Analysis of the B7 costimulatory pathway in allograft rejection.

BACKGROUND: Blockade of the B7/CD28 costimulation pathway with the fusion protein, CTLA4-Ig, has been shown to prolong allograft survival in numerous rodent models, suggesting that this pathway is functionally important in the allograft rejection response. This pathway is complex and consists of at least the B7-1, B7-1a, B7-1cyt II, and B7-2 molecules on the antigen-presenting cell and CD28 and CTLA4 molecules on the T cell. METHODS: The intragraft transcript expression of the B7 molecules and their counterreceptors was defined using reverse transcriptase-polymerase chain reaction in the vascularized mouse cardiac allograft model. In addition, the functional significance of these molecules was investigated both in vitro in the mixed leukocyte response (MLR) and in vivo in the vascularized mouse cardiac allograft model. RESULTS: Intragraft expression of B7-1, B7-1a, B7-1cyt II, B7-2, CD28, and CTLA4 transcripts is up-regulated in allografts when compared with both normal untransplanted hearts and syngeneic transplants at between 5 and 12 days after transplant. Both anti-B7-1 and anti-B7-2 monoclonal antibodies alone inhibited T-cell proliferation in the MLR, however, equivalent maximal inhibition was obtained by a combination of these agents or by CTLA4-Ig. Likewise, in the mouse cardiac allograft model, both anti-B7-1 and anti-B7-2 modestly prolonged graft survival. However, an increased survival was obtained with either a combination of anti-B7-1 and anti-B7-2 or CTLA4-Ig. Blockade of the B7/CD28 pathway in the MLR using T cells from CD28 knockout mice had no effect on the proliferative response. Likewise, blockade of the B7/CD28 pathway did not effect the rate of rejection of cardiac allografts by CD28 knockout recipients. CONCLUSIONS: These data suggest that both B7-1 and B7-2 have an important role in allograft rejection in the mouse vascularized cardiac allograft model.

Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

IL-12-deficient mice are defective but not devoid of type 1 cytokine responses.

Interleukin-12 (IL-12) has been described as a pivotal molecule in the immune response based in part on its ability to influence the differentiation of T helper (Th) cells into a type 1 (Th1) phenotype. This event is crucial in that appropriate differentiation of naive T cells can determine susceptibility or resistance to given pathogens by influencing the balance between cellular and humoral immunity. In order to further delineate the role of IL-12 in the immune response, we generated mice deficient for this cytokine. IL-12 knockout mice were viable, fully fertile, and displayed no obvious developmental abnormalities. Upon immunological analysis, these mice demonstrated an impaired ability to effect a Th1 response as well as an impaired ability to produce interferon-gamma in response to endotoxin in vivo. These data establish an essential role for IL-12 in the generation of optimal Th1 responses in vivo, but weak responses can occur independently of IL-12.

IL-12-deficient mice are defective in IFN gamma production and type 1 cytokine responses.

IL-12 is a cytokine that can exert regulatory effects on T and NK cells and promote Th1 responses. To delineate further the physiologic role of IL-12 in immunity, mice deficient for this cytokine were generated. IL-12-deficient mice were impaired but not completely lacking in the ability to produce IFN gamma following endotoxin administration and to mount a Th1 response in vivo, as measured by antigen-induced IFN gamma secretion by immune lymph node cells in vitro. In contrast, secretion of IL-4 was enhanced, while proliferation and secretion of IL-2 and IL-10 were normal following antigen stimulation. DTH responses were significantly reduced in IL-12-deficient mice, but no defect in allogeneic CTL responses was observed. These results indicate that IL-12 plays an essential role in regulating IFN gamma production and in facilitating normal DTH responses. However, other phenomena associated with Th1 responses and cell-mediated immunity, i.e., IL-2 secretion and CTL generation, were not compromised in the absence of IL-12.

Monoclonal antibody 2D10 recognizes a novel T cell costimulatory molecule on activated murine B lymphocytes.

We have developed a panel of rat mAbs against dibutyryl cAMP-activated 5C2 cells. In this panel, one mAb, 1G10, recognized murine B7. Another mAb designated 2D10 did not bind to murine B7 but could recognize a surface molecule expressed only on dibutyryl cAMP-activated 5C2 mouse B lymphoma cells or on LPS-stimulated splenic B cells. This new molecule is referred to as early T cell costimulatory molecule-1 (ETC-1). From both activated 5C2 cells and splenic B cells, mAb 2D10 immunoprecipitated a 59- to 60-kDa protein, which was different from the 47- to 55-kDa murine B7 protein precipitated from the same cell populations. FACS analysis showed that, in contrast to B7, the expression of ETC-1 on 5C2 cells was induced by lower concentrations of dibutyryl cAMP and displayed a faster kinetics. LPS-stimulated splenic B cells expressed relatively low levels of B7 and much higher levels of ETC-1. Importantly, in an Ag presentation assay using activated 5C2 cells as APC, the secretion of IL-2 by C8A3 T hybrids was partially inhibited by mAb 2D10 alone and completely blocked by combination use of mAbs 2D10 and 1G10 in a dose-dependent and synergistic fashion. In a one-way primary MLR, mAb 2D10 alone at 0.1 to 1 microgram/ml inhibited T cell proliferation by 19 to 56%. However, an additive blocking effect (up to 76%) was observed when two mAbs were added in combination. Thus, our data have demonstrated that a novel T cell costimulatory molecule is present on activated murine B cells, which, in cooperation with B7, may play a critical role in optimal T cell activation.

Expression and functional analysis of murine B7 delineated by a novel monoclonal antibody.

The B cell surface molecule designated B7 has been shown to be expressed by activated human B cells and monocytes and to be a ligand for the CD28 and CTLA-4 molecules on T cells. B7/CD28 interactions can provide a second signal to T cells (in addition to occupancy of the T cell antigen receptor) that is needed for T cell activation. COS cells transfected with the mouse homologue of B7 have been demonstrated to provide a stimulatory signal to murine and human T cells. In this report we describe a rat anti-mouse B7 mAb designated 1G10. Scatchard and/or FACS analyses utilizing 1G10 demonstrated that B7 was not expressed on resting splenic T cells or B cells, but could be induced at high levels on B cells cocultured with a syngeneic I-Ak-restricted autoreactive T cell hybridoma. Furthermore, activation of B cells with dibutyryl-cAMP (db-cAMP), a second messenger for class II MHC signaling, or with LPS induced the expression of B7 and the two agents showed additive effects. In contrast to B cells, freshly isolated mouse peritoneal macrophages constitutively expressed B7. Antibody-blocking experiments indicated that anti-B7 antibody partially inhibited T cell proliferative responses to primary antigenic stimulation but had no effect on the responses of previously activated T cells to antigenic restimulation.

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo.

IL-12 is a heterodimeric cytokine that has been shown to enhance natural killer (NK) and cytotoxic T lymphocyte (CTL) responses, and to induce IFN-gamma production in vitro. In this study, we have examined the effects in vivo of administering purified murine rIL-12 to normal mice. Daily injections of rIL-12 i.p. (1 ng to 10 micrograms/day) caused dose-dependent enhancement of NK cell lytic activity in the spleens and livers of treated mice. Histologic examination of the livers of IL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studies indicated that the livers of IL-12-treated mice contained increased numbers of NK cells, CD8+ T cells, and monocytes. Liver and splenic lymphoid cells from IL-12-treated mice, unlike liver and splenic lymphoid cells from control mice, spontaneously secreted IFN-gamma in vitro, suggesting that they had been induced by IL-12 to produce IFN-gamma in vivo. Consistent with this, IFN-gamma could be detected in the serum of IL-12-treated mice. In mice which had been immunized by footpad injection of allogeneic splenocytes, daily administration of rIL-12 i.p. was shown to enhance the specific CTL response in the draining lymph nodes. Thus, these studies demonstrate that IL-12 can enhance NK and CTL activity and induce IFN-gamma production in vivo, as well as in vitro, and suggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment of some tumors and infectious diseases.

Immunosuppression by discodermolide.

In summary, discodermolide, a novel, marine-derived compound, is a potent in vitro and in vivo immunosuppressive agent. Discodermolide blocks cellular proliferation in lymphoid and nonlymphoid cells. This blocking action is not due to cytotoxicity. Blockage of cell proliferation by discodermolide appears to occur at the G2/M interface of the cell cycle, similar to that observed with other types of antiproliferative drugs (i.e., doxorubicin). The cell cycle block appears to be reversible, as cells recover normal cycling patterns within 48 h after removal of the compound. Additional work with this compound is targeted towards determining the exact nature of discodermolide's mitotic block and is currently under way.

Failure of IL-1 receptor antagonist and monoclonal anti-IL-1 receptor antibody to inhibit antigen-specific immune responses in vivo.

rIL-1R antagonist (rIL-1ra) and 35F5, a neutralizing mAb, have been shown to inhibit the ability of IL-1 alpha and IL-1 beta to bind to type I but not type II murine receptors. Additionally, IL-1ra and 35F5 inhibit a variety of inflammatory responses in vitro and in vivo. In the present report we have evaluated the activity of human IL-1ra and 35F5 in murine Ag-specific cell-mediated and humoral immune response models. Administration of IL-1ra, either twice daily or as a continuous infusion, did not inhibit the cytolytic T lymphocyte response to allogeneic splenocytes. The CTL response was also not inhibited by daily administration of 35F5. The delayed type hypersensitivity response to oxazolone was similarly refractory to administration of IL-1ra and 35F5. In the humoral immune response models, neither the splenic plaque response to SRBC nor the IgG or IgM response to TNP-keyhole limpet hemocyanin was inhibited by treatment with IL-1ra or 35F5. These data suggest that signaling through the type I IL-1R is not required for these Ag-specific immune responses.

Analysis of retinoid-mediated immunosuppression in vivo. Effects of Ro23-6457 on cellular alloimmune responses.

We have investigated the immunosuppressive effects of a synthetic retinoid, Ro23-6457, on the in vivo development of cellular alloimmunity. We initially observed that treatment of C57B1/6 mice with 10 mg/kg/day Ro23-6457 drug could prolong the survival of DBA/2 cardiac allografts, thus verifying its immunosuppressive potential in murine experimental models. We next used the sponge matrix model of allograft rejection and limiting dilution analysis (LDA) of cytotoxic T lymphocyte (CTL) frequency to dissect this phenomenon further. In this experimental system we observed the following effects of Ro23-6457: (1) dose-dependent decrease in the number of LDA-detectable, donor-reactive CTL accumulating in sponge matrix allografts; (2) failure to interfere with in vitro assays of cellular alloimmunity, including LDA; and (3) antigen non-specific depression of LDA-detectable CTL in lymph nodes, spleen and especially in peripheral blood. For peripheral blood CTL, the drug eliminated LDA-detectable CTL, an effect that was reversible and could not be attributed to the activation of suppressor cells. Since Ro23-6457 has little effect on the number of peripheral blood Thy1.2+ cells, it appears that this drug does not physically eliminate CTL, but makes them temporarily hyporesponsive to antigen stimulation, and thus undetectable in functional assays like LDA.

Different T cell requirements for specific memory induction in normal and xid B cells.

The immune response to TTGG-A--L, a defined-sequence, branched-chain polypeptide, is regulated by MHC-linked Ir genes. TTGG-A--L specific B cells can be demonstrated in both normal and immune-defective low responder strains by activation to specific antibody secretion after immunization with TTGGAA-F gamma G, a conjugate of the hexapeptide TTGGAA and the immunogenic carrier fowl gamma-globulin. It is shown that immunization with TTGG-A--L induces specific memory B cells with equal efficiency in normal low and high responder strains but not in immune-defective low responder strains. We conclude that memory induction in xid B cells in contrast to normal B cells is dependent on MHC-restricted, carrier-specific helper T cells. Other observations also suggest a more stringent requirement for MHC-restricted, carrier-specific helper T cells in the induction of TTGGAA-specific antibody secretion by xid as compared to normal B cells. Both normal and immune-defective H-2k/b hybrids between the mutant CBA/N strain and TTGG-A--L high responder BALB.B are responders to TTGG-A--L. In contrast, normal but not immune-defective H-2k/d hybrids with responder BALB/c are responders to TTGG-A--L. This identifies H-2d as a TTGG-A--L high responder haplotype for normal B cells but a low responder haplotype for xid B cells, whereas H-2b is a high responder haplotype for both normal and xid B cells. This must reflect a quantitative or qualitative difference in Ir gene-mediated cellular interactions required for induction of antibody secretion in normal and xid B cells.

TTGG-A-L-specific memory B cells induced in low responder strains.

The immune response to TTGG-A--L, a defined-sequence, branched-chain polypeptide, is regulated by MHC-linked Ir genes. TTGG-A--L-specific B cells can be demonstrated in low responder strains by activation to specific antibody secretion after immunization with TTGGAA-F gamma G, a conjugate of the hexapeptide TTGGAA and the immunogenic carrier fowl gamma-globulin. It is shown that immunization with TTGG-A--L induces specific memory B cells with equal efficiency in low and high responder strains. This finding demonstrates that memory formation in a B cell subpopulation represented by TTGG-A--L-specific precursors is independent of carrier-specific, MHC-restricted helper T cells. This conclusion is further supported by the demonstration in an adoptive transfer model that immunization with TTGG-A--L induces equivalent levels of TTGG-A--L-specific memory B cells in T cell-deficient nude mice and their normal heterozygous littermates.

Origin and specificity of autoreactive T cells in antigen-induced populations.

We have characterized the major histocompatibility complex (MHC) specificity of autoreactive T cell clones arising from diverse donors after immunization with different antigens. The MHC fine specificity of autoreactive T cells for unique F1 hybrid determinants of BALB.K X BALB.B F1, and for the mutant I-Ab determinants of the B6.C-H-2bm12 (bm 12) strain is similar to that previously described for antigen-specific T cells. We find, furthermore, that the MHC specificity of autoreactive T cell clones selected from primed populations grown in the absence of Con A-stimulated supernatant factors reflects the predominant MHC restriction specificity of T cells specific for the immunogen. Thus, I-E subregion-specific autoreactive T cells are detected at a much higher frequency after immunization with the I-E-restricted antigen, GL (terpolymer of glutamic acid, lysine, and phenylalanine), than with the predominantly I-A-restricted antigen, keyhole limpet hemocyanin (KLH). These experiments strongly suggest that some autoreactive T cells are derived from antigen-stimulated precursors. This result contrasts with that obtained when autoreactive T cells are selected in bulk cultures, or in the presence of exogenous T cell factors. We conclude that, under optimal conditions, most autoreactive T cells are recruited from a relatively stable pool of predominantly I-A-specific precursors. Autoreactive precursors in this pool might themselves derive from previous antigenic stimulation, or be of independent origin.

Functionally distinct helper T cells enriched under different culture conditions cooperate with different B cells.

Helper T cells selected from KLH-primed lymph node cells under different in vitro conditions were analyzed for the ability to cooperate in secondary hapten-specific antibody responses. It is shown that carrier-specific, MHC-restricted helper T cells selected in low-density cultures cooperate with equal efficiency in either PC-specific or DNP-specific responses. In the presence of both PC-specific and DNP-specific B precursors, however, such T cells are limited to participate in only one helper event in vitro. In contrast, the predominant helper T cell in a second population maintained in high-density lymph node cultures cooperates, presumably through a soluble factor, with multiple B precursors. These properties correspond to those previously described for specific and nonspecific effector functions. The experiments reported here further demonstrate that different B cells respond in vitro to carrier-specific and nonspecific helper signals.