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Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not become part of standard cancer therapy because of factors that include their inherent immunogenicity, which limits the duration of therapy. To address this issue, we evaluated in preclinical models the concomitant use of the immunosuppressive agent CTLA4Ig and BR96 sFv-PE40, a single-chain immunotoxin that binds to carcinoma cells expressing Le(y). Cotreatment with CTLA4Ig, an inhibitor of the CD28/CTLA4-CD80/CD86 costimulation pathway, blocked the production of Abs against BR96 sFv-PE40 in immunocompetent rodents and dogs. It also blocked hypersensitivity reactions in rats carrying colon carcinoma allografts during a second course of BR96 sFv-PE40 therapy, and the cotreatment with CTLA4Ig resulted in enhanced antitumor activity. Cotreatment with CTLA4Ig also prevented hypersensitivity reactions induced by repeat dosing of BR96 sFv-PE40 (q3dx5) in dogs. The production of anti-BR96-sFv-PE40 Abs was decreased in CTLA4Ig-cotreated rodents and dogs resulting in increased plasma levels of BR96 sFv-PE40 relative to non-CTLA4Ig-cotreated animals. These data show that cotreatment of immunotoxins with CTLA4Ig, by inhibiting the production of anti-immunotoxin Abs, can extend the duration of BR96 sFv-PE40 therapy to give greater exposure, reduced toxicities, and increased efficacy.

Murine lupus in NZB/NZW F1 (B/W) mice can be retarded by sustained administration of CTLA4Ig and by brief treatment early in life with mAb that block CD40/gp39 interactions. We sought to determine whether brief therapy with CTLA4Ig could provide sustained benefit in B/W mice and whether a synergistic effect could be derived by blockade of both the B7/CD28 and the CD40/gp39 pathways. We found that a short course of CTLA4Ig at the onset of disease produced only short-term benefit. However, when CTLA4Ig was combined with anti-gp39, there was long-lasting inhibition of autoantibody production and renal disease. Ten months after the 2-wk course of therapy, 70% of these mice were alive, compared with only 18% and 0% of those that received only anti-gp39 or CTLA4Ig, respectively. These findings demonstrate that brief simultaneous blockade of the B7/CD28 and CD40/gp39 costimulation pathways can produce benefit that lasts long after treatment has been discontinued.

We tested the effects of blocking CD28-B7 T cell costimulation by using CTLA4Ig in an established transplantation model in which LBNF1 cardiac allografts are rejected in an accelerated manner (

Blockade of CD28-mediated T cell costimulatory signals produces effective immunosuppression of a variety of T cell-dependent in vivo immune responses, including autoimmune disorders and transplant rejection. The soluble fusion protein CTLA4Ig, which competitively blocks CD28 ligands B7-1 and B7-2, can prevent allograft and xenograft rejection and in some circumstances induce transplantation tolerance. To determine the relative roles of B7-1 and B7-2 in graft rejection, we have performed islet and cardiac allografts with normal and B7-1(-/-) mice in conjunction with selective blocking reagents. We found that the absence of B7-1 on donor or recipient tissues leads to a slight prolongation of islet allograft survival, but has minimal or no effect on cardiac allograft survival. Allograft function is further prolonged in the islet model when both donor and recipient lack B7-1, although cardiac allograft survival is not prolonged. In the cardiac model, treatment with CTLA4Ig induces long term survival in B7-1(-/-) recipients regardless of donor status. In contrast, anti-B7-2 mAb leads to indefinite allograft survival only when the recipient and donor both lack B7-1, indicating that even in the absence of available B7-2, B7-1 molecules on the donor or recipient cells alone are sufficient to induce graft rejection. These data also indicate that B7-1 and B7-2 are the only CD28 ligands relevant to cardiac allograft rejection in mice.

We have previously demonstrated that the development of allergen-induced airway hyperresponsiveness in a murine model is CD4+ T cell dependent. In the present study, we examined the role of the B7/CD28-CTLA4 costimulatory T cell activation pathway in the pathogenesis of allergen-induced airway hyperresponsiveness in this murine model. Sensitized A/J mice develop significant increases in airway responsiveness, bronchoalveolar lavage eosinophils, serum IgE levels, and Th2-associated cytokine production following aspiration challenge with OVA. Administration of CTLA4-Ig either before Ag sensitization or before pulmonary Ag challenge abolished Ag-induced airway hyperresponsiveness and pulmonary eosinophilia. Examination of cytokine protein levels in the bronchoalveolar lavage showed a significant decrease in the level of the Th2 cytokine, IL-4, after CTLA4-Ig treatment either before sensitization or before challenge, with no significant change in the concentration of the Th1 cytokine, IFN-gamma. Further, the Ag-specific Ab isotypes IgG1 and IgE were significantly decreased in animals treated with CTLA4-Ig before challenge, while there was no significant change in the IgG2a Ab isotype. These data demonstrate that administration of CTLA4-Ig is effective in ablating allergen-induced airway dysfunction concomitant with a significant reduction in the Th2 response. We conclude that B7/CD28-CTLA-4 costimulation is required for the development of many of the immunologic and physiologic features of asthma, possibly by promoting a pathologic type 2-associated response.

We examined the role of the CD28/B7 interaction in regulation of NK cell activity. Cells transfected with B7 enhanced IL-12-induced production of IFN-gamma by IL-2-activated, CD28+ NK cells, but not by resting CD28- NK cells. The ability of B7 transfectants to enhance NK cell production of IFN-gamma was dependent on the intracellular adhesion molecule-1/LFA-1 interaction and could be inhibited by TGF-beta, but not IL-10. Since IL-12-induced production of IFN-gamma by NK cells is associated with resistance to certain infections, we examined whether the CD28/B7 interaction regulated NK cell responses during infection. Infection of SCID mice with Toxoplasma gondii resulted in the appearance of a population of CD28+ NK cells, NK cell production of IFN-gamma, and increased NK cell cytolytic activity. Administration of CTLA4-Ig to SCID mice infected with T. gondii inhibited these latter two effects and resulted in a significant increase in parasite burden. The stimulus for CD28 expression by NK cells in SCID mice infected with T. gondii appeared to be independent of IL-2. However, mRNA for IL-15, a cytokine with properties similar to those of IL-2, was detected in tissues of SCID mice infected with T. gondii. In vitro experiments demonstrated that IL-15 could stimulate resting NK cells to express functionally active CD28 as well as enhance the production of IFN-gamma by SCID splenocytes stimulated with T. gondii. Together our data demonstrate that the interaction of CD28+ NK cells with B7 regulates NK cell production of IFN-gamma associated with resistance to infection and that IL-15 may be involved in these events.

Blocking the CD28-B7 T lymphocyte costimulatory pathway with the recombinant protein CTLA4Ig induces long term allograft survival in rodents. It has been suggested that this results from selective activation of the Th2 immune pathway. To test this hypothesis, we compared vascularized cardiac allograft survival in wild-type (IL-4 +/+) and homozygous IL-4 gene-knockout (IL-4 -/-) mice. We report in this study that long term survival (>100 days) of fully allogeneic grafts can be induced readily in IL-4 -/- recipients treated with a short course of CTLA4Ig. We also demonstrate that IL-4 -/- mice are deficient in Th2-type cytokine expression following in vitro or in vivo allostimulation. These results suggest that IL-4 production and subsequent generation of a Th2-type immune response are not obligatory for CTLA4Ig-induced long term acceptance of vascularized allografts.

CD22 is a key accessory molecule for Ag receptor signaling in B cells that becomes tyrosine phosphorylated in the signaling process. CD22 associates with sIg and strongly amplifies sIg-induced signals. During B cell development, CD22 is initially expressed intracellularly, with surface expression appearing with IgD expression. We used confocal laser-scanning microscopy and flow cytometry to analyze CD22 translocation responses to signaling events. Cross-linking surface IgM (sIgM) induced rapid movement of CD22 to the cell surface in both Ramos and Daudi B cells, with a 50 to 100% increase in surface expression observed within 5 min of stimulation. The increase in CD22 surface expression was specific in that CD19 expression was not affected. Both cell surface and intracellular CD22 were directed toward the site of sIgM stimulation. Treatment with the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV) also increased CD22 surface expression. The tyrosine kinase inhibitor tyrphostin A10 inhibited CD22 movement at concentrations that inhibited tyrosine phosphorylation of CD22 and other cellular proteins. In contrast to the B cell lines, mature peripheral blood B cells contained very little intracellular CD22 and showed no significant increase in surface expression following sIgM stimulation. The rapid directed movement of intracellular CD22 provides a new mechanism to dynamically regulate Ag receptor signaling, as well as CD22-mediated adhesion.

B7 molecules (CD80 (B7-1) and CD86 (B7-2/B70)) on APCs provide costimulatory signals for T cell proliferation. We previously described the presence of an alternatively spliced form of murine CD80 (previously termed MB7-2 and renamed as B7-1a) that completely lacks the second Ig-like domain coded by exon 3 in activated murine B cells. in this study, we first examined whether B7-1a mRNA can be detected in vivo by RNase protection assay. The expression of B7-1a mRNA was only detected in lymphoid organs although the level of expression was lesser than that of CD80 mRNA. However, we demonstrated that the expression of B7-1a mRNA like CD80 mRNA was considerably augmented in spleen cells treated with either LPS in vitro or OVA/CFA conjugate in vivo. We next determined the functional activity of B7-1a using Chinese hamster ovary (CHO) cells transfected by B7 genes. When resting T cells were cocultured with CHO cells expressing B7-1a molecules in the presence of PMA/ionomycin, T cell proliferation was not detected, while CHO cells either expressing CD80 or CD86 could promote the proliferation of resting T cells. in contrast to resting T cells, CHO cells expressing B7-1a could support the proliferation of activated T cells. Thus, costimulatory activity of B7-1a molecules was dependent upon the activation stage of T cells. Therefore the IgV-like region of CD80 contains a critical region for functional interaction with its ligands and can transduce a costimulatory signal for T cell proliferation.

In the BXSB autoimmune disease-prone mouse strain, male mice develop severe lupus-like symptoms and die early in life (4-6 mo), whereas females do not. We have previously demonstrated that profound phenotypic and functional changes occur with age in CD4+ cells from BXSB males. CD4+ cells from males (4 mo old) were predominantly CD44high, CD45RBlow, and MEL-14low (activated/memory phenotypes), while the reciprocal phenotypes characteristic of naive cells were prevalent in age-matched females and young adult males (2 mo old). CD4+ cells from older males proliferated less and produced less IL-2 and IFN-gamma than cells from either females or young males in response to immobilized anti-CD3 mAb. We tested the effect of CTLA4Ig treatment on the progression of disease in BXSB males. CD4+ cells from CTLA4Ig-treated mice at 4 mo of age were predominantly CD44low, CD45RBhigh, and MEL-14high phenotypes that were identical with those observed in CD4+ cells from young (3-mo-old) females. In contrast, control male mice treated with IgG2a accumulated the CD4+ memory phenotype. CD4+ cells from 4-mo-old male CTLA4Ig-treated mice proliferated and produced IL-2 at levels similar to those of cells from females in response to immobilized anti-CD3 mAb. Furthermore, in contrast to IgG2a-treated mice, female and CTLA4Ig-treated male mice at 4 mo of age produced no anti-chromatin Abs. Three of four male mice injected with CTLA4Ig until 6 mo of age appeared healthy at 8 mo of age, whereas all five of IgG2a-treated control males died by 6 mo of age. These 8-mo-old CTLA4Ig-treated males showed variable resistance to autoimmunity as well as function and phenotype marker expression, and a less striking glomerulonephritis than 4-mo-old untreated males. The results of this study demonstrate that the rampant T cell activation and T cell dysfunction that occur in male BXSB mice by 4 mo of age are abrogated by blocking the CTLA4-dependent costimulatory signal(s). They also show that treatment with CTLA4Ig can suppress the pathogenesis of disease and increase longevity.

We have examined the ability of CTLA4Ig to induce long-lasting Ag-specific tolerance to a T-dependent Ag. Treatment of mice with murine CTLA4Ig following immunization with SRBC induced immune unresponsiveness to SRBC that was only reversible upon repeated Ag exposure. This unresponsiveness was Ag specific, lasted > 90 days, was observed in thymectomized mice, and was maintained even during challenge with another strong T-dependent Ag. A third challenge with SRBC, however, led to an Ab response. Splenic T cells and B cells from unresponsive mice were functional upon transfer to irradiated hosts, indicating that they had been neither depleted nor rendered permanently "tolerant" by CTLA4Ig. The mechanism of immunosuppression by CTLA4Ig was investigated by measuring cytokine transcripts in spleens of immunized mice. CTLA4Ig treatment following primary immunization blocked the induction of IL-2 transcripts in splenic T cells and IL-4 transcripts in both T cells and non-B, non-T cells. Splenocytes from CTLA4Ig-treated, SRBC-unresponsive mice showed altered induction of IL-2 and IL-4 transcripts, but T cells nonetheless became primed for IL-4 mRNA synthesis despite the lack of a measurable Ab response. Anti-IL-4 mAb and human CTLA4Ig were synergistic in their ability to induce unresponsiveness, indicating that incomplete suppression of IL-4 production by CTLA4Ig limited its effectiveness.

The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A-sensitive pathway.

The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus.

B7-2 is a recently discovered, second ligand for the CTLA-4/CD28, T cell signaling system. Using the GL-1 rat monoclonal antibody (mAb), we monitored expression of B7-2 on mouse leukocytes with an emphasis on dendritic cells. By cytofluorography, little or no B7-2 was detected on most cell types isolated from spleen, thymus, peritoneal cavity, skin, marrow, and blood. However, expression of B7-2 could be upregulated in culture. In the case of epidermal and spleen dendritic cells, which become highly immunostimulatory for T cells during a short period of culture, the upregulation of B7-2 was dramatic and did not require added stimuli. Lipopolysaccharide did not upregulate B7-2 levels on dendritic cells, in contrast to macrophages and B cells. By indirect immunolabeling, the level of staining with GL-1 mAb exceeded that seen with rat mAbs to several other surface molecules including intercellular adhesion molecule 1, B7-1, CD44, and CD45, as well as new hamster mAbs to CD40, CD48, and B7-1/CD80. Of these accessory molecules, B7-2 was a major species that increased in culture, implying a key role for B7-2 in the functional maturation of dendritic cells. B7-2 was the main (> 90%) CTLA-4 ligand on mouse dendritic cells. When we applied GL-1 to tissue sections of a dozen different organs, clear-cut staining with B7-2 antigen was found in many. B7-2 staining was noted on liver Kupffer cells, interstitial cells of heart and lung, and profiles in the submucosa of the esophagus. B7-2 staining was minimal in the kidney and in the nonlymphoid regions of the gut, and was not observed at all in the brain. In the tongue, only rare dendritic cells in the oral epithelium were B7-2+, but reactive cells were scattered about the interstitial spaces of the muscle. In all lymphoid tissues, Gl-1 strongly stained certain distinct regions that are occupied by dendritic cells and by macrophages. For dendritic cells, these include the thymic medulla, splenic periarterial sheaths, and lymph node deep cortex; for macrophages, the B7-2-rich regions included the splenic marginal zone and lymph node subcapsular cortex. Splenic B7-2+ cells were accessible to labeling with GL-1 mAb given intravenously. Dendritic cell stimulation of T cells (DNA synthesis) during the mixed leukocyte reaction was significantly (35-65%) blocked by GL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Infection of inbred strains of mice with Leishmania major is a well-characterized model for analysis of the development of effector CD4+ subsets of the Th1 and Th2 types in vivo. We co-administered a fusion protein, CTLA4Ig, that blocks the CD28-B7 costimulatory pathway important for optimal T cell activation, to assess the relative role for this pathway during maturation of Th1 and Th2 cells in vivo. Surprisingly, CTLA4Ig administered within the first week of infection completely abrogated progressive disease in susceptible BALB/c mice while having no effect on the protective immune response developed by resistant C57BL/6 mice. The protective effect in BALB/c mice was increasingly lost if administration of CTLA4Ig was delayed longer than 1 wk after infection. As in other protective interventions used in this model, control of infection was associated with down-regulation of IL-4 mRNA transcripts in lymph node cells recovered 5 wk after infection together with abrogation of IgE production and enhanced parasite-specific IgG2a relative to IgG1. Although a single dose of CTLA4Ig was protective, sustained delivery abolished the capacity of BALB/c mice to contain infection, suggesting that costimulation through this pathway is required at later stages of the immune response. Taken together, the data demonstrate that the priming of Th2 cells is more dependent upon the CD28-B7 pathway than the priming of Th1 cells, and suggest that the development of Th subsets in vivo may be influenced by limiting CD28-B7 costimulation.

Microbial superantigens (SAgs), by virtue of their binding to TCR V beta elements on T cells and to class II MHC molecules on accessory cells (AC), trigger T cell activation. Although anti-CD3 mAb (which also trigger T cell activation via surface CD3/TCR) can readily induce T cell-dependent B cell differentiation in unmanipulated PBMC cultures, induction of Ig production in SAg-stimulated cultures has usually required special manipulation of the T cells, such as irradiating them or treating them with mitomycin C. We now demonstrate that eight different staphylococcal SAgs, typically at concentrations 10- to 100-fold lower than those required for proliferation, can each trigger unmanipulated peripheral blood and tonsil T cells to drive polyclonal B cell differentiation. Such SAg-induced T cell-dependent generation of Ig-secreting cells (IgSC) requires T cells and B cells only and occurs in the absence of monocytes as long as there are adequate numbers of B cells to serve as (DR+) AC. Physical contact among T cells, responder B cells, and AC (when different from the responder B cells) is required. The fusion protein CTLA4Ig inhibits SAg-induced IgSC generation in a dose-dependent fashion, whereas a control fusion protein has no such effect. In contrast, CTLA4Ig has, at best, only modest effects on SAg-induced T cell proliferation, indicating that CD28 (CTLA4)/B7 (B7-like) interactions play a more prominent role in SAg-induced IgSC generation than in SAg-induced T cell proliferation. These results establish SAg-induced T cell-dependent B cell differentiation as a useful model for T cell/B cell interactions, inasmuch as no other cell types are necessary for successful B cell differentiation; these results also demonstrate the importance of CD28 (CTLA4)/B7 (B7-like)-dependent mechanisms in this process.

Activation of Ag-specific T cells often requires costimulatory signals in addition to the primary signal mediated through the TCR. The CD28-B7 interaction provides one important costimulatory signal. Previous studies have shown that a soluble CD28 homologue, CTLA4lg, binds B7 with high affinity and can inhibit CD28-B7-mediated costimulation in vitro and in vivo. In this study we examined the ability of soluble human CTLA4lg to inhibit autoantibody production in vivo. For this purpose we used a novel transgenic (Tg) model of autoantibody production. Hepatitis B eAg-expressing Tg mice can be induced to produce autoantibody to the circulating autoantigen (HBeAg) by the injection of a T cell recognition site that fails to elicit T cell tolerance in these mice. Autoimmunity in this model is quantitative because serum autoantibody and autoantigen concentration are inversely correlated and easily measurable by ELISA. In this system a single regimen of CTLA4lg treatment significantly suppressed primary autoantibody production and variably led to long term unresponsiveness. Furthermore, in vivo treatment with CTLA4lg inhibited both T cell activation and T cell-B cell interactions.

Transplant rejection requires not only T cell receptor/CD3 complex activation by foreign MHC, but also additional costimulatory signals, as T cell receptor activation alone is insufficient for induction of the immune response. The CD28 receptor on helper T cells, interacting with its ligand B7 on activated B cells or macrophages, provides this costimulus to support T cell activity. CTLA4Ig (a soluble CD28 receptor analog), binds B7 and inhibits CD28 activation. As cyclosporine (CsA) has many side effects and CTLA4Ig alone has a significant benefit upon cardiac allograft survival, we theorized that allograft survival could be improved by using CTLA4Ig with lowered dose CsA. In vitro, high-dose CTLA4Ig inhibited the mixed lymphocyte culture reaction (MLR) between MHC-incompatible rat strains. Furthermore, there was synergistic suppression of MLR by low-dose CTLA4Ig combined with low-dose CsA. In vivo studies used a cervical heterotopic transplant model. Control recipients received no immunotherapy. Experimental recipients received low-dose CsA (1.5 mg/kg/day im) × 14 days after transplant or CTLA4Ig (10, 50, or 150 μg IP × 7 days). Combination animals received both CTLA4Ig and CsA. These studies showed that low doses of CsA and CTLA4Ig were additive in vivo, although no additional benefit was seen when CsA was combined with high-dose CTLA4Ig. These data suggest that the combination of low-dose CsA plus CTLA4Ig may prove useful in clinical transplantation to maximize immunosuppression and minimize side effects.

We tested whether the in vivo infusion of recombinant, soluble CTLA4 fused with Ig heavy chains, as a surrogate ligand used to block CD28/CTLA4 T-cell costimulation, could prevent efficient T-cell activation and thereby reduce graft-versus-host disease (GVHD). Lethally irradiated B10.BR recipients of major histocompatibility complex disparate C57BL/6 donor grafts received intraperitoneal injections of human CTLA4-Ig (hCTLA4-Ig) or murine CTLA4-Ig (mCTLA4-Ig) in various doses and schedules beginning on day -1 or day 0 of bone marrow transplantation (BMT). In all five experiments, recipients of CTLA4-Ig had a significantly higher actuarial survival rate compared to mice injected with an irrelevant antibody control (L6) or saline alone. Survival rates in recipients of hL6 or PBS were 0% at 29 to 45 days post-BMT. In recipients of CTLA4-Ig, survival rates were as high as 63% mice surviving 3 months post-BMT. However, protection was somewhat variable and recipients of CTLA4-Ig were not GVHD-free by body weight, clinical appearance, and histopathologic examination. There were no significant differences in the survival rates in comparing injection dose, injection duration, or species of CTLA4-Ig (hCTLA4-Ig v mCTLA4- Ig). Splenic and peripheral blood flow cytometry studies of long-term hCTLA4-Ig-injected survivors showed a significant peripheral B-cell and CD4+ T-cell lymphopenia, consistent with GVHD. A kinetic study of splenic reconstitution was performed in mice that received hCTLA4-Ig and showed that mature splenic localized CD8+ T-cell repopulation was not significantly different in recipients of hCTLA4-Ig compared with hL6, despite the significant increase in actuarial survival rate in that experiment. These data suggest that the beneficial effect of hCTLA4-Ig on survival is not mediated by interfering with mature donor- derived T-cell repopulation post-BMT. Neither hCTLA4-Ig nor mCTLA4-Ig interfered with hematopoietic recovery post-BMT. We conclude that CTLA4- Ig (most likely in combination with other agents) may represent an important new modality for GVHD prevention.

Dendritic cells (DC) play a critical role in the initiation of T cell-mediated immune responses, and express costimulatory molecules that are required for optimal activation of unprimed T cells. Studies on the regulation of the costimulatory molecules on DC have produced evidence from several systems that GM-CSF can up-regulate expression of CTLA4 counter receptor (CTLA4-CR) (but not intercellular adhesion molecule 1 (ICAM-1) and heat stable Ag (HsAg)) on DC. This is demonstrated on splenic DC, Langerhans cells, kidney DC in culture, and in a skin-explant culture system, in which the increased expression of CTLA4-CR on Langerhans cells (LC) occurs concomitantly with their migration out of skin. Interestingly, despite the ability of both GM-CSF and IFN-gamma to increase CTLA4-CR and maintain similar levels of ICAM-1, HsAg, and MHC molecule expression, the functional consequences of these cytokines on splenic DC are distinctly different. GM-CSF enhances the ability of DC to stimulate both T cell proliferation and cytokine release, whereas IFN-gamma causes no increase in immunostimulatory function. Further analysis of the CTLA4-CR on these cell populations by using the GL-1 and IG10 mAbs has shown that GM-CSF-cultured DC express high levels of both B7-1 and B7-2, whereas IFN-gamma-cultured DC express increased levels of only B7-2. These results suggest that optimal stimulation of unprimed T cells to proliferate and release cytokines may require participation of both of these CTLA4 counter receptors, and confirm the importance of GM-CSF for the maturation of DC into potent stimulators of T cell activation.

Costimulation by the CD28 ligand B7/BB1 plays an important role during T cell proliferation primarily by augmenting synthesis of IL-2 and other cytokines. Resting CD4+ T cells express CD28 but not CTLA-4 on their surface. Costimulation of T cells with ICAM-1 or VCAM-1 induced CTLA-4 expression and up-regulated CD28 expression. CD28 and CTLA-4 were independently distributed on the surface of activated T lymphoblasts. When co-immobilized with anti-TCR mAb both anti-CD28 and anti-CTLA-4 mAb augmented T cell proliferation. Although anti-CD28-mediated augmentation of T cell proliferation was stronger than that seen with anti-CTLA-4 mAb, together these two mAb caused supraadditive augmentation of T cell proliferation. The augmentation of the effects of anti-CD28 mAb by anti-CTLA-4 mAb was greater at low occupancy of CD28 by anti-CD28 mAb. Costimulation of CD28+ CTLA-4+ T cells with anti-CTLA-4 caused three- to fivefold increase in IL-2 production, whereas similar treatment with anti-CD28 caused > 40-fold increase. The costimulatory effect of B7 on primed T cells was partially inhibited by Fab anti-CD28 mAb. Anti-CTLA-4 mAb alone did not inhibit B7-induced response but caused modest increase in the inhibitory effect of anti-CD28 Fab. On integrin-mediated costimulation, Ag-specific CD4+ T cell lines also up-regulated their CTLA-4 expression, and proliferation of these cells was augmented by anti-CTLA-4 mAb. Unlike that of CD28, ligation of CTLA-4 alone failed to mobilize intracellular [Ca2+]. However, coligation of CTLA-4 and TCR induced stronger [Ca2+] response in Ag-specific T cell lines than that seen with TCR alone. These results suggest that integrin-costimulated T cells express CTLA-4 and can be costimulated via CTLA-4. Optimal development of various immune functions may involve combined costimulation via both CD28 and CTLA-4.

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.

A fusion protein consisting of the extracellular domain of CTLA4 and an Ig C gamma 1 chain (CTLA4-Ig) was used to examine the distribution of the ligands for CTLA4 within the murine thymus and to characterize the nature of these ligands. Two-color immunofluorescence of thymus tissue revealed binding of the fusion protein to medullary thymic epithelial cells and dendritic cells within the corticomedullary and medullary areas of the thymus. Medullary cells binding the fusion protein also expressed MHC class II products and ICAM-1. Thymus tissue sections treated with cross-linking fixatives, such as glutaraldehyde, paraformaldehyde, or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide no longer bound the CTLA4 fusion protein, indicating that binding was very sensitive to the tertiary structure of the tissue ligand. The ability of thymic tissue to bind the fusion protein was developmentally regulated. At day 14 of gestation, only scattered single cells were labeled. Clusters of labeled cells, which were detected by day 16 of gestation, increased in frequency with advancing gestational age. Consistent with the in situ labeling studies, CTLA4-Ig also labeled several thymic epithelial cell lines previously shown to have a medullary phenotype. Polymerase chain reaction analysis of mRNA extracted from these cells indicated they contained mRNA for B7, a known counter receptor for CTLA4 and CD28. Immunoprecipitation of 125I-labeled thymic epithelial cells with the CTLA4-Ig detected a M(r) 65,000 to 70,000 species under reducing conditions, consistent with previous studies of B7. These data suggest that the ligand for CTLA4 expressed by thymic epithelial cells in vitro is B7 and that the expression of this ligand in situ is largely restricted to the medullary compartment and is associated with epithelial cells and dendritic cells.

Integrin ligands intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) can efficiently costimulate proliferation of resting T cells but not that of Ag-specific T cells. In contrast, CD28 ligand B7 and CD2 ligand leukocyte function-associated Ag (LFA-3) can support IL-2 synthesis and proliferation of Ag-specific T cells more efficiently than that of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. In this study, using mAb and soluble IgC gamma 1 chimeras of these adhesion molecules, we demonstrate that coligation of the TCR and CD11a/CD18 (LFA-1/beta 2 integrin) or CD29/CD49d (very late activation Ag-4/beta 1 integrin) using anti-TCR mAb and either ICAM-1 or VCAM-1 induces activation-dependent death of DRw6-specific CD4+ T cells. Similar coligation of the TCR with CD2 or CD28 using either mAb or ligands LFA-3 or B7 not only lacked the ability to induce death but also failed to reverse or inhibit integrin-facilitated death of DRw6-specific T cells. Each of these ligands augmented anti-TCR mAb-induced transcription of IL-2 and IL-4 genes. Exogenous addition of IL-2 and IL-4 did not reverse the integrin-supported T cell death. The death-promoting costimulatory effects of ICAM-1 and VCAM-1 were observed with Ag-specific chronically stimulated T cells but not with either resting T cells or those activated in short-term cultures. Treatment of T cells with cyclosporin A or a protein tyrosine kinase inhibitor herbimycin A inhibited ICAM-1 or VCAM-1-promoted activation-induced T cell death. The Ag-specific T cells that survived death-promoting effects of ICAM-1 or VCAM-1 proliferated efficiently upon restimulation with these ligands. Exposure of DRw6-specific T cells to DRw6+ B7+ ICAM-1+ LFA-3+ VCAM-1+ APC but not DR3+ B7+ ICAM-1+ LFA-3+ VCAM-1+ APC induced death of these T cells. This effect was blocked by pretreatment of T cells with mAb directed at CD18 or CD29 but not with those against CD2 or CD28. Taken together, these results suggest that TCR-directed engagement of integrins by their ligands ICAM-1 or VCAM-1 induces activation-dependent death of some perhaps more differentiated Ag-specific T cells and this may be an important homeostatic mechanism by which functional expression of Ag-specific T cells is regulated during an ongoing immune response.

We have previously demonstrated that the inhibitory effects of IL-10 on ConA-induced T cell proliferation or IL-2 production by resting murine T cells were only observed when macrophages, but not when activated B cells, dendritic cells, or L cells, were used as accessory cells. To further elucidate the mechanism of action of IL-10 on the inhibition of macrophage costimulatory activity, we have used a system in which macrophages can develop into effective costimulator cells and the effect of IL-10 on this process can be studied in the absence of T cells. After fixation, resting macrophages have no costimulatory activity for soluble anti-CD3-induced T cell proliferation nor do they express the activation Ag B7/BB1. In contrast, macrophages activated by culture alone, or by culture with IFN gamma or LPS for 24 h, and then fixed, were effective accessory cells, expressed B7, and their costimulatory activity correlated with their level of cell surface B7 expression. Addition of IL-10 during the process of macrophage activation resulted in both a marked reduction in costimulatory activity and in B7 expression. IL-4 and transforming growth factor-beta that suppress many macrophage functions did not inhibit the induction of B7 expression. The inhibitory effect of IL-10 on the up-regulation of B7 was selective because the up-regulation of intercellular adhesion molecule-1 and MHC class II Ag was not affected. Direct evidence that the lack of B7 is the relevant limiting defect for IL-10-treated macrophage accessory cell function was obtained from studies in which the costimulatory capacity of IL-10-treated macrophages could be completely restored by the addition of B7 transfected, but not nontransfected, L cells to the assays.

Costimulatory molecules on the APC regulate T cell growth by providing signals that regulate responses to TCR occupancy. One such molecule is B7/BB-1, which triggers a T cell activation pathway by binding the CD28 and/or CTLA-4 cell-surface molecules. Expression and signaling activity of CD28 have been shown to increase after T cell activation by various polyclonal activators. Here we show that CD28 expression and signaling activity in activated T cells decrease after ligand binding to CD28. Stimulation of CD28 on PHA- or PMA-activated T cells by cross-linked mAb 9.3 or by co-culture with B7+ Chinese hamster ovary (CHO) cells caused a marked reduction of CD28 mRNA levels within 4 h. The decrease in CD28 mRNA was transient, and by 24 h of CD28 stimulation, CD28 mRNA was found at approximately initial levels. In contrast, CTLA-4 mRNA levels were usually up-regulated by CD28 triggering. Cell-surface expression of CD28, but not CD2 or CD3, decreased by 12 to 24 h after addition of B7+ CHO cells, but returned to initial levels or higher by 48 h. The ability of CD28 cross-linking on PMA-activated CD4+ cells to trigger calcium mobilization was also reduced by treatment with B7+ CHO cells, and remained reduced even after cell-surface expression of CD28 returned to normal levels. Thus, engagement of the CD28 receptor by its natural ligand B7/BB-1 leads to a transient down-regulation of CD28 synthesis and a prolonged unresponsiveness to CD28 signaling. This represents a novel mechanism for regulation of costimulatory signals delivered by interactions of CD28 with the B7/BB-1 counter receptor.

The B7 molecule is expressed by APC that can costimulate T cells by binding the T cell surface receptors CD28 and CTLA-4. The human epidermal Langerhans cell (LC) is one of the most potent APC, yet B7 expression by this cell type has not previously been assessed. We used a CTLA4-Ig fusion protein that binds B7 with high avidity to probe cell surface expression of B7 by cultured and noncultured LC. LC cultured for 1 or more days were specifically stained with biotinylated CTLA4-Ig and fluorescent streptavidin. In contrast, binding of CTLA4-Ig to freshly isolated LC was not detected. The cell surface distributions of B7 and of HLA-DR on cultured LC differed, as CTLA4-Ig binding was localized to discrete foci, whereas anti-DR mAb uniformly stained the LC plasma membrane. Analyses of epidermal cell (EC) mRNA indicated that the B7 gene is expressed by these cells. Thus, B7 gene probes specifically hybridized to polymerase chain reaction-amplified B7 mRNA isolated from cultured and noncultured EC. As LC are the only normal epidermal cell type that induces proliferation of allogeneic T cells, the role of B7 in this LC function was studied by coculturing highly purified resting CD4+ T cells and allogeneic EC in the presence of CTLA4-Ig, anti-CD54 (RR/1, anti-intercellular adhesion molecule-1) mAb, or both. CTLA4-Ig and RR/1 each inhibited CD4+ T cell responses to freshly isolated allogeneic EC, and cooperative inhibition of more than 90% was observed in cultures treated with both CTLA4-Ig and RR/1 at 5 micrograms/ml. CTLA4-Ig inhibited stimulation by either fresh EC or cultured EC, suggesting that the increased potency of cultured LC vs noncultured LC may reflect the time needed for noncultured LC to express cell surface B7 in vitro. These studies indicate that B7 is expressed on the cell surface of cultured LC, and that LC B7 costimulates the proliferation of resting allogeneic CD4+ T cells.