The classical complement pathway in transplantation: unanticipated protective effects of C1q and role in inductive antibody therapy.

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.

Transforming growth factor beta-induced connective tissue growth factor and chronic allograft rejection.

Late loss of allograft function is primarily attributed to chronic rejection (CR). There are no effective treatments for CR and the underlying cause of the disease is unknown. This study compared events that occurred within cardiac allografts placed in mice that received either anti-CD4 therapy and develop CR or anti-CD40L therapy and do not develop CR. Both TGFbeta and connective tissue growth factor (CTGF), which is induced by TGFbeta, were expressed in grafts with CR but were not expressed in grafts without CR. TGFbeta transfection of allografts in anti-CD40L-treated recipients resulted in CTGF expression and CR. However, TGFbeta transfection of syngeneic grafts did not result in CTGF expression or CR. These data indicate that TGFbeta alone is insufficient to induce CR and that CTGF is required. Further, antigenic stimulation is required for TGFbeta induction of CTGF. Thus, CTGF may serve as a therapeutic target for CR.

Interleukin-18 production following murine cardiac transplantation: correlation with histologic rejection and the induction of INF-gamma.

Interleukin-18 (IL-18) and IL-12 have been shown to play an important role in the induction of interferon-gamma (IFN-gamma). IFN-gamma induces the proliferation of T cells and natural killer (NK) cells and augments the Th1 immune cascade. The role of IL-18 and IL-12 in the induction of IFN-gamma following allogeneic heart transplantation has not been described. We sought to characterize the IL-12 and IL-18 response to murine allogeneic heart transplantation, particularly with respect to IFN-gamma production and histologic transplant rejection. Forty-eight heterotopic heart transplants were performed in two groups of mice: syngeneic C3H/HeN to C3H/HeN mice and allogeneic BALB/C to C3H/HeN mice. Transplants were followed out to 2, 6, 10, and 14 days. Six transplants were performed in each group. Serum and splenic samples were used to evaluate the cytokine response by ELISA. Explanted heart tissue was processed for evidence of histologic rejection, and RT-PCR was performed to evaluate the IL-12, IL-18, and IFN-gamma signal qualitatively. Analysis of variance (ANOVA), Fisher's projected least significant difference (PLSD) was used for statistical analysis. Transplant rejection occurred in the allogeneic group histologically by day 6 and clinically by day 10. Serum IFN-gamma levels rose significantly by day 6 in the allogeneic group and then continued to rise in the splenocyte cultures. Serum IL-18 also rose significantly in the allogeneic group at day 6 compared with syngeneic group. RT-PCR revealed that the allogeneic tissue contained an increased signal for IL-12, IL-18, and IFN-gamma beginning at day 6 and peaking at day 10 after transplant. Beginning 6 days after transplantation, IL-12 and IL-18 appear to play a significant role in the induction of IFN-gamma in allogeneic heart transplants.

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions.

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.

DNA-liposome versus adenoviral mediated gene transfer of transforming growth factor beta1 in vascularized cardiac allografts: differential sensitivity of CD4+ and CD8+ T cells to transforming growth factor beta1.

We have developed a model of transforming growth factor (TGF)beta1 gene transfer into mouse vascularized cardiac allografts to study the use of gene transfer as an immunosuppressive therapy in transplantation. Donor hearts were perfused with either DNA-liposome complexes or adenoviral vectors that encode the active form of human TGFbeta1. DNA-liposome mediated transfection prolonged allograft survival in approximately two-thirds of transplant recipients, while adenoviral delivery of TGFbeta1 was not protective. Protective TGFbeta1 gene transfer was associated with reduced Th1 responses and an inhibition of the alloantibody isotype switch. The protective effects of TGFbeta1 gene transfer were overridden by exogenous interleukin-12 administration. Interestingly, alloreactive CD4+ and CD8+ cells exhibited distinct sensitivities to TGFbeta1 gene transfer: CD4+ Th1 function was abrogated by this modality, although CD8+ Th1 function was not. Transient depletion of recipient CD8+ cells markedly prolonged the survival of grafts transfected with either DNA-liposome complexes or adenoviral vectors. Transgene expression persisted for at least 60 days, and Th1 responses were not detectable until CD8+ T cells repopulated the periphery. However, long-term transfected allografts appeared to exhibit exacerbated fibrosis and neointimal development. These manifestations of chronic rejection were absent in long-term transfected isografts, suggesting that long-term expression of active TGFbeta1 alone is not sufficient to induce fibrosis of the grafts. Collectively, these data illustrate the utility of immunosuppressive gene therapy as a treatment for transplantation when combined with additional conditioning regimens. Further, they illustrate that alloreactive CD4+ and CD8+ cells may be differentially influenced by cytokine manipulation strategies.

Microphthalmic mice display a B cell deficiency similar to that seen for mast and NK cells.

The microphthalmic mouse (mi) possesses a 3-bp deletion of the Mi gene that alters the DNA binding site of the transcription factor gene product. This animal has diminished numbers of NK and mast cells (MC) and is osteopetrotic due to a lack of the normal complement of functional osteoclasts. The reduction of MC has been proposed to be due to the lack of adequate c-Kit expression that is required for MC differentiation. However, data from other labs has questioned this interpretation. In this report, we present data suggesting bone marrow-derived deficiencies of the mi mouse are not due to a lack of c-Kit expression and function, but instead due to an inhospitable environment within the bone marrow itself. Specifically, we have found that such animals also lack virtually all B cell precursors within the marrow and rely upon other lymphatic sites, such as the spleen, for B cell development and maturation. Although the animal has depressed numbers of NK cells, B cells, and MC, it still possesses a normal thymus and peripheral T cells. Therefore, the block in cellular differentiation must be within the marrow environment, which is essential for maturing B cells, NK cells, and MC but not T cells.

Tissue-specific consequences of the anti-adenoviral immune response: implications for cardiac transplants.

The immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. This would limit the use of adenovirus-mediated gene transfer in disease states in which long-term gene expression is required. While studying the effect of the anti-adenoviral immune response in transplantation, we found that transgene expression persisted in cardiac isografts transfected with an adenovirus encoding beta-galactosidase. Transfected grafts remained free of inflammation, despite the presence of an immune response to the vector. Thus, adenovirus-mediated gene transfer may have therapeutic value in cardiac transplantation and heart diseases. Furthermore, immunological limitations of adenoviral vectors for gene therapy are not universal for all tissue types.

Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process.

BACKGROUND: Transient depletion of CD4+ T cells in cardiac allograft recipients prolongs allograft survival; however, grafts exhibit signs of chronic rejection characterized by collagen deposition and neointima development. Although it is believed that Th1 cells promote acute graft rejection, the role of these cells in chronic rejection remains unclear. Hence, our study evaluated whether Th1 cells are associated with the development of chronic cardiac allograft rejection. METHODS: Splenocytes obtained from C57BL/6 recipients bearing BALB/c hearts with signs of chronic rejection were adoptively transferred into C57BL/6 SCID cardiac allograft recipients. As a measure of Th1 function, interferon-y production was determined after restimulation of recipient splenocytes with donor alloantigens. RESULTS: Transfer of splenocytes in SCID allograft recipients resulted in accelerated chronic rejection in the majority of mice. Characterization of these cells before transfer revealed hyporesponsive Th1 function. However, donor-specific proliferative responses and precursor interleukin-2 producing helper and cytotoxic T lymphocyte frequencies were comparable to that of naive splenocytes. Further, splenocytes obtained from SCID recipients with advanced signs of chronic rejection remained deficient in Th1 function, suggesting that Th1 are not involved in this disease process. This possibility was further supported by the development of chronic rejection in IL-12 knockout recipients. Finally, when splenocytes used for adoptive transfer retained Th1 function, transfer of these cells into SCID recipients resulted in acute allograft rejection. CONCLUSIONS: We have established a model in which the mediators of chronic rejection may be further explored. In this system, the absence rather than the presence of donor-reactive Th1 is associated with chronic rejection. These data indicate that Th1-independent effector mechanisms are responsible for chronic rejection in this model.

Interleukin-12 (IL-12)-driven alloimmune responses in vitro and in vivo: requirement for beta1 subunit of the IL-12 receptor.

BACKGROUND: Interleukin-12 (IL-12) mediates its biologic activities via binding high-affinity receptors on T and natural killer cells. Although emphasis has been placed on the requirement for IL-12Rbeta2 in IL-12 bioactivity, the role of IL-12Rbeta1 is less well defined. The current study evaluated the effects of exogenous IL-12 on alloantigen-specific immune responses and determined the requirement for IL-12Rbeta1 in IL-12-mediated alloimmunity. METHODS: The mouse heterotopic cardiac transplant model was employed to evaluate the effects of IL-12 on alloantigen-specific immune responses in vivo. In addition, IFN-gamma production in mixed lymphocyte cultures (MLC) supplemented with IL-12 was measured to assess the effects of IL-12 on Th1 function in vitro. Mice deficient in IL-12Rbeta1 (IL-12Rbeta1-/-) were used to determine the requirement for this receptor component in IL-12-driven alloimmune responses. RESULTS: Addition of IL-12 to MLC consisting of wild-type splenocytes enhanced alloantigen-specific proliferative responses and Th1 development. In contrast, IL-12 did not alter these in vitro immune parameters in IL-12Rbeta1-/- MLC. Treatment of wild-type cardiac allograft recipients with IL-12 resulted in high concentrations of serum interferon-gamma (IFN-gamma) and a 10-fold increase in IFN-gamma production by recipient splenocytes after restimulation in vitro. However, this fulminate Th1 response did not accelerate allograft rejection. Importantly, IL-12 had no effect on serum IFN-gamma or in vivo priming of Thl in IL-12Rbeta1-/- recipients. Finally, administration of IL-12 to WT allograft recipients resulted in a bimodal alloantibody response: antibody production was suppressed at high doses of IL-12, and enhanced at lower doses. CONCLUSIONS: IL-12 markedly enhances alloantigen-specific immune function; however, these exaggerated Th1-driven responses do not culminate in accelerated allograft rejection. Further, these data indicate that IL-12Rbeta1 is essential for the enhancement of both in vitro and in vivo alloimmune responses by exogenous IL-12.

Alloantigen-reactive Th1 development in IL-12-deficient mice.

IL-12p70, a 70- to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits, enhances Th1 development primarily by its ability to induce IFN-gamma production by NK and Th1 cells. Although homodimers of the p40 subunit of IL-12 are potent IL-12 receptor antagonists in some systems, we have reported that p40 homodimer may accentuate alloreactive CD8+ Th1 function. To test the role of endogenously produced p40 in alloimmunity, Th1 development was assessed in either IL-12 p35 knockout (p35-/-) mice, the cells of which are capable of secreting p40, or p40 knockout (p40-/-) mice. Compared with IL-12 wild-type controls, splenocytes obtained from both p35-/- and p40-/- mice produced markedly less IFN-gamma after in vitro stimulation with Con A or alloantigens. Interestingly, in vivo-sensitized Th1 were detected in both p35-/- and p40-/- cardiac allograft recipients. However, in vivo Th1 development was enhanced in p35-/- recipients compared with p40-/- animals, suggesting that endogenous p40 produced in p35-/- mice may stimulate alloreactive Th1. Indeed, neutralizing endogenous p40 with anti-IL-12 p40 mAb reduced Th1 development in p35-/- allograft recipients to that seen in p40-/- mice. To determine whether Th1 development that occurred in the absence of IL-12p70 and p40 required IFN-gamma, p40-/- allograft recipients were treated with anti-IFN-gamma mAb. Neutralizing IFN-gamma did not inhibit in vivo Th1 development in p40-/- recipients and resulted in a unique pathology of rejection characterized by vascular thromboses. Collectively, these data suggest that 1) endogenous p40 may substitute for IL-12p70 in alloantigen-specific Th1 sensitization in vivo and 2) in vivo alloreactive Th1 development may occur independent of IL-12 and IFN-gamma, suggesting an alternate Th1-sensitizing pathway.

Distinct characteristics of resistance to Borrelia burgdorferi-induced arthritis in C57BL/6N mice.

Studies of mice infected with Borrelia burgdorferi have indicated that the severity of arthritis is influenced by the genetic composition of the host: the C3H mouse develops severe arthritis while BALB/c and C57BL/6 mice develop mild arthritis. In this study, the effects of increasing infectious dose on the severity of arthritis were determined in these three mouse strains. C3H/He mice developed severe arthritis at all infectious doses, with 100% infection requiring 200 spirochetes. In BALB/cAnN mice, arthritis severity was dependent on infectious dose; symptoms were mild with infection by 200 B. burgdorferi and progressively more severe with increasing infectious dose. Infection of BALB/cAnN mice with 2 x 10(4) B. burgdorferi resulted in arthritis with severity identical to that in C3H/He mice. Spirochete levels in rear ankle joints of C3H/HeJ and C3H/HeN mice were relatively high, as detected by PCR, and did not increase with infectious dose. Spirochete levels in joints from BALB/cAnN mice increased with increasing infectious dose to levels found in severely arthritic C3H/He mice. Thus, resistance to severe arthritis in BALB/cAnN mice was conditional: it could be overcome by high infectious dose and the arthritis became severe when high levels of B. burgdorferi were present in joints. A unique response to increasing infectious dose was seen in C57BL/6N mice, which displayed mild to moderate arthritis at all doses of B. burgdorferi tested, up to 2 x 10(5). At all infectious doses, the levels of spirochetes in ankle joints of C57BL/6N mice were high, equivalent to those found in the severely arthritic C3H/He mice. The arthritis observed in infected (C57BL/6N x C3H/HeN)F1 mice was of severity intermediate between those of the two parental strains. The finding that resistance to severe arthritis in C57BL/6N mice could not be overcome by high infectious doses and was independent of spirochete levels in joints suggested that it was mediated by a distinct mechanism from that operating in BALB/cAnN mice.

Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gamma-producing cells.

The role of IL-12 role in regulating Th1/Th2 balance is attributed in part to the ability of this cytokine to induce IFNgamma production by NK and Th1 cells, which in turn promotes Th1 and inhibits Th2 development. In the present study, the requirement for IL-12 in the development of alloantigen-reactive Th1 was assessed by adding neutralizing anti-IL-12 Abs or the IL-12 receptor antagonist p40 homodimer to primary MLC. The resulting cell populations were assessed for Th1 development by measuring IFN-gamma production upon restimulation with alloantigens. While the addition of anti-IL-12 Abs to primary MLC did not influence subsequent cytokine production, addition of p40 homodimer markedly enhanced, rather than decreased, Th1 development. To determine which T cell population produced enhanced levels of IFN-gamma in response to p40 homodimer, CD4+ or CD8+ T cells were depleted from the MLC. While p40 homodimer was inhibitory to selected CD4+ Th1 development, it enhanced IFN-gamma production by CD8+ T cells. To test the in vivo relevance of these findings, mouse heterotopic cardiac allograft recipients were treated with either p40 homodimer, anti-CD8 mAb, or with both p40 homodimer and anti-CD8 mAb. Treatment of allograft recipients with p40 homodimer had no effect on the in vivo sensitization of IFN-gamma-producing cells and resulted in accelerated allograft rejection relative to unmodified recipients. However, p40 homodimer markedly prolonged allograft survival in mice depleted of CD8+ T cells. Hence, p40 homodimer stimulates CD8+ Th1 development in vitro but inhibits CD4+ T cell function both in vitro and in vivo.

IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection. Evidence against a dominant protective role for T helper 2 cytokines in alloimmunity.

IL-12 promotes Th1 development and inhibits the generation of Th2 by inducing IFN-gamma production. In several experimental models, Th2 are preferentially induced in the absence of IL-12. It was proposed that the preferential induction of Th2 by IL-12 antagonism would inhibit Th1-driven rejection responses, thereby promoting allograft acceptance. To test this possibility, mouse cardiac allograft recipients were treated with either anti-IL-12 Abs, or with the IL-12 receptor antagonist p40 homodimer. Unmodified rejection is characterized by a Th1-dominated response, with Th2 cytokines being absent or only weakly expressed within the allograft. Though both forms of IL-12 antagonism induced Th2 cytokine expression within the allograft, these treatments surprisingly exacerbated graft rejection relative to control animals. Interestingly, IL-12 antagonism did not inhibit IFN-gamma gene expression or in vivo sensitization of IFN-gamma-producing cells. Similar observations were made when IL-12 p40 knockout mice were used as allograft donors and recipients, verifying that IL-12 was not required for Th1 development. Further, IL-12 antagonism was associated with strong expression of p40 and weak expression of p35 within the graft. Neither p35 nor p40 mRNAs were detectable in control allografts. These data indicate that while IL-12 antagonism does induce Th2 cytokine expression within cardiac allografts, Th2 cytokines do not play a dominant protective role in the rejection process. Further, the Th2-inducing activity of IL-12 antagonism is not related to decreased IFN-gamma production, but may reflect altered regulation of IL-12 itself.

Dissociation of mouse cardiac transplant rejection and donor alloantigen-specific T cell responsiveness.

Mouse hearts transplanted into MHC disparate donors are usually rejected 1 week after placement. It is widely accepted that alloantigen-reactive helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) are the key mediators of acute allograft rejection. This report demonstrates that the presence or absence of 'traditional' graft-reactive HTL and CTL is not necessarily related to allograft survival. In these studies, donor/recipient combinations disparate only at MHC or only at minor histocompatibility (mH) loci were employed. Allograft survival was monitored, donor-reactive IL-2 (interleukin-2) producing HTL and CTL were quantified by modified limiting dilution analysis, and serum levels of cytolytic alloantibody were determined. C57BL/10 hearts (H-2b) transplanted into B10.BR (H-2k) recipients (full MHC disparity) enjoyed prolonged survival despite massive infiltration of the allograft by donor-reactive HTL and CTL. IgM, but not IgG, donor-reactive alloantibody was present in the sera of these mice. Hence, traditional IL-2 producing HTL and CTL were not capable of mediating acute graft rejection, nor of providing help for alloantibody isotype switching in this MHC disparate combination. In contrast, C3H/HeN (H-2k) hearts transplanted into B10.BR (H-2k) recipients (mH disparity only) were acutely rejected. Donor-reactive HTL and CTL were rare or not detectable in these recipients, and cytolytic alloantibody was not detectable. Similar observations were made when B10.BR hearts were transplanted into C3H/HeN recipients. Interestingly, treatment of recipients with anti-CD4 monoclonal antibody prevented rejection of mH disparate allografts. Therefore, CD4+ T cells were required for rejection of mH disparate allografts, but this process was independent of detectable IL-2 production or CTL function. Hence, the significance of monitoring 'traditional' T cell functions should be questioned in certain donor/recipient combinations.

In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection.

A current hypothesis states that Th1 cytokines promote allograft rejection and that Th2 cytokines promote graft acceptance. We present data that question the tolerogenic activity of Th2 cytokines, and we suggest that Th2 cytokines may evoke allograft rejection by recruitment of alternate effector mechanisms. Unmodified rejection of mouse heterotopic cardiac allografts is associated with the accumulation of large numbers of donor-reactive CD8+ CTL within the allograft, which is indicative of a Th1-driven cellular response. However, when recipients are depleted of CD8+ CTL, rejection still occurs and is associated with an aggressive cellular infiltrate rich in eosinophils, large mononuclear cells, and fibroblast-like cells. Eosinophils, which are responsive to the Th2 cytokines IL-4 and IL-5, are not present in unmodified rejecting allografts. Differential production of Th1 versus Th2 cytokines was further suggested by altered levels of IgG2a (promoted by IFN gamma) and IgG1 (promoted by IL-4) alloantibody in the sera of these mice; IgG2a dominated the alloantibody response in unmodified allograft recipients, whereas IgG1 levels increased in recipients depleted of CD8+ CTL. Altered intragraft cytokine gene expression was verified by RT-PCR; Th1 (IL-2, IFN gamma), but not Th2 (IL-4, IL-5, IL-10), cytokine mRNAs were readily detectable in the allografts of unmodified recipients. In contrast, both Th1 and Th2 cytokine genes were expressed in the allografts of mice depleted of CD8+ CTL. These data suggest that donor-reactive CD8+ CTL inhibit intragraft production of Th2 cytokines, thereby promoting a Th1 dominated-rejection response. Elimination of CD8+ cells allows Th2 cytokine production, which may have deleterious, rather than protective, effects.

Helper T lymphocyte unresponsiveness to cardiac allografts following transient depletion of CD4-positive cells. Implications for cellular and humoral responses.

Initial treatment of heterotopic cardiac transplant recipients with anti-CD4 mAb promotes long-term (> 60 days) allograft survival. We have used modified limiting dilution analysis to quantitate donor alloantigen-reactive helper T lymphocytes (HTL) and CTL in mice bearing long-term cardiac allografts. Despite repopulation of lymphoid tissues with CD4+ T cells, donor alloantigen-reactive IL-2 producing and IL-4-producing HTL were rare or not detectable in lymphoid tissues or in the graft. While donor-reactive precursor CTL were present in lymphoid tissues, modified limiting dilution analysis revealed that stimulated ("antigen-conditioned") CTL were not detectable, and few CTL were present in the graft. In addition, antibodies reactive with donor alloantigens were not detectable in the sera of mice bearing long-term cardiac allografts. To determine whether additional in vivo stimulation with donor alloantigens would elicit an immune response, sponge allografts were implanted into mice bearing long-term cardiac allografts. Previous reports from this laboratory have demonstrated that T cell infiltration of sponge allografts is dependent upon antigen-driven cytokine production. While third-party sponge allografts were readily infiltrated by third-party-reactive HTL and CTL, sponge allografts of the same strain as the cardiac allograft were not infiltrated by T cells. However, donor strain sponge allografts induced an IgM (but not IgG) alloantibody response. These data indicate that IgM alloantibody could be induced in the absence of a cellular response to the sponge allograft. Kinetic studies revealed that a transient IgM (but not IgG) response was induced by the initial cardiac transplantation in the absence of CD4+ cells. These IgM alloantibodies disappeared by day 21 despite the persistence of the allograft. These observations indicate that transient depletion of CD4+ T cells induces allograft-specific T cell tolerance, but does not eliminate the ability to mount an allograft-specific IgM response. To our knowledge, this is the first report of a transient humoral response to alloantigens that occurs in the absence of CD4+ T cells, and can be reinduced in "tolerant" animals that fail to mount a cellular immune response. Potential mechanisms involved in the development and maintenance of anti-CD4 mAb-induced tolerance are discussed.

CD4-positive helper T lymphocytes mediate mouse cardiac allograft rejection independent of donor alloantigen specific cytotoxic T lymphocytes.

Mouse heterotopic cardiac allograft recipients were depleted of CD4+ or CD8+ T lymphocytes in vivo to assess cellular requirements for graft infiltration, tissue damage, and acute allograft rejection. Modified limiting dilution analysis was employed to quantitate IL-2-producing Th lymphocytes (HTL) and CTL infiltrating the graft. Results were correlated with graft function and histologic evidence of tissue damage. In unmodified recipients, large numbers of donor alloantigen-specific CTL infiltrated the graft, overshadowing a modest number of HTL. CTL infiltration coincided with tissue damage and loss of graft function, suggesting a key role for CTL in rejection. In vivo treatment with anti-CD4 mAb inhibited both HTL and CTL infiltration, and no histologic evidence of tissue damage was observed. This observation suggested that HTL, although few in number, regulated the development of effector CTL and/or entry of these CTL into the graft. Reconstitution of HTL-depleted recipients with IL-2 resulted in graft infiltration by stimulated CTL, as assessed by modified limiting dilution analysis. However, these stimulated CTL failed to mediate tissue damage, and graft survival was prolonged. Unlike CTL obtained from unmodified recipients, graft-infiltrating CTL of IL-2-reconstituted mice were incapable of directly lysing donor cells in a 51Cr release assay. Hence, while IL-2 facilitated partial CTL differentiation and mobilization to the graft, additional signals appear necessary for maturation into lytic CTL. Furthermore, in recipients depleted of CTL by treatment with anti-CD8 mAb, HTL infiltrating the allograft, though few in number, were associated with extensive tissue damage and loss of graft function. These data suggest a less important role for CTL in the rejection process, and indicate that graft-infiltrating CTL are insufficient as sole mediators of cardiac allograft rejection. Potential mechanisms by which CD4+ HTL mediate cardiac allograft rejection independent of CTL are discussed.

Mobilization of T lymphocytes following cardiac transplantation. Evidence that CD4-positive cells are required for cytotoxic T lymphocyte activation, inflammatory endothelial development, graft infiltration, and acute allograft rejection.

Modified limiting dilution analysis (LDA) techniques were used to evaluate the mobilization of antigen-stimulated helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) following allogeneic heterotopic cardiac transplantation. These modified LDA techniques allow a quantitative comparison of T cells that have been stimulated by antigen in vivo versus unstimulated precursor T cells of the same antigen specificity. Endothelial changes associated with mononuclear cell infiltration of the transplant were studied using endothelia-specific monoclonal antibodies and immunohistochemistry. Early (day 3) infiltration of cardiac allografts was characterized by a prevalence of donor alloantigen-specific HTL over CTL. Immunohistology revealed that the day-3 infiltrate was associated with areas of differentiated vascular endothelium, located primarily in the subepicardial region. Though donor-specific precursor HTL and CTL were present in the peripheral lymphoid tissues and blood, very few of them had been stimulated at this early time. During the latter phases of the response (days 6-9), antigen-stimulated HTL and CTL were present in the rejecting heart with CTL dominating the response. Accumulation of large numbers of donor-specific CTL in the allograft correlated with extensive inflammatory endothelial development, myocyte destruction, and loss of graft function by day 9. Stimulated HTL and CTL were detectable in peripheral lymphoid tissues at days 6 and 9. In addition, a marked increase in the number of donor-specific precursor CTL, but not precursor HTL, was observed in the lymphoid tissues at the peak of the response. Depletion of class II MHC-restricted T cells by in vivo treatment with anti-CD4 mAb eliminated HTL activity in all lymphoid compartments assessed and markedly reduced the number of CTL infiltrating the allograft. In addition, no stimulated CTL were detectable in lymphoid tissues, and the number of precursor CTL was not increased. In anti-CD4-treated recipients, cardiac allografts remained functional with minimal histological evidence of rejection for at least 21 days. Though graft-associated inflammatory endothelia were absent in anti-CD4-treated recipients at day 6, endothelial differentiation was observed in day 21 allografts in anti-CD4-treated recipients. These observations indicate that inflammatory endothelial development may precede T cell infiltration and subsequent loss of the cardiac allograft function. Thus, CD4-positive HTL are required for (1) graft-associated inflammatory endothelial development; (2) CTL activation in peripheral lymphoid tissues; (3) CTL accumulation in allografted tissues; and (4) acute cardiac allograft rejection.