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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.

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.

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.

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.

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.

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.

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.

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.

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.

Antigen-specific T-cell activation depends initially on the interaction of the T-cell receptor with peptide/major histocompatibility complex (MHC). In addition, a costimulatory signal, mediated by distinct cell surface accessory molecules such as CD28, is required for complete T-cell activation. One essential element of the CD28 costimulatory system that makes it an attractive target for immunotherapy is the selective effect of CD28 antagonists on activated T cells. Only cells encountering antigen presenting cells (APCs) without the appropriate CD28 ligand will be rendered functionally inactive as desired for any next-generation immuno-suppressive drug. This brief review will focus on the role of CD28/B7 interactions in regulating organ graft rejection. In vitro and in vivo studies will describe the use of a soluble fusion protein antagonist of CD28/B7 (CTLA-4Ig), anti-B7 MAbs, and genetically altered CD28 "knockout" mice to study immune responses. The studies suggest that: 1) CTLA-4Ig induces long-term, antigen-specific unresponsiveness in vivo; 2) two distinct ligands for CD28, B7-1 and B7-2, are differentially regulated during immune responses; and 3) both B7-1 and B7-2 costimulatory molecules are active, in vivo, although B7-2 plays a clearly dominant role in murine allograft rejection.

Cognate recognition of antigen-presenting cells by antigen-specific T cells is critically dependent on non-cognate adhesive interactions. For instance, several studies have shown that in vivo anti-LFA-1 plus anti-ICAM-1 mAb treatment results in prolongation of allograft survival. We have developed a xenogeneic islet transplant model to investigate the role of various adhesion interactions in the xenogeneic response and study the effect of pretreating donor tissue with immunosuppressive drugs. Pancreatic islet cells were pretreated in vitro with anti-human ICAM-1 mAb, transplanted under the renal capsule of diabetic B6 mice in the absence of systemic immunosuppression and examined for long-term xenograft acceptance. The survival of human islets pretreated with anti-human ICAM-1 was significantly prolonged (MST = 53 days, with 40% of grafts surviving100 days). In contrast, the survival of human islets pretreated with the control antibody was similar to those of nontreated islets (MST = 7 days). A massive lymphocyte infiltrate into control xenografts was observed at 5 days post-transplant. In contrast, a lymphocyte infiltrate did not appear in the anti-ICAM-1-treated islets for at least 11 days. Only mAbs specific for the LFA-1 binding epitope of ICAM-1 were found to inhibit a mixed islet/lymphocyte reaction in vitro and block graft rejection in vivo. However, graft prolongation is not accompanied by systemic tolerance. Mice transplanted simultaneously with human islet cells treated with control Ig (left kidney) or anti-ICAM-1 (right kidney) rejected the control islets but not anti-ICAM-1-treated islets. These results suggest that the LFA-1/ICAM-1 interaction is a critical component for xenograft rejection and, more important, that pretreatment of islet tissue with anti-adhesion molecule antibodies can profoundly alter graft recognition and rejection in the absence of any systemic drug therapy. However, graft prolongation is not accompanied by systemic tolerance induction.

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.

Ag-pulsed B cells are potent APCs, in part, because of the ability of the Ig receptor to mediate rapid and specific Ag uptake. However, it is also known that full T cell activation requires signals delivered by costimulatory molecules, which naive B cells seem to lack. This study examines the effect Ig receptor engagement has on the expression and function of a new CD28 counter-receptor, B7-2. Unlike B7-1 (B7), B7-2 was rapidly induced on the cell surface of B cells after engagement of the Ig receptor by either anti-Ig mAbs or hen egg lysozyme (HEL) on normal and HEL-specific B cell receptor transgenic B cells, respectively. Furthermore, B7-2 expression was up-regulated on tolerant B cells isolated from HEL/anti-HEL double transgenic mice after Ag stimulation, although at lower levels than on nontolerant transgenic B cells. No significant cell surface levels of B7-1(B7) were observed under these conditions. Finally, the B7-2 molecules induced by Ig cross-linking costimulated T cell proliferation in a CD28-dependent manner, independent of B7-1(B7) expression. Thus, the effectiveness of Ag-specific B cells as APCs depends on both their enhanced Ag uptake, mediated by the B cell receptor, and immediate up-regulation of a potent costimulatory molecule, B7-2.

Despite intensive efforts, the general rules for gamma delta T cell recognition remain undefined. Here, we take advantage of the detailed knowledge of the molecular structure and biosynthetic pathways of major histocompatibility complex (MHC) molecules to analyze the recognition properties of the gamma delta T cell clones LBK5 (specific for the class II MHC, IEk) and G8 (specific for the nonclassical class I MHC, TL10b). We find that the activation of these clones requires neither class I nor class II antigen-processing and that peptides do not confer specificity. Epitope mapping also shows that the topology of gamma delta T cell receptor interaction with the MHC is distinct from that of alpha beta T cells. These results suggest that the molecular nature of gamma delta T cell recognition is fundamentally different than that of alpha beta T cells.

Fetal thymus organ culture system (FTOC), a well-known model used for the study of TCR alpha beta development, was employed to study TCR gamma delta cell development. It was found that different waves of TCR gamma delta cells develop from precursors within the fetal thymi at the time in vitro culture. Subsets of fetal thymocytes were analyzed by flow cytometry and 2-D gel biochemical analysis was performed. After 5 days in FTOC, V gamma 3+ and V gamma 2+ cells were dominant. By day 12 FTOC, the absolute number of V gamma 3+ cells decreased while V gamma 2+ and V gamma 4+ cells became dominant. These observations suggest that the thymic micro-environment affects the thymic waves of TCR gamma delta subsets. Furthermore, the effect of TCR/antigen interaction in the development of TCR gamma delta cells was examined with anti-TCR mAbs added into the FTOC. Anti-CD3 mAb added to day 5 and day 12 FTOC inhibited TCR gamma delta development, especially V gamma 4+ cells. On the other hand, V gamma 2+ cells were relatively resistant to the addition of anti-TCR mAb. The reduction of TCR gamma delta+ thymocytes was not due to the modulation of TCR molecules and could be reversed by Cyclosporin A (CsA). These results suggest that TCR ligation negatively regulates the development of TCR gamma delta cells in a V gamma-specific manner.

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.

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.

To investigate the role of CD3-positive T cells in allogeneic marrow rejection in mice and to examine the effects of anti-CD3 monoclonal antibody (MoAb) on allogeneic marrow engraftment, a hamster MoAb, 145-2C11, with specificity for the CD3 epsilon portion of the murine T-cell receptor complex was administered to B6 (H-2b) mice that had been sublethally irradiated with 626 cGy and injected with 10 x 10(6) T-cell-depleted B6C3F1 (H-2b/k) bone marrow cells. Lymphoid chimerism status was assessed by flow cytometric analysis of peripheral blood lymphocytes using H-2k-specific MoAb 5 to 6 weeks after bone marrow transplantation. When hosts were treated with 400 micrograms of anti-CD3 MoAb at the time of marrow injection, the percentage of donor-type cells was 75.2% +/- 15.0%, while it was 1.9% +/- 1.2% in untreated mice. It was demonstrated that anti-CD3 MoAb not only suppressed T-cell function but also induced colony-stimulating factors in host mice, and that enhancement of marrow engraftment in anti-CD3 MoAb-treated mice was associated with factor release as well as suppression of host T-cell function. Results were consistent with engraftment being enhanced by a differential response of donor (rather than host) marrow to serum factors in association with host T-cell immunocompromise.

Antibodies that possess the Ag-binding regions of OKT3 within the context of a human framework (Hu-OKT3 Ab) offer distinct advantages for optimizing anti-CD3 mAb therapy. First, manipulation of Ab genes to produce humanized Ab that retain Ag-binding activity may circumvent antigenicity problems. Second, Ab gene engineering provides a means for modifying functional properties, including T cell activation and immune suppression. The purpose of this study was to determine the functional properties of Hu-OKT3 Ab and to compare the functional properties and idiotypes of Hu-OKT3 Ab to those of murine OKT3. Three Hu-OKT3 IgG4 Ab, a chimeric OKT3 antibody (cOKT3-1) (grafted sequences comprising all OKT3 VH and VL regions) and two complementarity determining region (CDR)-grafted antibodies, gOKT3-5 and gOKT3-6 (grafted sequences comprising only OKT3 VH and VL CDR and some framework amino acids, were analyzed. Initial studies demonstrated that the cOKT3 and gOKT3-5 Ab bound selectively to T cells and competitively inhibited OKT3-FITC binding with avidities similar to that of murine OKT3. Binding avidity of the gOKT3-6 Ab was markedly less than that of the other two Hu-OKT3 Ab. Serologic analysis suggested that cOKT3 and gOKT3-5 Ab possess idiotypes (combining sites) similar to murine OKT3. T cell activation potency of all three Hu-OKT3 Ab was assessed by proliferation, induction of activation marker expression (IL-2R and Leu 23), and lymphokine production (TNF-alpha and IFN-gamma). The cOKT3 and gOKT3-5 Ab demonstrated T cell activation potencies similar to murine OKT3 as assessed by each parameter. CD3 coating and modulation by these two Ab was effective but somewhat less potent than that observed with OKT3. Finally, cOKT3 and gOKT3-5 Ab both inhibited CTL activity comparably to murine OKT3. In conclusion, these studies indicate that gOKT3-5 and cOKT3 Ab possess immune modulating properties similar to murine OKT3 and thus offer attractive alternatives to murine OKT3 for in vivo therapy.

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.

One of the major problems in clinical immunity is that neoplastic and virally infected cells are insufficiently immunogenic to trigger an immune response. During the past several years, our laboratory has explored the use of T-cell-specific reagents including monoclonal antibodies (MAbs) and staphylococcal enterotoxins to amplify immune responses. This report summarizes our efforts to augment immunity with these agents.

The alpha beta and gamma delta T cell receptors for antigen (TCR) delineate distinct T cell populations. TCR alpha beta-bearing thymocytes must be positively selected by binding of the TCR to major histocompatibility complex (MHC) molecules on thymic epithelium. To examine the requirement for positive selection of TCR gamma delta T cells, mice bearing a class I MHC-specific gamma delta transgene (Tg) were crossed to mice with disrupted beta 2 microglobulin (beta 2M) genes. The Tg+beta 2M- (class I MHC-) offspring had Tg+ thymocytes that did not proliferate to antigen or Tg-specific monoclonal antibody and few peripheral Tg+ cells. This is evidence for positive selection within the gamma delta T cell subset.

Several problems remain associated with anti-CD3 monoclonal antibody therapy, including first-dose reactions, recurrent rejection, and the host humoral response to the xenogeneic mAb. One approach toward optimization of anti-CD3 mAb therapy involves the use of anti-CD3 mAbs of defined idiotype, isotype, and epitope specificity, so that the desired T cell activation and suppression properties are obtained and neutralization by antiidiotypic antibody is avoided. The purpose of the present study was to define the contribution of mAb isotype and epitope specificity in determining T cell activation and suppression potencies and to evaluate the idiotypic relationships among ten anti-CD3 mAbs and one anti-TCR alpha beta mAb selected for this study. Epitope mapping by flow cytofluorometry indicated that one mAb, OKT3D, possesses an epitope specificity distinct from that of other anti-CD3 mAbs. Analysis of early T cell activation, proliferation, and lymphokine production (TNF-alpha, gamma-IFN, and GM-CSF) indicated that mAb isotype exerted a profound effect on activation potency (IgG2a much greater than IgG1 greater than IgG2b), whereas epitope specificity exerted a minor effect. CTL inhibition studies demonstrated an epitope effect with highest inhibition potencies observed with OKT3D IgG1 and OKT3D IgG2b mAbs. Idiotypic analysis of nine mAbs indicated that all anti-CD3 mAbs except OKT3E possess idiotypes distinct from that of OKT3. Thus, two anti-CD3 mAbs, OKT3D IgG1 and OKT3D IgG2b, possess high immune suppression potency, low activation potency, and idiotypes distinct from OKT3. In conclusion, selection of anti-CD3 mAbs of defined idiotype and appropriate T cell activation and suppression properties may provide a means for mitigating problems associated with OKT3 therapy.

T cells expressing the gamma delta receptor heterodimer can recognize a broad array of different antigens, including classical and non-classical major histocompatibility complex (MHC) proteins, MHC-like CD1 antigens, and bacterial antigens such as the mycobacterial heat shock proteins and staphylococcal enterotoxins. Reactivity to self antigens including autologous stress proteins implicates TCR gamma delta T cells in autoimmune disease. It is as yet unclear whether the responses of gamma delta T cells specific for soluble proteins are restricted by conventional or non-classical MHC molecules. Correlations of TCR gamma delta usage with specificity suggest that, like TCR alpha beta, sequences encoded within both the V regions and the V(D)J junctions are important in determining receptor specificity.

Within murine epidermis exists a population of Thy-1+ DEC which express membrane Thy-1 antigen, but lack CD8 or CD4 antigen. We examined freshly obtained non-cultured Thy-1+ DEC both by immunofluorescence and by biochemical techniques to identify the protein products of the T cell receptor (TCR) and the associated CD3 complex on these cells. Virtually all of the Thy-1+ DEC are brightly positive in CD3 expression with immunofluorescence using the monoclonal antibody 145-2C11. By immunoprecipitation, using this same antibody and polyclonal anti-TCR-gamma antibody, the only TCR heterodimer detected on the freshly isolated Thy-1+ DEC is the gamma delta heterodimer. These findings suggest that in the phenotype and TCR expression, Thy-1+ DEC are analogous to CD8-, CD4- early fetal thymocytes.

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.

In this report, we have characterized T cell ontogeny in mice transgenic for the V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) TCR chain gene by analyzing the T cell surface phenotype and microarchitecture of the transgenic lymphoid organs during development. The first wave of V gamma 3J gamma 1C gamma 1 TCR+ thymocytes that appear in the thymus at day 14 of gestation were virtually absent in the TCR gamma 4 transgenic mice. However, comparable levels of total gamma delta+ thymocytes were present in the TCR gamma 4 transgenic mice, suggesting that these thymocytes expressed a transgene receptor. In addition, the appearance of significant numbers of TCR alpha beta+ T cells occurred earlier (day 17 of fetal development) in the transgenic thymus. After birth, thymuses from TCR gamma 4 transgenic mice were characterized by a consistent increase in the number of TCR gamma delta+ thymocytes and a transient increase (between 2 and 4 wk of age) in the absolute number of TCR+ thymocytes (2- to 4-fold) compared with thymuses from normal mice. Immunohistologic analysis of the thymus, spleen, and lymph nodes from 2-day and 3-wk-old transgenic mice showed significant differences to controls only in regions harboring mature, functional T cell subsets which may be the result of bidirectional signaling between lymphocyte subsets (influenced by transgene expression) and stromal elements. The results demonstrate that striking differences, leading to the earlier appearance of mature T cells, occurred in both the fetal and neonatal thymus and periphery of mice that expressed a TCR gamma 4 transgene. These findings are consistent with the hypothesis that expression of the TCR gamma 4 chain gene, early in ontogeny, has a positive influence on the development of the T cell compartment.

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.

The genes encoding a gamma delta T-cell receptor specific for a major histocompatibility complex class I molecule encoded by the TIa locus have been inserted into the mouse germ line. In mice that do not express the TIa-encoded determinant, transgenic gamma delta T cells are a functional component of the CD4-CD8- 'double-negative' T cells in the thymus and peripheral lymphoid organs. In mice that express the TIa-encoded determinant, there are no transgenic gamma delta T cells in peripheral lymphoid organs, and there are no thymocytes expressing normal levels of the transgenic gamma delta T-cell receptor.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.

The present study focused on attempts to correlate the expression of T cell differentiation antigens and development. A series of mAb was developed that recognized an Ly-6.2-linked Ag, Ly-6C.2. These mAb reacted with a subset of peripheral Lyt-2+ T lymphocytes but not Lyt-2+ thymocytes. Functional analysis of the Lyt-2+, Ly-6C.2- and Lyt-2+, Ly-6C.2+ subsets demonstrated that both cell types responded in class I and class II allogeneic MLC as well as in virus-specific cell-mediated cytolysis assay, although precursor frequency analysis suggested that the majority of precursor CTL were Ly-6C.2+. The marker did not appear to represent an activation Ag because Ly-6C.2-, Lyt-2+ precursor CTL generated Ly-6C.2-, Lyt-2+ CTL. Finally, virtually all splenic Lyt-2+ T cells from athymic (nu/nu) mice were Ly-6C.2+ whereas all thymic CTL were Ly-6C.2-. These results suggested that Ly-6C.2+, Lyt-2+ T cells may represent a distinct lineage of Lyt-2+ T cells that can develop in the absence of thymic influence.

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.

Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.

Reconstitution of lethally irradiated mice with a mixture of T cell-depleted syngeneic plus T cell-depleted allogeneic bone marrow (B10 + B10.D2----B10) leads to the induction of mixed lymphopoietic chimerism, excellent survivals, specific in vivo transplantation tolerance to subsequent donor strain skin grafts, and specific in vitro unresponsiveness to allogeneic donor lymphoid elements as assessed by mixed lymphocyte reaction (MLR) proliferative and cell-mediated lympholysis (CML) cytotoxicity assays. When B10 recipient mice received mixed marrow inocula in which the syngeneic component had not been T cell depleted, whether or not the allogeneic donor marrow was treated, they repopulated exclusively with host-type cells, promptly rejected donor-type skin allografts, and were reactive in vitro to the allogeneic donor by CML and MLR assays. In contrast, T cell depletion of the syngeneic component of the mixed marrow inocula resulted in specific acceptance of allogeneic donor strain skin grafts, whether or not the allogeneic bone marrow was T cell depleted. Such animals were specifically unreactive to allogeneic donor lymphoid elements in vitro by CML and MLR, but were reactive to third party. When both the syngeneic and allogeneic marrow were T cell depleted, variable percentages of host- and donor-type lymphoid elements were detected in the mixed reconstituted host. When only the syngeneic bone marrow was T cell depleted, animals repopulated exclusively with donor-type cells. Although these animals had detectable in vitro anti-host (B10) reactivity by CML and MLR and reconstituted as fully allogeneic chimeras, they exhibited excellent survival and had no in vivo evidence for graft-vs-host disease. In addition, experiments in which untreated donor spleen cells were added to the inocula in this last group suggest that the presence of T cell-depleted syngeneic bone marrow cells diminishes graft-vs-host disease and the mortality from it. This system may be helpful as a model for the study of alloresistance and for the identification of syngeneic cell phenotypes, which when present prevent engraftment of allogeneic marrow.

The thymus is the major site for T-cell receptor (TCR) gene rearrangement and T-cell maturation. The specific antigen recognition structure (TCR) on murine T cells has been shown to be dependent on a polymorphic set of disulphide-linked heterodimers, containing two integral membrane glycoprotein chains, TCR alpha and TCR beta, expressed in non-covalent association with an invariant complex of proteins, CD3 (T3). Recently, a novel TCR/CD3 complex, that includes the product of the TCR gamma gene, has been identified on a subset of both peripheral cells and thymocytes. Here we examine the expression of TCR/CD3 complexes in fetal ontogeny and in the adult thymus. The results demonstrate that CD3+4-8-(T3+,L3T4-,Lyt2-)cells are detected in day-15 fetal thymi, throughout fetal development and in adult thymus. In situ hybridization studies indicate that these early CD3+ cells express high levels of TCR gamma-specific RNA, low levels of TCR beta-specific RNA and no detectable TCR alpha-specific RNA. Day-16 CD3+,4-,8- fetal thymocytes can be activated to proliferate and demonstrate cytolytic activity when cultured in the presence of anti-CD3 monoclonal antibodies and interleukin-2 (IL-2). These results suggest that CD3-bearing cells, present early in thymic ontogeny, express a functional TCR and may, therefore, be important in repertoire development.

A new T cell molecule defined by the mAb 143-4-2 has been identified that is involved in T cell activation. The expression of the 143-4-2-defined epitope is linked to the previously characterized Ly-6 locus and restricted to bone marrow cells and to a subset of peripheral Lyt-2+ cells. In comparison to other anti-Ly-6.2 mAb, the 143-4-2 mAb appears to be directed at an allogeneic determinant of the Ly-6.2C molecule. The anti-Ly-6.2C antibody can promote the lysis of antigen-non-bearing target cells by alloreactive CTL clones, and in the presence of cofactors (PMA or IL 2) induces a subset of Lyt-2+ cells to proliferate, perhaps through an autocrine pathway. Although the antibody described has antigen-like effects as described for anti-TcR complex reagents, studies performed with a recently derived anti-murine T3 mAb suggest that the Ly-6.2C molecule is not associated on the cell surface with components of the TcR complex. Nevertheless, cell surface expression of the TcR complex is required for optimal triggering of T cells via the Ly-6.2C molecule. Because Ly-6.2C determinants are expressed in bone marrow and not in the thymus, the possibility is considered that expression of this molecule identifies a distinct subset of extrathymically derived T cells.

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.

Adherent spleen cells from late (greater than or equal to 18 days post inoculation) tumor-bearing BALB/c mice suppressed lymphoproliferative and effector immunity as evaluated by the mixed leukocyte culture and cell-mediated lympholysis assays. Procedures that eliminated T-cells or B-cells while enriching for macrophage populations significantly augmented the suppression, whereas removal of phagocytic and adherent cells abrogated the suppressive effect. We concluded that the cells responsible for suppression of cell-mediated immune responses in late tumor-bearing mice were of the monocyte-macrophage series. Furthermore, the suppressive influence was not due merely to the increased number of macrophages in tumor-bearing animals. Experiments clearly showed that the splenic macrophages, even at low concentrations, demonstrated a suppressive function.