The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4+ T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4+ T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4+ T cells. METHODS: Transgenic RAG2-/- mice expressing I-Ab-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2k male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2k male allograft and presented by the recipient's own I-Ab APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-Ab MHC class II molecules. This observation suggested that graft endothelium replacement by I-Ab-positive cells of recipient origin could stimulate the rejection of male H-2k graft by I-Ab-restricted H-Y-specific T cells. To investigate further this possibility, hearts from H-2b-into-H-2k irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-Ab-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2k male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4+ T cells.     

Visualization of the in vivo generation of donor antigen-specific effector CD8+ T cells during mouse cardiac allograft rejection: in vivo effector CD8+ T cell generation during allograft rejection

BACKGROUND: An adoptive transfer system was used to study the fate of alloreactive CD8+ H-2Kb-specific TCR transgenic (DES+) T cells in vivo after transplantation. METHODS: A trace population of 2.0x10(6) CD8+DES+ T cells were adoptively transferred into syngeneic CBA.Ca (H-2k) mice 24 hr before transplantation of an H-2Kb+ or H-2Kb- cardiac allograft. RESULTS: H-2Kb specific T cells proliferated and produced interleukin-2 and interferon-gamma in response to H-2Kb+, but not H-2Kb- cardiac allografts. CD8+DES+ T cells that infiltrated the H-2Kb+ cardiac allografts developed a distinct cell surface and cytokine phenotype compared with the CD8+DES+ T cells that remained in the periphery. H-2Kb-specific graft infiltrating T cells (a) underwent a larger number of cell divisions (> =3), (b) increased in size, (c) up-regulated CD69, and (d) down-regulated CD62L. CONCLUSIONS: These results demonstrate that alloantigen-specific T cells can be monitored in vivo during the immune response to an allograft and that the fate of CD8+ T cells specific for the allogeneic class I molecules expressed by the graft is different between cells in the periphery and those that infiltrate the graft.     

Effects of noninherited maternal antigens on allotransplant rejection in a transgenic mouse model

We studied the influence of noninherited maternal antigen (NIMA) on allotransplant rejection using a mouse transgenic model. CBK transgenic (CBA [H-2k] expressing K(b) MHC class I transgene) mice were used as donors in heart transplantation experiments. Offspring of BM3.3 (CBA anti-K(b) TCR transgenic) male mice and (CBA x CBK)F1 females were used as NIMA (offspring that did not inherit K(b)) and IMA (offspring that inherited K(b) maternal antigen) recipient mice. Survival of allografts was monitored and the alloimmune response evaluated using an ELISPOT assay. IMA mice accepted CBK heart allografts and displayed no alloresponse to K(b+) cells. In contrast, mice never exposed to K(b) (offspring of BM3.3 males and CBA females) acutely rejected their grafts within 18 days posttransplantation and exhibited potent inflammatory alloresponses to K(b+) cells. NIMA mice displayed prolonged survival of allotransplants (MST >60 days). Although no deletion of anti-K(b) TCR transgenic cells was detected in these mice, they had a marked reduction in the frequency of activated alloreactive T cells producing type 1 (IFN-gamma and IL-2) cytokines and concomitant expansion of type 2 (IL-4) cytokine-secreting cells. Finally, depletion of CD4+ T cells from NIMA mice restored acute rejection of CBK hearts. This study is the first demonstration of the tolerogenic effects of NIMA on alloimmunity and allotransplant rejection in a transgenic model. It is shown that, although the NIMA tolerogenic effect is not due to deletion of alloreactive T cells, it is mediated by CD4+ T cells producing type 2 cytokines.     

Viral infection abrogates CD8(+) T-cell deletion induced by costimulation blockade

BACKGROUND: Treatment with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (mAb) prolongs skin allograft survival in mice. It is known that prolongation of allograft survival by this method depends in part on deletion of alloreactive CD8(+) T cells at the time of tolerance induction. Recent data suggest that infection with lymphocytic choriomeningitis virus (LCMV) abrogates the ability of this protocol to prolong graft survival. METHODS: To study the mechanism by which viral infection abrogates allograft survival, we determined (1) the fate of tracer populations of alloreactive transgenic CD8(+) T cells and (2) the duration of skin allograft survival following treatment with DST and anti-CD154 mAb in the presence or absence of LCMV infection. RESULTS: We confirmed that treatment of uninfected mice with DST and anti-CD154 mAb leads to the deletion of alloreactive CD8(+) T cells and is associated with prolongation of skin allograft survival. In contrast, treatment with DST and anti-CD154 mAb in the presence of intercurrent LCMV infection was associated with the failure to delete alloreactive CD8(+) T cells and with the rapid rejection of skin allografts. The number of alloreactive CD8(+) cells actually increased significantly, and the cells acquired an activated phenotype. CONCLUSIONS: Interference with the deletion of alloreactive CD8(+) T cells mediated by DST and anti-CD154 mAb may in part be the mechanism by which viral infection abrogates transplantation tolerance induction.     

In vivo mechanisms of alloreactivity. II. Allospecificity of cytotoxic T lymphocytes in sponge matrix allografts as determined by limiting dilution analysis

We have examined the frequency and alloantigen specificity of CTL that accumulate in sponge allografts (sponges seeded with allogeneic splenocytes) in sponge isografts (sponges seeded with syngeneic splenocytes), and in splenocyte-free sponge implants. Using limiting dilution analysis (LDA), we observed that sponge isografts and splenocyte-free sponge implants from C57BL/6 (H-2b) mice usually acquire small numbers of CTL (less than 250 cells per graft) with DBA/2 (H-2d)-reactivity or C3H/HeJ (H-2k)-reactivity. These alloreactive CTL are not detectable in conventional 51Cr-release assays, presumably because they are too infrequent and/or because they are inactive CTL precursors. When we examined the accumulation of alloreactive CTL in sponge allografts, we observed that DBA/2 sponge allografts from C57BL/6 recipients accumulate 10 to 100 times more DBA/2-reactive CTL than alloantigen-free sponge grafts. Nonetheless, these donor-reactive CTL rarely constitute more than 0.5% of the T cells recovered from sponge allografts, even at the peak of the rejection response. This raises questions concerning the remaining 99.5% of the allograft-infiltrating T cells. We were unable to detect by LDA any host-reactive CTL in sponge allografts, thus excluding the possibility that some of the remaining T cells were host-reactive CTL of donor origin which diluted graft-reactive T cells. However, using LDA we did detect a significant number of third-party (C3H/HeJ)-reactive CTL in sponge allografts, suggesting that the intense immune response at a graft site might facilitate indiscriminate recruitment of T lymphocytes. Alternatively, this enhanced third-party alloreactivity might reflect the proliferation of donor-reactive CTL with incidental crossreactivity for C3H/HeJ alloantigens. While testing these two alternatives, we observed that LDA cultures designed to detect third-party-reactive CTL could also support the growth of the in vivo-activated, donor-reactive CTL from sponge allografts; This compromised enumeration by LDA of the less frequent, third-party-reactive CTL by LDA. Although LDA is the only method that detects the growing population of third-party-reactive CTL in sponge allografts, technical restraints exclude LDA as a method of determining whether donor-reactive CTL and third-party-reactive CTL are separate or overlapping CTL subpopulations. Hence, it remains unclear if third-party-reactive CTL are a significant or insignificant proportion of the CTL that infiltrate sponge allografts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allopeptide-Specific CD4+ T Cells Facilitate the Differentiation of Directly Alloreactive Graft-Infiltrating CD8+ T Cells

To investigate the mechanism of CD4(+) T-cell help during the activation and differentiation of directly alloreactive CD8(+) T cells, we examined the development of obliterative airways disease (OAD) following transplantation of airways into fully mismatched recipient mice deficient in CD4(+) T cells. BALB/c trachea allografts became fibrosed significantly less frequently in B6 CD4(-/-) recipients as compared to wildtype controls. Furthermore, class I-directed cytotoxicity failed to develop in the absence of CD4(+) T cells. The infiltration of graft tissue by primed L(d)-specific directly alloreactive 2C CD8(+) T cells was not found to depend on the presence of CD4(+) T cells. Nevertheless, graft-infiltrating 2C CD8(+) T cells failed to express CD69 and granzyme B when CD4(+) T-cell help was unavailable. Importantly, reconstitution of B6 CD4(-/-) recipient mice with graft peptide-specific TCR-Tg CD4(+) T cells (OT-II or TEa) capable of recognizing antigen only on recipient APC allowed for full expression of CD69 and granzyme B by the directly alloreactive CD8(+) T cells and restored the capacity of recipients to reject their allografts. These results demonstrate that indirectly alloreactive CD4(+) T cells ensure the optimal activation and differentiation of graft-infiltrating directly alloreactive CD8(+) T cells independent of donor APC recognition.     

Evidence that long-term cardiac allograft survival induced by anti-CD4 monoclonal antibody does not require depletion of CD4+ T cells.

Monoclonal antibodies that deplete cells carrying their target antigen are being used increasingly for immunosuppression in clinical and experimental transplantation. We have characterized a panel of rat antimouse CD4 monoclonal antibodies with the aim of establishing, in a vascularized organ transplant model, whether prolonged graft survival can be induced without recipient T cell depletion. The spatial relationship of the epitopes recognized by the anti-CD4 mabs was established. Mabs of the IgG2b isotype were found to profoundly deplete CD4+ T cells in vivo, whereas IgG2a mabs did not. The IgG2b anti-CD4 mab YTS191 and the IgG2a mab KT6 both blocked proliferation of C3H/He leukocytes in mixed leukocyte culture. Potent suppression of rejection and indefinite survival of cardiac allografts, mismatched for both major and multiple minor histocompatibility antigens (C57BL/10, H-2b into C3H/He, H-2k), was achieved with the IgG2b anti-CD4 mab YTS191 that depleted CD4+ T cells, (n = 9, median survival time (MST) greater than 100 days, P less than 0.001). The non-depleting IgG2a anti-CD4 mab, KT6, which had been shown to recognize and epitope on the CD4 molecule closely related to that recognized by YTS191 and to block comparably in MLC, was also shown to be capable of producing long-term cardiac graft survival in this strain combination (n = 6, MST greater than 100 days P less than 0.001). The kinetics of the KT6 therapy on the blocking of the CD4 molecule in vivo were investigated and shown to correlate with the effectiveness of the mab in prolonging graft survival.     

Mechanisms of Tolerance Induced by Donor-Specific Transfusion and ICOS-B7h Blockade in a Model of CD4+ T-Cell-Mediated Allograft Rejection

The inducible co-stimulatory molecule (ICOS) has been shown to play a critical role in T-cell activation and differentiation, and the regulation of alloimmune responses in vivo. Using an MHC class II mismatched model of CD4(+) T-cell-mediated rejection, we found that treatment of mice with DST and ICOS-B7h blockade induced long-term skin allograft survival and donor-specific transplantation tolerance. ICOS blockade, either during antigen priming or during the effector phase, previously shown to alter the outcome of the immune response, had a similar effect on graft survival. DST and anti-B7h mAb reduced the frequency of IFN-gamma-producing allospecific cells but did not produce deviation to a T(H)2 phenotype. In an adoptive transfer model using ABM TCR transgenic mice directly reactive to I-A(bm12), DST and anti-B7h mAb reduced the number of allospecific CD4(+) T cells and increased CD4(+) T-cell apoptosis. These data demonstrate that DST and anti-B7h mAb induces transplantation tolerance to MHC class II mismatched skin grafts by a reduction of the alloreactive clone size that is, at least in part, dependent on apoptosis of host alloantigen-specific CD4(+) T cells.     

Islet allograft rejection by contact-dependent CD8+ T cells: perforin and FasL play alternate but obligatory roles.

Though CD8(+) T lymphocytes are important cellular mediators of islet allograft rejection, their molecular mechanism of rejection remains unidentified. Surprisingly, while it is generally assumed that CD8(+) T cells require classic cytotoxic mechanisms to kill grafts in vivo, neither perforin nor FasL (CD95L) are required for acute islet allograft rejection. Thus, it is unclear whether such contact-dependent cytotoxic pathways play an essential role in islet rejection. Moreover, both perforin and CD95L have been implicated in playing roles in peripheral tolerance, further obscuring the role of these effector pathways in rejection. Therefore, we determined whether perforin and/or FasL (CD95L) were required by donor MHC-restricted ('direct') CD8(+) T cells to reject islet allografts in vivo. Islet allograft rejection by primed, alloreactive CD8(+) T cells was examined independently of other lymphocyte subpopulations via adoptive transfer studies. Individual disruption of T-cell-derived perforin or allograft Fas expression had limited impact on graft rejection. However, simultaneous disruption of both pathways prevented allograft rejection in most recipients despite the chronic persistence of transferred T cells at the graft site. Thus, while there are clearly multiple cellular pathways of allograft rejection, perforin and FasL comprise alternate and necessary routes of acute CD8(+) T-cell-mediated islet allograft rejection.     

The influence of MHC and non-MHC genes on the nature of murine cardiac allograft rejection. I. Kinetic analysis of mononuclear cell infiltrate and MHC-class I/class II expression in donor tissue

While normal cardiac tissue expresses low levels of MHC-class I, undetectable levels of MHC-class II antigens, and no mononuclear cell infiltrates, posttransplantation allogeneic donor cardiac tissue demonstrates dramatic increases of MHC-class I/class II expression coincident with the infiltration of the tissue with mononuclear cells. Results of this study demonstrate that the kinetics of MHC-class I/II antigen expression and the phenotype of mononuclear cell infiltrate are influenced, to a great degree, by the genetic H-2, intra-H-2 and non-H-2 incompatibility between donor and recipient strains of mice. Increases of MHC-class I precede class II expression in cells from donor cardiac tissue from completely allogeneic BALB/c, H-2-disparate B10.D2, B10.BR, and K, I-A and I-E-disparate B10.T (6R) strains of mice implanted in B10 recipients. In contrast, increase in the level of MHC-class II precedes MHC-class I increases in donor cardiac tissue from H-2-identical but non-H-2-incompatible A.By and the I-E + H-2D end-different B10.A(5R) donor tissue. The completely allogeneic, H-2-disparate or K, I-A, I-E-disparate donor cardiac tissue induced the infiltration of predominantly CD8+ T cells, whereas the non H-2 and I-E + H-2D end-different donor cardiac tissue induced the infiltration of predominantly CD4+ T cells. Finally, whereas bm1 donor cardiac tissue is rejected by B6 recipients by day 32, the (bm1 x bm12)F1 allografts are rejected by day 20, and both express MHC-class I antigens followed by MHC-class II antigens, and contain predominantly CD8+ T cells. In contrast, bm12 allografts are not rejected by B6 recipients, express chronic low levels of both MHC-class I and II antigens, and contain predominantly CD4+ T cells. Of interest is our preliminary finding that bm12 allografts placed in one ear of B6 recipients appear to modify the kinetics of MHC antigen expression and the predominant phenotype of mononuclear cell infiltrates in bm1 allografts placed in the opposite ear. Cumulatively, these data suggest that the type of genetic disparity between cardiac donor and recipient greatly influences the quantitative and qualitative host responses.     

Rejection of cultured thyroid allografts by the transfer of sensitized LYT 2+ T cells

Cultured BALB/c (H-2d) thyroid allografts, which are accepted permanently in fully immunocompetent allogeneic CBA/H (H-2k) recipients, are rejected following the transfer of immune spleen cells generated by immunization of recipient-strain mice with P815 (H-2d) which, like the cultured thyroid, expresses only class I major histocompatibility (MHC) antigens. Depletion of Lyt 2+ T cells from the transferred population abolished the capacity of immune spleen cells to trigger rejection, whereas depletion of L3T4+ T cells was without effect. Transfer of in-vitro-activated H-2d specific Lyt 2+ T cells also triggered rejection of cultured thyroid allografts. The results are consistent with previous studies showing that sensitized Lyt 2+ T cells are required for rejection of cultured pancreatic islet allografts, which also express only class I MHC antigens. These results are in accord with the notion that Lyt 2+ T cells are activated by and are active against class I MHC antigens.     

Expression of CR1/2 receptor on alloantigen-stimulated mouse T cells

Antibodies can mediate injury of organ transplants by several mechanisms, including complement activation and interaction with Fc receptors on cells. We tested the hypothesis that antibodies could also cause up-regulation of complement receptors on cells to increase the responses to complement activation by interaction with split products of C3. In our experimental model, B10.A (H-2(a)) cardiac transplants survive significantly longer in C57BL/6 (H-2(b)) immunoglobulin knockout recipients (IgKO) than in their wild-type counterparts. Passive transfer of specific antibodies to donor MHC class I to IgKO recipients of cardiac allografts at the time coinciding with a vigorous cellular infiltration reconstituted acute rejection. We tested the effects of alloantibodies on CR1/2 expression by alloantigen-stimulated T cells. Both CD4(+)/CR1/2(+) and CD8(+)/CR1/2(+) populations of T cells were expanded in C57BL/6 splenocytes stimulated by B10.A alloantigen in 7-day MLR after coculture with endothelial cells sensitized with IgG1 and IgG2b mAb specific to MHC. Endothelial cells sensitized with antibodies also caused an expansion of CD8(+) T cells expressing CR1/2 in lymph node lymphocytes harvested from a C57BL/6 recipient of a B10.A cardiac allograft. These data suggest that antibodies can augment the cellular rejection process through expanding the population of T cells interacting with complement split products.     

Delayed rejection of MHC class II-disparate skin allografts in mice treated with farnesyltransferase inhibitors

Farnesyltransferase inhibitors (FTIs), developed as anti-cancer drugs, have the potential to modulate immune responses without causing nonspecific immune suppression. We have investigated the possibility that FTIs, by affecting T cell cytokine secretion, can attenuate alloreactive immune responses. The effects of FTIs on murine alloreactive T cells were determined both in vitro, by measuring cytokine secretion or cell proliferation in mixed lymphocyte cultures, and in vivo, by performing skin allografts from H-2(bm12) mice to MHC class II-disparate B6 mice. We found that two different FTIs, ABT-100 and L-744,832, blocked secretion of IFN-gamma, IL-2, IL-4, and TNF-alpha from na?ve T cells in vitro. ABT-100 and L-744,832 blocked cytokine production from both CD4(+) and CD8(+) na?ve T cells stimulated with CD3 and CD28 antibodies, but only if the cells were pretreated with the FTIs for 48 h. Proliferation of alloreactive T cells in mixed lymphocyte cultures was blocked by either FTI. We also found that the proliferation of enriched T cells stimulated with IL-2 was blocked by ABT-100 treatment. In mice with an MHC class II-disparate skin graft, rejection of primary allografts was significantly delayed by treatment with either ABT-100 or L-744,832. Secondary rejection in mice previously primed to the alloantigen was found to be unaffected by L-744,832 treatment. We have shown that FTIs can block T cell cytokine secretion and attenuate alloreactive immune responses. The ability of FTIs to block secretion of cytokines, including IFN-gamma and IL-4, from na?ve T cells provides a likely biological mechanism for the specific suppression of class II MHC-mediated allorejection. These results demonstrate that FTIs may have useful immunomodulatory activity due to their ability to delay priming to alloantigens.     

Syenergistic effect of 15-deoxyspergualin with costimulation blockade on alloimmune response

INTRODUCTION: A virally induced alloreactive memory seems to represent a potent barrier to tolerance induction but the combination of 15-deoxyspergualin (DSG), an inhibitor of NFkB translocation, with costimulation blockade (CB)-based chimerism as an induction regimen can overcome a preformed anti-donor memory response. In this study, we investigate the ability of DSG with CB to inhibit a naive alloimmune responses. METHODS: A BALB/c (H-2d) skin or heart was transplanted into a C57BL/6 (H-2b) recipient treated with anti-CD154 mAb (MR1; 500 mcg/d on days 0, 2, 4, 6) alone, DSG (5 mg/kg/d, days 0 to 7) alone, or both agents. Proliferation of alloreactive T cells after each treatment was also examined using a graft-versus-host disease (GvHD) model using the fluorescent dye CFSE. RESULTS: Treatment with DSG alone induced prolonged survival of the cardiac allografts (median survival time [MST]: 97.5 days). MR1 alone induced indefinite survival of cardiac allografts, although at 150 days after transplantation, the histology showed changes characteristic of chronic rejection, including interstitial fibrosis, infiltration of mononuclear cells, and intimal hyperplasia in coronary vessels. Combined treatment with DSG and MR1 induced donor-specific unresponsiveness in all recipients, graft histology showed only minimal infiltration. Treatment with DSG and MR1 also significantly prolonged the survival of skin allografts (MST: 31 days) compared with that of DSG or MR1 alone (MST: 17 and 14 days, respectively). In the GvHD model assessed with CFSE, the combined treatment was the more effective to suppress proliferation of alloreactive T cells while DSG alone inhibited proliferation more than MR1 alone. CONCLUSION: DSG potentiates anti-CD154 therapy to suppress the alloimmune response.     

Alloantibody Production is Regulated by CD4+ T Cells'' Alloreactive Pathway, Rather Than Precursor Frequency or Th1/Th2 Differentiation

Although CD4(+) T cells play an important role in the regulation of allograft rejection, the exact mechanisms by which they operate and the actual contribution of direct and indirect alloreactivity pathways remain to be fully characterized. Previous studies have established a possible relationship between the indirect alloreactivity pathway and antibody production, but interpretation of these results have been complicated by shortcomings inherent to the models used in these studies. To address this issue, we have developed a model based on TCR transgenic mice derived from a CD4(+) T-cell clone which recognize specific alloantigens by both alloreactivity pathways. Skin allografts on alphabeta T-cell deficient mice adoptively transferred with transgenic CD4(+) T cells were rejected without significant delay between the two alloreactivity pathways. No IgG alloantibody was produced following allograft rejection by the direct alloreactivity pathway alone. Importantly, production of antibodies against alloantigens of the direct pathway was shown to require help from CD4(+) T cells activated by the indirect pathway. These results indicate that the events leading to the initiation of immune responses responsible for graft rejection are clearly dependent on the population of antigen-presenting cells involved in T- and B-lymphocyte activation.     

Alloantigen-induced, T-cell-dependent production of nitric oxide by macrophages infiltrating skin allografts in mice

The immunological rejection reaction occurring after organ or tissue transplantation is characterized by a strong infiltration of the graft by T cells and macrophages. Since the rejection reaction is highly specific, we tested the role of T cells in the activation of macrophages and in the induction of nitric oxide (NO) production during graft rejection. The rejection of both MHC and non-MHC antigen-disparate skin allografts was associated with a significantly increased production of NO in the graft. The kinetics of NO production after transplantation correlated with the rejection reaction and with the fate of the allograft. A significant reduction in NO production was found in immunologically hyporeactive mice treated with cyclosporine, and no specific production of NO was found in tolerated skin allografts from neonatally tolerant mice. The production of NO was completely suppressed in graft explants from mice with depleted CD4(+) cells, but remained at a normal level in skin allografts from mice treated with anti-CD8 monoclonal antibody. The treatment of recipients of fully allogeneic skin grafts with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NO synthase, resulted in a significant prolongation of graft survival. The results thus show CD4(+) T-cell-dependent, alloantigen-induced production of NO by graft-infiltrating macrophages and the role of NO in the rejection reaction. We suggest that this pathway may represent one of the local effector mechanisms of graft rejection.     

Acceptance of Third-Party Cardiac but not Skin Allografts Induced by Neonatal Exposure to Semi-Allogeneic Lymphohematopoietic Cells

Neonatal tolerance is exclusively donor-specific when assessed by skin allograft survival and in vitro alloreactivity assays. In contrast, we reported previously that acceptance of primarily vascularized cardiac allografts was not donor-specific in C3H/He (C3H, H-2(k)) mice treated as neonates with BALB/c-derived (BALB, H-2(d)) lymphohematopoietic cells, but included third-party C57BL/10 (B10, H-2(b)) allografts. The present study examined whether this unusual pattern is limited to heart grafts in this strain combination, and defined the relative importance of the donor cell H-2(d) haplotype for third-party cardiac allograft acceptance. C3H neonates were injected with (C3HxBALB)F1 bone marrow and spleen cells. Tolerance was assessed at age 8-10 weeks by transplantation of heart or skin allografts from several donor strains, and by in vitro assays of proliferation and cytotoxicity. Additionally, cells from H-2(d) and H-2(b)-expressing strains on BALB or non-BALB minor histocompatibility (miH) antigen backgrounds were tested as tolerizing inocula. Prolonged survival of cardiac grafts from all donor strains was observed in neonatally treated mice, whereas skin grafting and in vitro assays demonstrated donor-specific hyporesponsiveness. Both H-2(d) haplotype and non-H-2 miH background of graft donor and tolerizing cell donor were important to third-party cardiac allograft acceptance. These results suggest that the functional alteration in alloreactivity induced by neonatal alloantigen exposure depends partly on method of assessment.     

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4(+) T cells

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A(u), were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20-100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from nai;ve animals. The proliferative response was primarily mediated by CD4(+) T cells recognizing the RT1.A(a) molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4(+) T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.     

Influence of direct and indirect allorecognition pathways on CD4+CD25+ regulatory T-cell function in transplantation

While both direct and indirect allorecognition are involved in allograft rejection, evidence to date suggests that tolerance is primarily dependent on indirect pathway-triggered CD4+CD25+ T cell-mediated immunoregulation. However, the precise influence of these two pathways on CD4+CD25+ T-cell function has not been addressed. In the current study, we have utilized an adoptive transfer model to assess selectively how the absence of either direct or indirect allorecognition affects CD4+CD25+ T-cell function. The effects of the loss of the direct pathway were assessed by transplanting skin grafts from minor histocompatibility mismatched B10.D2 (H-2d) donors onto Balb/c (H-2d) recipients, or by placing bone marrow chimeric DBA/2 (H-2d/H-2b) allografts onto C57BL/6 (H-2b) hosts. The requirement for indirect allorecognition was tested by grafting DBA/2 skin allografts onto either C57BL/6- or MHC-II-deficient C57BL/6 recipients. We report here that although CD4+CD25+ regulatory T cells can suppress both directly and indirectly generated alloresponses, immunoregulation is favored when indirect presentation is the sole mechanism of allorecognition. Hence, in the absence of indirect presentation, net CD4+CD25+ T cell-dependent immunoregulation is weak, and high ratios of CD4+CD25+ to CD4+CD25 T cells are required to ensure graft survival.