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A murine CTLA4/Fc gamma2a heavy chain (mCTLA4-Fc) chimeric fusion molecule was used in B6AF1 recipients of BALB/c pancreatic islet allografts to study the induction and maintenance of tolerance following inhibition of the CD28-B7 pathway for T cell activation. Donor-specific tolerance was achieved by administering 100 microg of mCTLA4-Fc on alternate days for 14 days (8 total doses) or a single 500 microg dose of mCTLA4-Fc on day 2 after transplant. Tolerance was mediated by long-lived peripheral lymphocytes and showed features of organ and alloantigen specificity. Whereas tolerance could not be established in allograft recipients receiving simultaneous mCTLA4-Fc and rIL-2, previously tolerant animals did not reject their grafts when given IL-2, suggesting that the induction and maintenance phases of tolerance were distinct and separate. The maintenance of donor-specific tolerance was an active immunologic process that was CD4+ T cell dependent and could be adoptively transferred to naive lymphocytes, but could not be explained by apoptosis or deletion of alloreactive T cells. Although an IL-2-sensitive mechanism such as anergy may contribute toward the induction of tolerance, its maintenance involves active suppression.

CD4-targeted therapy with a nondepleting RIB-5/2 mAb abrogates accelerated (< 36 h) rejection in presensitized LEW rats and results in permanent acceptance of LBNF1 cardiac allografts in conjunction with the features of infectious tolerance. This study examined the role and functional significance of the Th1 and Th2 cytokine network and systemic host allospecific Ab (allo-Ab) responses in the development of the infectious tolerance pathway in this model. Long term survival of cardiac transplants in rats treated with the tolerizing RIB-5/2 mAb regimen was accompanied by profound depression of Th1 (IL-2 and IFN-gamma) and Th2 (IL-4, IL-10) cytokines at the graft site, as shown by competitive template reverse transcription-PCR and immunohistochemistry. In contrast, the expression of Th2-type cytokines was selectively up-regulated after transfer of infectious tolerance by spleen cells into new generations of primary and secondary test recipients. Donor-specific circulating IgM allo-Ab responses were diminished throughout, and the switch from IgM to IgG allo-Ab was completely prevented in tolerant hosts, as shown by flow cytometry. The demonstration that treatment with cytolytic anti-CD4, but not anti-CD8, mAb recreated rejection of test cardiac allografts with simultaneous down-regulation of IL-4 mRNA/protein expression underlines the importance of this cytokine in the development of infectious tolerance. Hence, this report documents distinct cytokine elaboration patterns in animals tolerized by CD4-targeted therapy compared with those rendered tolerant by putative regulatory Th2-like cells. The mechanism of tolerance in anti-CD4 mAb-treated hosts appears distinct from that operating in the absence of mAb, when the tolerant state is being transferred in an infectious manner to new cohorts of test recipients.

The IL-2 pathway is portrayed often as central to allograft rejection. To test this hypothesis, we studied IL-2-deficient mice as allograft recipients. IL-2 gene knockout (KO) mice reject islet allografts and demonstrate a classical mononuclear leukocytic infiltrate, containing CD4+ and CD8+ T cells, surrounding and invading the islet allografts. Moreover, allograft rejection in the IL-2 KO mouse is associated with intragraft expression of certain cytokine and CTL attack molecule genes (e.g. IFN-gamma, IL-4, IL-7, IL-10, and granzyme B). In separate experiments, IL-2 KO mice generated CTLs in response to in vivo challenge with allogeneic tumor cells. Although IL-2 KO mice reject allografts in vivo, spleen cells from immunologically naive IL-2 KO mice exhibit a diminished proliferative response to mitogens in vitro that could be restored largely by exogenous IL-2, IL-4, or IL-7. The paradoxical ability to execute graft rejection in vivo despite near absent T cell proliferative responses in vitro may result from the expression of IL-7 in vivo, but not in vitro. Con A-stimulated bulk spleen cell cultures derived from IL-2 KO mice were essentially devoid not only of IL-2 but also IL-7 gene transcripts. These data indicate that 1) IL-2 is not the sole T cell growth factor capable of supporting allograft rejection and 2) expression of IL-4, but not IL-2, during the allograft response does not lead inevitably to tolerance.

Numerous studies have suggested the potential application of IL-10 as an anti-inflammatory and as an antirejection agent. Unfortunately, cytokines have short circulating t1/2 We developed a murine IL-10/Fc gamma 2a immunoligand that possesses the biologic functions of IL-10 and the long circulating t1/2 in vivo, characteristic of Igs. We mutated the Fc gamma 2a fragment to render the immunoligand ineffective in directing Ab-dependent cell-mediated cytotoxicity and complement-directed cytolysis (noncytolytic IL-10/Fc (IL-10/Fc2-)). In terms of IL-10 activity, IL-10/Fc2- was as effective as rIL-10 mole per mole in preventing lethal septic shock, but the immunoligand had a prolonged period of efficacy in accord with its extended circulating half-life. Contrary to expectations, IL-10/Fc2- treatment tended to accelerate the destruction of islet cell allografts and increase the levels of granzyme B gene expression in local draining lymph nodes. These data suggest that the enhanced cytotoxic activity of allograft-destroying CTLs may contribute to the accelerated allograft rejection. Finally, our studies suggest that a noncytolytic IL-10/Fc fusion protein provides a useful tool to study the biologic effects of IL-10 in vivo and may provide a useful agent for the prevention and treatment of septic shock.

LEW rat recipients of (LEW X BN)F1 strain heterotopic cardiac transplants treated with cyclosporine A (CsA) (15 mg/kg/day intramuscularly, 7 days) retain grafts indefinitely despite drug withdrawal. Donor-specific suppressor T cells that are active in passive transfer experiments have been harvested from long-term CsA-treated hosts. Although CsA is known to inhibit in vitro cytokine release, the in vivo effects of the CsA on the lymphokine cascade are not known. We investigated the action of the drug upon spontaneous and mitogen-induced interleukin 1 (IL 1), interleukin 2 (IL 2), and interleukin 3 (IL 3) release by spleen cells obtained from the following groups of rats: 1) normal, i.e., untreated and ungrafted; 2) grafted, acutely rejecting; 3) grafted, actively treated; and 4) under CsA-induced state of "tolerance." The results demonstrate that in vivo CsA therapy inhibits monocyte (IL 1 release) as well as lymphocyte function (IL 2 and IL 3 release) only during the inductive phase (the 7 days of treatment). During the "tolerant" phase, mitogen (Con A and LPS)-induced release of interleukins was quantitatively similar to that noted in normal animals. In contrast, a remarkable increase in the spontaneous production of IL 3 was observed in the "tolerant" group. Because cytokine release is not inhibited in the "tolerant" state, our data strongly support the concept that maintenance of the state of unresponsiveness is governed by the emergence of suppressor cells. The correlation of increased spontaneous production of IL 3 during this period leads us to postulate that this interleukin may be implicated in the activation or clonal expansion of suppressor cells, and hence may play a role in graft tolerance.

Polymerase chain reaction-assisted reverse transcription was used to study the temporal course of rejection in unmodified recipients of murine pancreatic islet cell allografts (DBA/2-->B6AF1) by using syngeneic tissues as controls. The histologic appearance of the grafts was analyzed in parallel. Preproinsulin and constant region of the TCR-beta chain transcripts were studied as markers of graft integrity and infiltrating T cell mass, respectively. The participation of certain cytokines and CTL were analyzed by the detection of IL-2, IFN-gamma, IL-4, and CTL-specific serine protease (granzyme B) transcripts. The time-related disappearance of intragraft preproinsulin transcripts correlated with graft destruction, whereas the intensity of intragraft TCR-beta chain transcript levels correlated with the magnitude of mononuclear leukocyte infiltration in allografts. In unmodified allografts, the magnitude of IL-2 and IFN-gamma intragraft mRNA levels correlated with the intense mononuclear leukocyte infiltrate found on histologic examination at day 8. Only after stable IL-2 gene transcription on day 8 does evidence of graft destruction become apparent, indicating that IL-2 gene activation is closely related to and probably required for expression of alloimmune cytopathic processes. In contrast, IL-4 transcripts were absent or detected in low copy number throughout this time course. Intragraft expression of granzyme B mRNA, a CTL-specific transcript, peaked from day 8 to day 12 in allografts compared with syngeneic grafts or normal tissue. In syngeneic grafts IL-2 and/or IL-4 mRNA was essentially not detected. Although IFN-gamma and granzyme B transcripts were detected in syngeneic grafts, after 4 days the levels of detected transcripts were far less than those noted in allografts. In vivo detection of intragraft IL-2 transcripts in the relative absence of detectable IL-4 transcripts strongly suggests IL-2-dependent immune effector mechanisms are associated with, and perhaps responsible, for allograft rejection. Apparently IL-4-dependent effector mechanisms are not necessary for allograft rejection.

To test the hypothesis that blockade of B7-triggered costimulation by donor cells could preclude allograft rejection, we coated crude islet allograft preparations in vitro for 1 h with a murine CTLA4/Fc fusion protein. Murine CTLA4/Fc blocks the proliferative response in primary mixed lymphocyte cultures (MLC) and Con A-stimulated murine spleen cell cultures by 85 to 95%. Responder cells from a primary MLC containing mCTLA4/Fc were hyporesponsive upon restimulation to the same stimulator cells in a secondary MLC lacking mCTLA4/Fc. Because of mutations in the Fc gamma RI and C'1q binding sites of the Fc portion of the murine CTLA4/Fc fusion protein, the molecule binds to, but does not target, cells for Ab-dependent cellular cytotoxicity or complement-directed cytolysis. Although systemic immunosuppression was not applied, 42% (10 of 24) of B6AF1 recipients of islet allografts pretreated with CTLA4/Fc were permanently engrafted. Further, 50% of hosts bearing functioning islet allografts more than 150 days post-transplant were formally proved to be tolerant to donor tissues. A persistent CD4+ and CD8+ T cell infiltrate surrounding, but not invading, islet grafts in tolerant hosts was discerned. In control experiments, 89% (8 of 9) of islet allografts coated with mIgG3, and 100% (n = 10) pretreated with media alone were rejected. Thus, we conclude that 1) B7-triggered costimulation by donor APCs is an important element of rejection, and 2) blockade of the B7 pathway by in vitro allograft manipulation is able to induce tolerance.

We have been successful in our efforts to develop a long lived noncytolytic murine IL-10/Fc fusion protein. In the nonobese diabetic mouse (NOD) model, administration of IL-10/Fc from 5 to 25 wk of age completely prevented the occurrence of diabetes. Moreover, these mice remained disease-free long after cessation of IL-10/Fc therapy. Immunohistochemistry studies show that IL-10/Fc treatment inhibits expression of TNF-alpha, proinflammatory cytokine, as well as Th1-type cytokines, IL-2 and IFN-gamma, but promotes expression of IL-4 and IL-10, Th2-type cytokines, by islet-infiltrating leukocytes. In an adoptive transfer model of diabetes in NOD mice, we found that: 1) IL-10/Fc treated hosts bear leukocytes that block expression of diabetes and 2) these leukocytes persisted even 8 wk after cessation of IL-10/Fc treatment. The potent antidiabetogenic effects provided by IL-10/Fc in the NOD model, together with its apparent lack of systemic toxicity, are notable.

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.