective mechanism is not due to an accelerated Sendai virus-specific Th cell, CTL, or antibody response but to a strong NK cell response via the in vivo induction of lymphokines. Antibodies directed against the NK1.1 and asialo GM1 marker totally reversed the protective effect of anti-CD3 treatment. In vivo treatment with rIL-2 also induced NK activity and induced antiviral protection. Treatment with anti-CD3 protects when given in a narrow time window (1 day before until 1 day after Sendai virus inoculation), indicating that NK activity is protective in the early phase of virus infection.

We have analyzed the functional significance of the four amino acid differences between the parental H-2Kb and mutant H-2Kbm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2Kb variants was analyzed using monoclonal antibodies which bind to well-defined H-2Kb epitopes. No alterations introduced into the conformational structure of H-2Kb by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2Kb anti-H-2Kbm8, H-2Kbm8 anti-H-2Kb, or third party anti-H-2Kb. The alloreactivity between H-2Kb and H-2Kbm8 is dominated by the amino acid substitution at residue 24 (Glu----Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr----Phe). The H-2Kbm8 anti-Kb bulk CTL reacted with both variant H-2Kbm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2Kbm8 anti-Kb CTL clones indicated the complexity of the self Kbm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met----Ile) and/or 30 (Asp----Asn) did not impact significantly on the alloantigenic properties of Kbm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2Kb and H-2Kbm8.

Thirteen T cell hybridomas and two cell lines expressing gamma delta TCR were generated from B10 and B6 CD3+CD4-CD8- splenic T cells. At least three different types of gamma-chains (V gamma 2-C gamma 1, C gamma 2, and C gamma 4) were shown to be expressed in these cells. Analysis of V delta gene segment usage in these hybridomas was performed by Northern blot hybridizations using probes specific for the V delta 1 through V delta 6 gene families and electrophoretic analysis of polymerase chain reaction amplified DNA obtained using V region-specific oligonucleotide primers for the V delta gene families as well as for V alpha 10, V alpha 4, and V alpha 11. These analyses revealed that V delta 5 gene segments are used by a high percentage of splenic gamma delta CD4-CD8- T cells (53% of the cells in our panel). Other V delta segments expressed by our panel of cells were V delta 2, V delta 4, V delta 6, and V alpha 10, indicating that the V delta repertoire expressed in the spleen is similar, but possibly not identical to the adult thymus repertoire. Sequence analysis of the V-D-J joinings of the delta-chain messages revealed substantial diversity, indicating that the delta-chain repertoire expressed in peripheral lymphoid organs uses a significant portion of the potential diversity predicted for these chains. These results demonstrate that, in spite of the low numbers of gamma delta T cells in peripheral lymphoid organs, the diversity of their TCR is extensive enough to play an important role in the immune response.

The present study was undertaken to determine the mechanism of tumor rejection in mice treated with low dose anti-CD3 mAb. It was found that treated mice developed nonrestricted antitumor cytolytic spleen cells of the Thy-1+, asialo GM-1+, CD4-, CD8- phenotype. Although these cells might play a role in immunopotentiating some immune responses, in vivo depletion studies using anti-asialo GM-1 mAb demonstrated that these cells were not involved in the rejection of the progressor tumor, 1591-PRO4L, by anti-CD3 mAb-treated mice. Mice treated with anti-CD3 did develop lasting tumor specific immunity as demonstrated by their ability to reject PRO4L on tumor rechallenge while being unable to reject an unrelated UV-induced tumor. The specificity of this memory implicated T cells in the response to PRO4L in anti-CD3-treated mice. Using in vivo T cell subset depletion of anti-CD3-treated animals, it was shown that both CD4+ and CD8+ T cells are required for anti-CD3-induced tumor rejection. The CD4+ cells provide helper function and are only required in the early rejection period, whereas CD8+ cells are required throughout the immune response. In fact, examination of rejecting tumors from treated animals revealed the presence of tumor-specific CD8+ cytolytic T cells capable of cytolysis immediately after removal from the rejecting PRO4L tumor. Thus, in vivo treatment with anti-CD3 mAb likely results in the pan-stimulation of the entire T cell population, which enhances the generation of specific CD8+ T cells, which then eliminate the tumor.

Regulation of the immune response to self-antigens is a complex process that depends on maintaining self-tolerance while retaining the capacity to mount a robust immune response to foreign antigens. Autoreactive T cells specific for these autoantigens are present in most normal individuals but are kept under control by multiple diverse peripheral tolerance mechanisms. In the last few years, there has been a re-emergence of suppressor cells as among the most central of these regulatory mechanisms. These cells, which express CD4, CD25, and CD62L, develop in the thymus and survive in a CD28-dependent manner in the periphery to maintain the homeostatic equilibrium of immunity and tolerance. In this review, we will summarize studies of these regulatory cells as they relate to autoimmune diseases and more specifically to type 1 diabetes and attempt to address some of the many outstanding questions. Finally, evidence is provided to support the ability of anti-CD3 mAbs to stimulate the regulatory T cells and reset the rheostat of immune tolerance in an animal model of autoimmune diabetes, the NOD mouse.

T-cell receptor gammadelta cells (TCRgammadelta) are often found in increased numbers during the course of several viral infections in humans. Although these findings suggest an important role for this unique subset, their precise function has not been ascertained. Recent studies in murine models of both RNA and DNA virus infections have begun to shed new light on the potential function for TCRgammadelta cells in antiviral immunity. It is clear that TCRgammadelta cells participate in the immune response to human immunodeficiency virus (HIV), influenza, Sendai, coxsackie, vaccinia, vesicular stomatitis virus (VSV), and herpes simplex virus-1 (HSV-1) viral infections since they become activated and home to the sites of viral replication. In this review we will summarize current efforts to dissect the role of TCRgammadelta cells in these disease settings, emphasizing the effector functions utilized, the TCR repertoire, and the antigens recognized. Particular focus will be placed on HSV-1 infections where we have begun to address these issues and have shown that TCRgammadelta cells are sufficient for protection from lethal infection and are able to recognize the herpes virus antigen glycoprotein I.

Insulin-dependent diabetes mellitus (IDDM) is the second most prevalent chronic illness of children. Investigation of the treatment of IDDM is hindered by the lack of a reproducible and easily maintained non-human primate model of this disorder.We induced IDDM in 11 juvenile cynomolgus monkeys after a single (150 mg/kg) intravenous injection of streptozotocin (STZ). All diabetic monkeys were treated with insulin twice daily, based on a sliding scale. Subcutaneous vascular access ports were surgically placed in each monkey to facilitate serial blood sampling and drug administration. Allogeneic pancreatic islet cells from unrelated donors were subsequently transplanted into the mesenteric circulation of all STZ-treated monkeys.Mild, transient nausea and vomiting occurred in all animals after STZ injection; however, no additional signs of toxicity occurred. Within 36 hr, all monkeys required twice daily administration of exogenous insulin to maintain a non-ketotic state. Serum C-peptide levels decreased from1.2 ng/ml before STZ, to between 0.0 and 0.9 ng/ml after STZ, confirming islet cell destruction. Animals were maintained in an insulin-dependent state for up to 147 days without any observable clinical complications. Subcutaneous vascular access port patency was maintained up to 136 days with a single incidence of local infection. Islet cell transplantation resulted in normoglycemia within 24 hr. Serum C-peptide levels increased (range: 2-8 ng/ml) for 6 - 8 days in immune competent animals, and for 39-98 days after transplant in immunosuppressed monkeys.IDDM can be consistently induced and safely treated in juvenile cynomolgus monkeys. Chronic vascular access can be maintained with minimal supervision and complications. This model is appropriate for studies investigating potential treatments for IDDM including islet cell transplantation.

Murine anti-CD3 (OKT3, Muromonab-CD3) is a potent human T-lymphocyte mitogen. A previous clinical Phase I trial examined OKT3 as an immunomodulator for the treatment of cancer. However, the murine monoclonal antibody triggered a potent humoral response that neutralized the antibody activity during subsequent administration. Thus, a "humanized" form of OKT3 (hOKT3gamma4) was developed to minimize immunogenicity. The genetically engineered human anti-CD3 retained its binding activity and effectively activated T cells in vitro. Therefore, we evaluated the safety and activity of hOKT3gamma4 in a Phase I clinical trial. hOKT3gamma4 was administered as a 10-min i.v. infusion every 2 weeks for three injections (one course of therapy). Six dose levels ranging from 50 to 1600 microg/injection were evaluated. Headache and fever were common, transient toxicities but were not dose limiting. The dose-limiting toxicities were rigors and dyspnea at the 1600-microg dose level, which defined 800 microg as the maximally tolerated dose in this trial. A dose-dependent in vivo T-lymphocyte activation was produced by this treatment, and the most significant T-lymphocyte activation occurred in patients treated at the two highest dose levels (800 and 1600 microg). Persistent CD3 modulation occurred after administration of 1600 microg of hOKT3gamma4. Anti-idiotypic antibodies were detected in only 6 of 24 patients after multiple injections and were not associated with attenuation of T-lymphocyte activation. Malignant ascites resolved in three patients, one each with peritoneal mesothelioma, pancreatic adenocarcinoma, and ovarian adenocarcinoma. hOKT3gamma4 can induce T-lymphocyte activation in patients with cancer, and the immunogenicity of the "humanized" antibody is sufficiently reduced relative to its murine "parent" to permit immunostimulation by repetitive i.v. administration. The therapeutic potential of biweekly i.v. hOKT3gamma4 at a dose of 800 microg should be further evaluated.

Mice lacking CTLA-4 die at an age of 2-3 wk due to massive lymphoproliferation, leading to lymphocytic infiltration and destruction of major organs. The onset of the lymphoproliferative disease can be delayed by treatment with murine CTLA4Ig (mCTLA4Ig), starting day 12 after birth. In this study, we have characterized the T cells present in CTLA-4-deficient mice before and after mCTLA4Ig treatment. The T cells present in CTLA-4-deficient mice express the activation markers, CD69 and IL-2R; down-regulate the lymphoid homing receptor, CD62L; proliferate spontaneously in vitro and cannot be costimulated with anti-CD28 mAb consistent with a hyperactivated state. The T cells from CTLA-4-deficient mice survive longer in culture correlating with higher expression of the survival factor, Bcl-xL, in these cells. Most significantly, the CD4+ T cell subset present in CTLA-4-deficient mice secretes high levels of IL-4 and IL-5 upon TCR activation. Treatment of CTLA-4-deficient mice treated with mCTLA4Ig reverses the activation and hyperproliferative phenotype of the CTLA-4-deficient T cells and restores the costimulatory activity of anti-CD28 mAb. Furthermore, T cells from mCTLA4Ig-treated mice are not skewed toward a Th2 cytokine phenotype. Thus, CTLA-4 regulates CD28-dependent peripheral activation of CD4+ T cells. This process results in apoptosis-resistant, CD4+ T cells with a predominantly Th2 phenotype that may be involved in the lethal phenotype in these animals.

B7-1 (CD80)-transfected P815 tumor cells were previously shown to elicit tumor-eradicating immunity that leads to the regression of B7-1+ P815 tumors after transient growth in normal syngeneic (DBA/2) mice. Here, we show that not only the B7-1 molecule but also the B7-2 (CD86) molecule contributed to the eradication of B7-1+ P815 tumors. The B7-1 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed not only on the tumor cells but also on host APCs, including MAC-1+ cells. The B7-2 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed only on host APCs, such as B220+ cells, and not on the tumor cells. In spite of the fact that B7-expressing host APCs contributed to the eradication of B7-1+ P815 tumors, only CD8+ T cells without help from CD4+ T cells were important for tumor eradication. Taken together, these findings indicate that in addition to the ability of B7-1-transfected tumor cells to stimulate CD8+ T cell-mediated tumor-eradicating immunity directly, such tumor cells can also stimulate CD8+ T cell-mediated tumor-eradicating immunity indirectly as a result of cross-priming through B7-expressing host APCs.

In order to elucidate the mechanisms that ensure survival of the allogeneic fetus, we are investigating the expression pattern of genes that are involved in peripheral self-tolerance in tissues at the maternal-fetal interface. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T cell activation and may modulate peripheral self-tolerance. Previously, we reported the preferential transmission of maternally-inherited shorter alleles at a 3'-UTR microsatellite locus to liveborn children, but random transmission of paternally-inherited alleles, suggesting that CTLA-4 may be involved in the maintenance of tolerance at the maternal-fetal interface. In this report, we demonstrate that CTLA-4 mRNA and protein are indeed expressed in fetal tissues at the maternal-fetal interface throughout gestation.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.

We have previously shown that B7-2 (CD86)-transfected P815 tumor cells elicit tumor-eradicating immunity that leads to the regression of the B7-2+ P815 tumor after transient growth in normal DBA/2 mice. Here, we show that both the B7-2 and B7-1 (CD80) molecules contribute to the eradication of B7-2+ P815 tumors as treatment of the mice with both anti-B7-2 and anti-B7-1 mAb was required to prevent B7-2+ P815 tumor regression. The cells that expressed the B7-1 molecule following inoculation of B7-2+ P815 tumor cells into normal mice were not the tumor cells but rather host APCs including MAC-1+ cells present in the draining lymph nodes. Moreover, B7-1-expressing host APCs were found to be important for the rejection of B7-2+ P815 tumors as anti-B7-2 mAb alone, which was ineffective in preventing B7-2+ P815 tumor rejection by normal wild-type mice, was effective in preventing B7-2+ P815 tumor rejection by mice in which the B7-1 gene was disrupted. Finally, consistent with the importance of B7-1-expressing host APCs for the generation of tumor-eradicating immunity against B7-2+ P815 tumor cells, CD4+ T cells (not only CD8+ T cells) were found to participate in tumor-eradicating immunity against B7-2+ P815 tumor cells. Thus, in addition to eliciting tumor-eradicating immunity directly, B7-2+ P815 tumor cells elicit tumor-eradicating immunity indirectly through B7-1-expressing host APCs that present tumor-associated Ags to CD4+ T cells.

CTLA-4 blockade has been shown by other investigators [D. R. Leach, et al., Science (Washington DC), 271: 1734-1736, 1996; and Y-F. Yang, et al., Cancer Res., 57: 4036-4041, 1997] to retard tumor growth in selected tumor systems. Here, we show that CTLA-4 blockade alone was ineffective in retarding tumor growth in the murine MOPC-315 tumor system. Yet, CTLA-4 blockade offered significant therapeutic benefits to MOPC-315 tumor bearers when combined with a subtherapeutic dose of the chemotherapeutic agent melphalan, which was previously shown (L. Gorelik, et al., Cancer Immunol. Immunother., 39: 117-126, 1994) to shift the cytokine profile in the tumor bearers toward type-1 cytokines. In addition, we show here that anti-CTLA-4 monoclonal antibody enhanced antitumor cytotoxicity when the anti-CTLA-4 monoclonal antibody was added to stimulation cultures of spleen cells from low-dose melphalan-treated MOPC-315 tumor-bearing mice but not from untreated tumor-bearing mice. These results suggest that the therapeutic benefits of CTLA-4 blockade depend on the ability of drugs such as melphalan to promote an immunogenic environment by altering the cytokine profile of tumor-specific T cells.

Despite the description of numerous antigenic ligands recognized by TCRgammadelta cells, detailed information concerning the structural nature of these antigenic epitopes is lacking. In addition, the recent descriptions of human TCRgammadelta cells recognizing mycobacterium-derived low m.w. lipid molecules confirms that the spectrum and nature of biologic structures that are capable of being recognized by TCRgammadelta cells are unclear. We have previously described a murine TCRgammadelta cell clone, TgI4.4, that is reactive to herpes simplex virus (HSV)-1 glycoprotein I (gI). Unlike TCRalphabeta-mediated, MHC-restricted Ag recognition but similar to Ig Ag recognition, TgI4.4 recognizes purified gI directly, in the absence of Ag processing or presentation. Since gI is a complex glycoprotein, the nature of the antigenic epitope was investigated. First, gI recognition by TgI4.4 is conformationally dependent, as revealed by denaturation and proteolytic experiments. Secondly, the epitope recognized by TgI4.4 was mapped to the amino terminus by using insertion mutants of gI. Lastly, TgI4.4 recognizes the gI protein directly since completely deglycosylated forms of gI are efficiently recognized. Therefore, TCRgammadelta cells are capable of recognizing a variety of molecular structures, including proteins. The ability of TgI4.4 to recognize a nonglycosylated form of gI suggests that HSV-1 recognition by TCRgammadelta cells in vivo is not limited by cell-specific glycosylation patterns or glycosylation-dependent conformational influences.

Autoimmune arthritides are characterized by an imbalance between pro- and anti-inflammatory cytokines. Viral IL-10 (vIL-10) shares many of the anti-inflammatory properties of mouse and human IL-10, but lacks their immunostimulatory properties and may therefore offer superior immunosuppression. Viral IL-10 has a short half-life; however, genetic modification of cells in vivo offers a potential means of achieving prolonged therapeutic titers. To determine the effects on collagen-induced arthritis of vIL-10 gene transfer, DBA/1 mice were administered i.v. or intra-articular injections of Av(vIL-10), a replication-deficient adenovirus encoding vIL-10. The i.v. injection of Av(vIL-10) before disease onset delayed the onset and reduced the severity of collagen-induced arthritis, but treatment of established disease was ineffective. The preventative effects were not due to decreased anti-type II collagen Ab production. Rather, T cells from mice treated with Av(vIL-10) demonstrated a decreased in vitro proliferative response to type II collagen, and a delay was observed in up-regulation of synovial mRNA for the proinflammatory cytokines IL-2 and IL-1beta. Intra-articular injection of Av(vIL-10) into knee joints did not reduce arthritis in the knees, but inhibited the development of arthritis in the paws. Humoral and cellular immune responses against Av(vIL-10) were observed. These results demonstrate that vIL-10 can significantly alter the course of autoimmune arthritis and emphasize the complexities of using gene transfer as a method of drug delivery for arthritis.

B7/CD28-mediated costimulation is a promising target for therapeutic intervention in autoimmune diseases. However, studies addressing the differential functional roles of B7-1 and B7-2 in several autoimmune models have resulted in conflicting data, perhaps due to the temporal dynamics of B7-1 and B7-2 surface expression on different cell types and/or at different sites during an autoimmune response. We examined the temporal expression of B7 costimulatory molecules in the CNS and in various lymphoid organs during the course of murine relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE). Following immunization of SJL mice with the immunodominant proteolipid protein epitope, PLP139-151, surface expression of B7-1 was up-regulated on B cells, T cells, and macrophages, relative to B7-2, on CNS-infiltrating cells and on splenocytes. Similar enhancement in splenic B7-1 expression could be induced in SJL mice by the adoptive transfer of PLP139-151-specific cells or by immunization with CFA alone. These changes were not observed on lymph node cells, including those isolated from lymph nodes draining the immunization site, which maintained the predominant B7-2 expression pattern seen in naive mice. These phenotypic expression patterns correlated with the functional predominance of B7-1 in costimulating T cell activation when employing APCs from the spleen or CNS of mice with ongoing R-EAE, while B7-2 remained functionally predominant on lymph node APCs. Variation of phenotypic expression and functional dominance of costimulatory molecule expression in different lymphoid compartments during an active inflammatory autoimmune response has important implications in immune regulation, autoimmune pathogenesis, and therapeutic strategies.

To examine the effect of self Ag on activation requirements of TCR-alphabeta intestinal intraepithelial lymphocytes (IELs), we utilized the 2C transgenic (Tg) mouse model specific for a peptide self Ag presented by class I MHC, H-2Ld. CD8alpha alpha and CD4-CD8- IELs from syngeneic (H-2b, self Ag-) and self Ag-bearing (H-2b/d, self Ag+) strains were examined for their ability to respond in vitro to P815 (H-2d) cell lines expressing the endogenous antigenic peptide, p2Ca. Proliferation, cytokine production, and CTL activity were elicited in IEL T cells isolated from self Ag- H-2b mice when stimulated with P815 cells expressing basal levels of self Ag. These responses were enhanced following the addition of exogenous p2Ca peptide and ectopic expression of the costimulatory molecule, B7-1. By comparison, IEL from self Ag-bearing mice failed to respond to basal levels of self Ag presented by P815 cells even in the presence of B7-1-mediated costimulation. However, the addition of increasing amounts of exogenous p2Ca peptide induced a response from the in vivo "tolerized" T cells. These results suggest that exposure to self Ag in vivo increased the threshold of TCR activation of Ag-exposed self-reactive IELs. The dependence of increased signal 1 to activate self-reactive IELs suggests a defect in TCR signaling that may maintain self tolerance in vivo. These data suggest that conditions that overcome signal 1 IEL defects may initiate autoreactive responses in the intestine.

Anti-CD3 mAbs with low FcR affinity prolong graft survival in the absence of the cytokine-mediated toxicity observed with conventional anti-CD3 treatment. Previous studies have shown that FcR-nonbinding anti-CD3 mAbs suppress immune responses, at least in part, by delivering a partial signal resulting in Th1 unresponsiveness. In this study, the biochemical and functional consequences of FcR-nonbinding anti-CD3 treatment for various activated T cell populations were examined. In contrast to Th1 cells, FcR-nonbinding anti-CD3-treated Th2 cells secreted IL-4 and proliferated. Furthermore, Th2 cells cultured with the mAb were not rendered unresponsive. Mixed "Th0" populations responded to FcR-nonbinding anti-CD3 by producing IL-4, and showed a selective decrease in IL-2 production following preculture with the mAb. The stimulation of IL-4-producing cells did not reflect a more complete TCR signal, since similar defects in zeta, ZAP-70, and MAP kinase phosphorylation were observed in Th1 and Th2 cells. Despite the proximal signaling defects, FcR-nonbinding anti-CD3 induced nuclear translocation of NF-ATc. Thus, Abs that deliver partial TCR signals may promote development of a Th2 phenotype during the course of an immune response via selective effects on different Th subsets.

Cytotoxic T lymphocyte Ag-4 (CTLA-4; CD152) is an important T cell regulatory molecule. In vitro experiments have shown that the blockade of signals through CTLA-4 augments T cell expansion, while CTLA-4 cross-linking results in decreased T cell proliferation due to decreased IL-2 production. However, less is known about the role of CTLA-4 in regulating an ongoing immune response. In this study, we examined the role of CTLA-4 in the expansion, decline, tolerization, and differentiation of T cells following treatment with staphylococcal enterotoxin B (SEB). Anti-CTLA-4 treatment resulted in increased numbers of SEB-reactive T cells and blockade of subsequent tolerance induction. Further examination of the SEB-reactive cells from anti-CTLA-4-treated mice demonstrated that both the CD4+ and CD8+ Vbeta8+ T cells produced IL-4, providing evidence that not only do signals through CTLA-4 regulate T cell-tolerizing events, but they also play an important role in the differentiation of T cells in vivo.

Ag-specific T cell activation requires a CD28-mediated costimulatory interaction. This observation has suggested novel approaches to suppress donor-specific immunity, including the use of soluble CD28 antagonists, such as CTLA4Ig, which suppresses transplant rejection in small animal models. In this study, CTLA4Ig therapy was examined in a non-human primate model of allogeneic pancreatic islet transplantation. Two of five CTLA4Ig-treated monkeys showed prolonged graft survival, which correlated with donor-specific hyporesponsiveness in vitro. Humoral responses to the transplanted tissue were suppressed in all treated animals. These results suggest that CTLA4Ig is effective in suppressing both humoral and cellular immune responses in a non-human primate model of allogeneic transplantation.