Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freund's adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freund's adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.
The B7 family of costimulatory molecules provides the second signal necessary for activation of T cells. In the absence of the second signal, responding T cells become anergic. Although predominantly expressed on professional APCs, recent evidence shows that the B7 molecules are also expressed on T cells. To study the functions of B7 molecules on T cells, we transfected murine B7.1 (CD80) and B7.2 (CD86) cDNAs into the EL4 T cell thymoma cell line and examined the transfectants for their ability to costimulate T cell proliferation in vitro and to induce antitumor immunity in vivo. Here we show that although EL4-B7.1 cells costimulate T cells and induce tumor regression, EL4-B7.2 transfectants failed to costimulate T cell proliferation or induce tumor regression. To understand the cellular basis for this difference, we examined the binding of EL4-B7.1 and EL4-B7.2 to CTLA4 and CD28. Whereas EL4-B7.1 cells bound both CTLA4-Ig and CD28-Ig, EL4-B7.2 transfectants preferentially bound CTLA4-Ig, but not CD28-Ig. Similar binding data were obtained with freshly isolated murine T cells, which have been shown to constitutively express B7.2. Our data suggest, therefore, that B7.2 expressed on T cells may not costimulate but instead inhibit the T cell response by preferential binding to CTLA4.
Acute myeloid leukaemia (AML) is a major haematopoietic malignancy characterized by the proliferation of a malignant clone of myeloid progenitor cells. A reciprocal translocation, t(8;21)(q22;q22), observed in the leukaemic cells of approximately 40% of patients with the M2 subtype of AML disrupts both the AML1 (CBFA2) gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (refs 3-5). A chimaeric protein is synthesized from one of the derivative chromosomes that contains the N terminus of the AML1 transcription factor, including its DNA-binding domain, fused to most of ETO, a protein of unknown function. We generated mice that mimic human t(8;21) with a "knock-in' strategy. Mice heterozygous for an AML1-ETO allele (AML1-ETO/+) die in midgestation from haemorrhaging in the central nervous system and exhibit a severe block in fetal liver haematopoiesis. This phenotype is very similar to that resulting from homozygous disruption of the AML1 (Cbfa2) or Cbfb genes, indicating that AML1-ETO blocks normal AML1 function. However, yolk sac cells from AML1-ETO/+ mice differentiated into macrophages in haematopoietic colony forming unit (CFU) assays, unlike Cbfa2-/- or Cbfb-/-cells, which form no colonies in vitro. This indicates that AML1-ETO may have other functions besides blocking wild-type AML1, a property that may be important in leukaemogenesis.
T cell activation requires the engagement of the TCR as well as a second, costimulatory signal. In this study, we demonstrate that MRC OX-2 (OX-2) mediates a previously unrecognized T cell costimulatory signal leading to enhanced T cell proliferation. One extensively studied costimulatory pathway, the B7/CD28 pathway, delivers its signal when CD28 is engaged by either of two ligands, B7-1 or B7-2, expressed on APC. Recent data have suggested that an additional ligand may exist in this pathway. This possibility prompted us to search previously cloned genes with both structural and expression characteristics similar to B7-1 and B7-2. Our search yielded OX-2, a rat lymphocyte activation marker, as a promising candidate gene. We now report that Chinese hamster ovary cell transfectants expressing the OX-2 protein can costimulate murine CD4+ T cells to proliferate in an Ag-independent fashion using anti-CD3, as well as an Ag-dependent fashion using peptide. In contrast to B7-1-mediated costimulation, OX-2 does not result in detectable levels of IL-2, IL-4, or IFN-gamma. In addition, OX-2 transfectants do not bind the soluble receptor reagents of the B7/CD28 pathway (CD28-Ig and CTLA4Ig). Furthermore, OX-2 costimulation is not inhibited by CTLA4Ig, as is B7-1-mediated costimulation, but is readily inhibited with an anti-OX-2 mAb. Thus, OX-2 is a T cell costimulatory ligand that acts through a non-B7/CD28 pathway, which leads to functionally distinct consequences, as reflected by the resulting cytokine profile.
For T cells to be optimally activated, recognition of Ag/MHC complexes by the TCR must be accompanied by a second, costimulatory signal that can be provided efficiently by the related costimulatory molecules CD80 (B7-1) and CD86 (B7-2). Recently, CD80 and CD86 have been implicated as differential determinants of Th1- vs Th2-type cytokine profiles. However, this remains a controversial issue since conflicting results have been obtained in different experimental models both in vivo and in vitro. To investigate the role of CD80 and CD86 in Th subset differentiation, we have examined the cytokine profiles induced in TCR transgenic T cells stimulated by peptide in association with splenic APCs obtained from knockout mice that selectively lack expression of either the CD80 or the CD86 molecule. Our data suggest that CD86, and to a lesser extent CD80, can make significant contributions to the production of both IL-4 and IFN-gamma. However, neither molecule plays an obligatory role in priming for the production of either effector cytokine. Furthermore, CD80 and CD86 contribute to the magnitude of T cell activation, but do not appear to selectively regulate Th1 vs Th2 differentiation.
Mice lacking CTLA-4 develop a fatal spontaneous lymphoproliferative disease with massive lymphocytic infiltrates and tissue destruction in many organs. CTLA-4-deficient (-/-) splenocytes and lymph node cells proliferate without added stimuli in vitro. We report here that CTLA4Ig treatment of CTLA-4 -/- mice prevents lymphoproliferation and fatal multiorgan tissue damage in vivo and proliferation of CTLA-4 -/- splenocytes and lymph node cells in vitro. Therefore, stimulation via CD28-B7 interactions appears necessary for CTLA-4 -/- T cell proliferation and the production of lymphoproliferative disease in vivo. When CTLA4Ig treatment is terminated, CTLA-4 -/- T cells become activated and lymphoproliferative disease develops. The lack of long term protective effects of CTLA4Ig treatment suggests that CTLA-4 is needed for the induction and or maintenance of tolerance.
T cell activation and tolerance are regulated by interactions between CD28 or CTLA-4 on T cells and B7 costimulatory molecules on APCs. We have generated transgenic mouse strains that constitutively express B7-1 (CD80) at high levels on B cells or T cells or express B7-2 (CD86) on T lymphocytes to examine the consequences of dysregulated B7 expression on T cell responses. The transgene-derived B7 molecules are functional, because B7-1 transgenic B cells are more efficient APCs than are wild-type B cells, and the activation of B7 transgenic T cells is less dependent on exogenous costimulation than that of wild-type T cells. In vivo, constitutive expression of B7 molecules leads to the elimination of immature B cells. The expression of B7 molecules on thymocytes results in the down-regulation of CD28 expression. However, B7 transgenic mice have normal numbers of mature lymphocytes and mount normal T cell responses following immunization with protein Ag. Neither anergy induction nor superantigen-mediated deletion of T cells is altered by the dysregulated expression of B7-1 or B7-2 on B or T lymphocytes in these transgenic strains. Therefore, functionally significant levels of B7 expressed constitutively on mature lymphocytes are not, by themselves, sufficient to abrogate T cell tolerance or induce autoimmune disease.
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.