Partial characterization of cytotoxic cells infiltrating sponge matrix allografts.

Adapting the Roberts and Hayry sponge allograft model, we have demonstrated the presence of an enriched, specifically cytotoxic population of cells which infiltrate rejecting sponge allografts. The number of cells infiltrating a rejecting sponge allograft peaks on day 14 after transplantation. Utilizing a short-term 51chromium cytotoxicity assay, peak antiallogeneic killing was demonstrable on day 14 also. Only T cell killing was apparent for the first 15 days after transplantation. After day 20, specific cytolysis was present which was not sensitive to anti-theta serum and complement. The infiltrating cytotoxic cells are large, specifically cytotoxic, do not proliferate in culture, do not respond to mitogen, and do not respond in mixed lymphocyte culture even to the same alloantigen to which the animal had been sensitized. In contrast, spleens from sponge-bearing animals kill poorly, respond to mitogen, and respond vigorously in mixed lymphocyte culture to specific and nonspecific alloantigens. The following hypotheses are set forth with regard to the cytotoxic lymphocytes (1) Such cells may be end stage and cannot proliferate. (2) The cytotoxic cells may kill the stimulator cells more rapidly than they can be stimulated to proliferate. (3) The sponge cell population may be enriched for nonspecific supressor cells. (4) The sponge cells may be devoid of helper T cells.     

Suppression of anti-skin-allograft response by photodamaged effector cells--the modulating effects of prednisolone and cyclophosphamide.

Using a murine model of skin allotransplantation, we have demonstrated previously that inhibition of specific response to alloantigen is inducible by immunization of the host with intravenously administered photoinactivated antigraft effector T cells. This hyporesponsiveness, which was demonstrated by specific inhibition of mixed leukocyte culture (MLC), inhibition of cytotoxic T lympholysis (CTL), specific suppression of the delayed type hypersensitivity (DTH) response, and prolongation of specific skin allograft survival, was adoptively transferable by CD8+ radiosensitive T lymphocytes. In this study, we extend those results to evaluate the effects of an immunosuppressive agent (prednisolone) and an alkylating drug (cyclophosphamide) on the induction of this specific suppressive cellular response. Our results reveal that the administration of prednisolone reduces the induction of the specific hyporesponsiveness to alloantigen, as demonstrated by maintenance of the DTH response to alloantigen and continued accelerated rejection of skin allografts. In contrast, the administration of cyclophosphamide augmented this specific suppressive response to alloantigen in the DTH assay and in prolongation of specific skin allograft survival. These results indicate that adjuvant immunomodulating chemotherapy alters the immune response to photoinactivated effector T cells.     

Suppression of the elicitation of the immune response to alloantigen by ultraviolet radiation

Previous studies have established that exposure of mice to ultraviolet radiation followed by injection of alloantigen can suppress the induction of delayed hypersensitivity and the rejection of allografts in an antigen-specific manner. In the clinical situation, however, UV irradiation several days prior to transplantation may prove impractical due to the difficulty in predicting when a donor organ will be available. Thus, the purpose of this study was to determine if exposure to UV radiation can suppress the elicitation of the immune response in mice sensitized with alloantigen. The data demonstrate that exposure of mice to UV radiation 1, 3, or 5 days after the injection of alloantigen can significantly suppress the delayed hypersensitivity response to that alloantigen. Present in the spleens of these mice are suppressor T lymphocytes. These suppressor cells are specific for the antigen originally used to sensitize the mice, in that they do not suppress the response to an irrelevant alloantigen. In addition, spleen cells from mice sensitized with alloantigen and exposed to UV radiation 1, 3, or 5 days later are unable to proliferate in response to the alloantigen in a mixed lymphocyte response. These cells do respond to irrelevant third-party cells, demonstrating again the specificity of the suppression. These data demonstrate that exposure of mice in vivo to UV radiation can inhibit the elicitation of the immune response to alloantigen. Since the immunosuppression is specific for the sensitizing antigen, these data suggest that this may provide a novel method of suppressing the immune response to tissue allografts.     

Comparison of in vivo and in vitro immune response to purified hepatocytes

We have previously reported that freshly isolated percoll-purified hepatocytes are MHC class I+, Class II- and stimulate allospecific cytotoxicity in mixed lymphocyte hepatocyte culture. In this report we determined the immunogenicity of purified hepatocytes in vivo using a modification of the sponge matrix allograft model. We found that hepatocytes were immunogenic in vivo because they stimulated the development of cytolytic effectors in allogeneic but not syngeneic hepatocyte sponge matrix allografts. These cytolytic effectors were not macrophages (because cytotoxicity was intact after nylon wool depletion of the bulk sponge effectors) but rather were Ly2+, L3T4- T cells. Cytolytic effectors in both MLHC and hepatocyte sponge matrix allografts demonstrated marked specificity for allogeneic MHC class I antigen of stimulator and donor hepatocytes, respectively. Allospecific cytolytic T cells developed in both MLHC and hepatocyte sponge matrix allografts across an isolated MHC class I genetic disparity but not across an isolated class II genetic disparity. Finally, sequential phenotypic analysis of host cells infiltrating allogeneic hepatocyte sponge matrix allografts demonstrated the presence of macrophages, Ly2+, L3T4- and Ly2-, L3T4+ T cells.     

The kinetics of tolerance induction by nondepleting anti-CD4 monoclonal antibody (RIB 5/2) plus intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. The monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating the recipient T cells. We have successfully induced tolerance to rat heart allografts by recipient pretreatment with a single dose of RIB 5/2 plus intravenous administration of donor splenocytes. In this study, we explored whether this potent regimen could induce tolerance to the more resistant kidney and skin allografts. Furthermore, we examined the kinetics and requirements for tolerance to be met by a single dose of RIB 5/2 plus i.v. alloantigen. METHODS: The efficacy of a single i.p. dose of 20 mg/kg RIB 5/2 plus i.v. donor antigen (25x10(6) splenocyte) pretreatment 0, 21, or 40 days before receipt of an MHC-mismatched Lewis (RT1l) to Buffalo (RT1b) rat cardiac, renal, or skin allograft was studied. Another group of Buffalo recipients treated with RIB 5/2 plus an i.v. alloantigen +/-thymectomy received kidney transplants after 40 days. Attempts to prevent tolerance used interleukin-2 or prior sensitization. Mixed lymphocyte cultures, cytotoxic assays, and precursor frequencies of helper and cytotoxic cells, by limiting dilution analysis, serially measured in vitro cell-mediated immunity. RESULTS: RIB 5/2 administration combined with i.v. alloantigen 21 days before induced tolerance to heart and kidney allografts but did not prolong skin graft survival. In contrast, kidney allografts delayed for 40 days after pretreatment were acutely rejected and survival was not affected by the thymectomy. MLC, CTL, and pTH, and pCTL precursor frequencies from recipients of long-term grafts were specifically suppressed to donor, but not third party, alloantigen. CONCLUSION: A single dose of the nondepleting anti-CD4 monoclonal antibody, RIB 5/2, plus i.v. alloantigen is a potent inducer of tolerance to heart and kidney, but not skin, allografts. The RIB 5/2-induced donor unresponsiveness to a delayed kidney or cardiac allograft is time dependent but can be prolonged if specific alloantigen is present. Suppression of cell-mediated allo-immune responsiveness correlates with allograft acceptance.     

Allograft tolerance induction in adult mice associated with functional deletion of specific CTL precursors

Rejection of H-2 class I bm 1 mutant skin allografts by B6 recipient mice is mediated by a population of CD8+ anti-bm1 cytotoxic T-lymphocytes that produces and consumes its own T helper factor in response to bm1 skin allografts (dual function CTL). Previously we have demonstrated that transfusion of allogeneic lymphocytes across an H-2 class I disparity induces specific long-lasting skin allograft survival. We now show that intravenous injection of allogeneic spleen cells across the bm1 mutant disparity results in a temporary decrease of donor-reactive CTLp in the spleen of recipient mice, lasting for approximately five weeks. The sponge matrix allograft model was used to show that allograft tolerance is caused by a specific functional clonal deletion of CTLp within the allograft. We propose that dual function CTL are vetoed by donor T cells, resulting in skin allograft tolerance.     

Specific suppression of allograft rejection after treatment of recipient mice with ultraviolet radiation and allogeneic spleen cells

Sensitization of UV-irradiated mice with alloantigen results in suppression of the immune response against that antigen. The induction of delayed hypersensitivity and the proliferative response of spleen cells isolated from the UV-irradiated mice are significantly suppressed. We demonstrate here that the rejection of tissue allografts is also suppressed after treatment of the recipient animals with UVR. Two signals, UVR and antigenic sensitization are required to suppress allograft rejection, exposure to UVR alone is not sufficient. The resulting immunosuppression is specific for the antigen used to sensitize the UV-irradiated animal. The induction of graft-versus-host disease was also significantly suppressed when spleen cells from UV-irradiated alloantigen sensitized mice were used to reconstitute lethally irradiated allogeneic mice. Our interpretation of these data is that the antigen-specific suppressor T cells present in the spleens of the UV-irradiated alloantigen sensitized mice are suppressing the immune response against the alloantigen. Thus, the induction of suppression by UVR serves as a novel method of suppressing graft rejection.     

Prevention of the induction of allospecific cytotoxic T lymphocyte and delayed-type hypersensitivity responses by ultraviolet irradiation of corneal allografts

The effect of ultraviolet radiation (UVR) on the immunogenicity of corneal allografts was examined in a mouse model. Corneal allografts differing from the host at the entire MHC and multiple minor H loci were subjected to 200 mJ/cm2 of UVB irradiation immediately prior to heterotropic transplantation. Analysis of cytotoxic T lymphocyte and delayed-type hypersensitivity responses revealed that UVR treated corneal grafts failed to induce either CTL or DTH responses in C57BL/6 recipients. UVB treatment abolished the immunogenicity of highly immunogenic corneal grafts containing either resident or infiltrating donor-specific Langerhans cells. Sequential grafting experiments demonstrated that UVB-treated grafts rendered the hosts anergic to subsequent immunization with highly immunogenic corneal limbus grafts that contained dense concentrations of Ia+ Langerhans cells of donor origin. The results indicate that UV treatment not only reduces the immunogenicity of the corneal allograft but may also render it tolerogenic.     

Comparative immunohistologic studies in an adoptive transfer model of acute rat cardiac allograft rejection

It has been shown that fulminant acute rejection of rat cardiac allografts across a full haplotype disparity may occur as a direct result of adoptive transfer of sensitized W3/25+ MRC OX8- SIg- T helper/DTH syngeneic spleen cells to sublethally irradiated recipients. In order to establish the immunohistologic parameters of this form of rejection, allografts and recipient lymphoid tissue were analyzed using a panel of monoclonal antibodies of known cellular distribution. These data were compared with those obtained following reconstitution of irradiated allograft recipients with unseparated sensitized spleen cells, with unreconstituted irradiated donor recipient pairs, with unmodified first-set rejection, and with induced myocardial infarction of syngeneic heart grafts transplanted to normal and to sublethally irradiated recipients. Rejecting cardiac allografts transplanted to all reconstituted irradiated recipients were characterized by extensive infiltration with MRC OX8+ (T cytotoxic-suppressor, natural killer) cells even when this subset was virtually excluded from the reconstituting inocula. A similar proportional accumulation of MRC OX8+ cells observed at the infarct margins of syngeneic heart grafts transplanted to irradiated unreconstituted recipients greatly exceeded that present in normal nonirradiated controls. These data provide evidence that under conditions of heavy recipient irradiation, MRC OX8+ cells may be sequestered within heart grafts in response to nonspecific injury unrelated to the rejection process. Although there was no significant degree of MRC OX8+ cellular repopulation within organized secondary lymphoid tissues of irradiated animals over the study period, the density of ileal mucosal MRC OX8+ lymphocytes approximated normal at 7 days post-irradiation, raising the possibility that these cells could share a common origin with those sequestered within the heart grafts. Carbon+ MRC OX6+ macrophages were a significant component of the infiltrate in all rejecting cardiac allografts, as well as in all infarcted syngeneic heart grafts--providing further evidence that macrophage "activation" with expression of class II determinants may occur in response to nonspecific injury. In unmodified first-set rejection there was an intense B cell reaction in recipient spleens and lymph nodes. In the adoptive transfer model, marked B cell expansion was exclusively confined to the parathymic lymph nodes of irradiated allograft recipients reconstituted with the sensitized W3/25+, MRC OX8-, SIg- T helper/DTH donor cell inocula.(ABSTRACT TRUNCATED AT 400 WORDS)     

Limiting dilution analysis for alloreactive, TCGF-secretory T cells. Two related LDA methods that discriminate between unstimulated precursor T cells and in vivo-alloactivated T cells

We have developed a rapid limiting dilution analysis technique for quantitating alloantigen-specific TCGF-secretory T lymphocytes (operationally defined as helper T lymphocytes or HTL) in murine lymphoid populations. A simple permutation of this technique allows the distinction between HTL that have encountered antigen in vivo and the large population of naive precursor HTL (pHTL) with the same alloantigen specificity. We present evidence to validate these LDA techniques, and show that lymphoid tissue from mice bearing sponge matrix allografts, but not isografts, contain HTL that have been activated in vivo. In addition, we demonstrate a requirement for restimulation with alloantigen for secretion of lymphokine by the in vivo-activated cells. We suggest that this differential LDA technique could serve as a valuable tool in evaluating in vivo immunity and/or the in vivo efficacy of immunosuppressive, as well as immunopotentiating, therapies.     

Detection of minor alloantigen-specific cytotoxic T cells after rejection of murine orthotopic corneal allografts: evidence that graft antigens are recognized exclusively via the "indirect pathway".

BACKGROUND: The immune mechanisms by which corneal allografts are rejected in normal ocular graft beds have not been identified. Both acceptors and rejectors of these types of grafts display donor-specific delayed hypersensitivity and in vitro proliferating primed T cells, yet neither develop conventional, donor-specific cytotoxic T cells. We wished to determine whether unconventional donor-specific cytotoxic T cells are generated in rejector mice that recognize donor minor alloantigens presented by recipient major histocompatibility complex (MHC) molecules. METHODS: BALB/c mice received orthotopic corneal allografts from C57BL/10 donors in normal eyes. At 4 weeks (when 50% of grafts can be designated as rejected), primed cytotoxic T lymphocyte (CTL) activity in draining lymph nodes and spleen was assayed on targets selected to present donor-type minor H antigens on recipient MHC molecules. Control mice received heterotopic corneal allografts and were similarly examined. RESULTS: Lymphoid organs of recipients that rejected orthotopic or heterotopic corneal allografts contained CTL that lysed targets expressing donor-type minor H antigens presented by recipient MHC molecules. By contrast, no CTL activity was detected from lymphoid cells of recipients that accepted orthotopic corneal allografts. Rejection of orthotopic corneal allografts placed into normal mouse eyes correlates directly with the generation of donor-specific CTL that recognize minor H antigens in the context of recipients MHC molecules. CONCLUSIONS: These results indicate the indirect pathway of alloantigen presentation is the only pathway operative in the process by which orthotopic corneal allografts are rejected. The roles of emigrant Langerhans cells and corneal lymphatics in the indirect pathway are discussed.     

Impairment of allograft tumor immunity by isotype-like suppression of antigen-specific T cell factors

PCl-F is an antigen-binding factor that is derived from T cells of picryl chloride (PCl) contact-sensitized mice. Intravenous transfer of PCl-F into naive recipients, followed immediately by PCl challenge, results in the ability to elicit an immediate hypersensitivity-like cutaneous response. This PCl-F-dependent early response is an obligatory step in the mediation of a PCl-specific delayed-type hypersensitivity reaction by late-acting, antigen/MHC-restricted effector T cells. These latter cells recruit a local infiltrate of inflammatory cells by producing chemoattractant lymphokines. Injection of PCl-F also induces a T cell-dependent feedback circuit that ultimately suppresses production of PCl-F, and thus suppresses DTH to PCl. This form of regulation is not antigen-specific, since PCl-F induces suppression of DTH to PCl and to other antigens via suppression of the production of antigen-binding T cell factors necessary for initiation of DTH. This form of regulation does not affect classic, late-acting, lymphokine-producing effector T cells of DTH, or helper T cells for antibody responses. We now report that injection of PCl-F is also capable of inducing suppression of cutaneous hypersensitivity responses to allogeneic and syngeneic tumor cells, and of immune resistance to an allogeneic tumor graft. Our results suggest, therefore, that antigen (tumor)-specific T cell factors play a role in initiation of hypersensitivity responses to tumor cells. Injection of PCl-F suppressed, besides tumor-specific cutaneous hypersensitivity, production of the tumor-specific T cell factor that renders macrophages cytotoxic to tumor cells (i.e., specific macrophage-arming factor or SMAF). Thus, PCl-F injection may impair immune resistance to tumor cells by suppressing initiation of hypersensitivity responses that recruit macrophages, and also by inhibiting production of SMAF that renders macrophages cytotoxic. It is therefore tempting to conclude that the antigen-specific T cell factors that initiate DTH, such as PCl-F and SMAF, belong to the same isotype or group of antigen-specific T cell products that can be regulated by a form of feedback suppression that is isotype-like and inhibits production of these related, antigen-specific T cell factors.     

Immunohistochemical analyses of skin graft rejection in mice. Kinetics of lymphocyte infiltration in grafts of limited immunogenetic disparity

We have examined the nature and kinetics of T cell infiltration into murine first-set skin allografts across a variety of histocompatibility differences. Both Ly 1+2- and Ly 2+ cells migrate into grafts that differ with respect to discrete class I, class II, or minor H barriers. The lymphoid cells in the allograft did not express MEL 14 or the B cell antigen B220. The cells in the graft progress from the vascular anastomosis region into the dermis with apparent homing and clustering about hair follicles and epithelial cells at the dermoepidermal junction. Microscopic necroses appear in a patchy distribution in these areas. The intensity of the infiltrate is related to the nature of the alloantigen recognized but it does not correlate with the tempo of rejection. Class II-disparate allografts evoke a peak response (mean 62 +/- 34 T cells/0.1 mm2) that is 2-6-fold greater than class I-disparate allografts or syngeneic control peak responses (mean 14 +/- 2.4 T cells/0.1 mm2). Although a difference at a minor H antigen evoked an intense peak T cell response (192 +/- 33 T cells/0.1 mm2), the mean survival time of the allografts was significantly longer than that observed with class I or class II differences. The IL-2 receptor+ subset of activated T cells is extremely rare in syngeneic grafts, but ranges from 0 to 56% of infiltrating T cells in allografts. We conclude that all major T cell subsets contribute to the in situ response to all classes of alloantigens, but at the apparent exclusion of the MEL 14+ T cell pool and B cells.     

Participation of the liver in generation of a vigorous anti-donor response after inoculation of donor spleen cells.

BACKGROUND: There is a general agreement that a preferential accumulation of alloantigens within the liver could induce hyporesponsiveness to the inoculated antigens. Entrapment of antigens in the liver may evoke an unique immune response in the organ and play a key role in determination of the fate of the transplanted grafts. To understand the immune response in the liver after inoculation of allogeneic donor antigens, we examined the immune response to systemically inoculated alloantigen in rats whose sensitized liver was replaced with that of naive rats or in naive rats whose liver was replaced with that of sensitized rats. METHODS: Using implantation of syngeneic liver (alloantigen-accumulated/naive) in rats (naive/alloantigen-sensitized), we compared the immune responses to alloantigen between rats with hepatic/extrahepatic alloantigen at 24 hr after alloantigen inoculation. This was called sensitized-liver-grafted (SLG)/sensitized-liver-removed (SLR) treatment. The immune response to donor alloantigen in this model was evaluated by survival of skin or heart grafts, complement-dependent cytotoxicity (CDC) titer and delayed-type hypersensitivity (DTH) response. RESULTS: Compared with the mean survival time (MST) in donor spleen cell inoculated (DSI) rats (skin and heart, MST: 8.2+/-1.1 and 10.7+/-2.3 days), SLG rats rejected allografts in an accelerated fashion (skin and heart, MST: 5.5+/-0.5 and 4.2+/-0.8 days), associated with higher CDC titer and DTH response. In contrast, allograft survival was moderately prolonged in SLR (skin and heart, MST: 16.5+/-2.6 and 29.5+/-3.7 days) associated with suppressed CDC titer and DTH response. The survival of third-party allograft after SLG or SLR treatment (skin, MST: 9.3+/-1.5 or 9.7+/-0.6 days) indicated that immunological hyper/hyporesponsiveness was donor-specific. CONCLUSIONS: A strong anti-donor immune response was induced by the transfer of donor antigen-baring liver to naive rats 24 hr after alloantigen inoculation, whereas removal of the liver suppressed alloimmune response. Our results indicate that vigorous anti-alloimmune response occurred in the liver after systemic inoculation of donor spleen cells.     

Allograft Acceptance Despite Differential Strain-Specific Induction of TGF-β/IL-10-Mediated Immunoregulation

We examined the immune approaches that C57BI/6 and BALB/c mice take when treated to accept cardiac allografts. C57BI/6 mice accept DBA/2 cardiac allografts when treated with gallium nitrate (GN) or anti-CD40L mAb (MR1). These allograft acceptor mice fail to mount donor-reactive delayed type hypersensitivity (DTH) responses, and develop a donor-induced immunoregulatory mechanism that inhibits DTH responses. In contrast, BALB/c mice accept C57BI/6 cardiac allografts when treated with MR1 but not with GN. These allograft acceptor mice display modest donor-reactive DTH responses, and do not develop donor-induced immune regulation of DTH responses. Real-time PCR analysis of rejecting graft tissues demonstrated no strain-related skewing in the production of cytokines mRNAs. In related studies, C57BI/6 recipients of cytokine and alloantigen educated syngeneic peritoneal exudate cells (PECs) failed to mount DTH responses to the alloantigens unless neutralizing antibodies to transforming growth factor-beta (TGF-p were present at the DTH site demonstrating regulation of cell-mediated alloimmune responses. In contrast, BALB/c recipients of cytokine-and alloantigen-educated PECs expressed strong DTH responses to alloantigens demonstrating a lack of regulated alloimmunity. In conclusion, C57BI/6 mice respond to immunosuppression by accepting cardiac allografts and generating TGF-beta-related regulation of donor-reactive T cell responses, unlike BALB/c mice that do not generate these regulatory responses yet still can accept cardiac allografts.     

Characteristics of rejection of orthotopic corneal allografts in the rat

We have employed a rat model of orthotopic corneal transplantation to study the characteristics of rejection and development of systemic immunity in the host. Lewis (LEW) rats underwent a true penetrating keratoplasty using Wistar-Furth (WF) donor corneas. A rejection incidence of 55% with a mean survival time (MST) of 17.1 days was observed using these untreated allogeneic corneas. Animals undergoing rejection of these allografts developed cytotoxic T lymphocytes (CTL) capable of lysing WF lymphoblasts in a standard 51-chromium release assay. These same rats did not have delayed-type hypersensitivity (DTH) responses when compared to skin grafted controls. Rats with clear allografts had no demonstrable CTL or DTH activity. As expected, LEW rats that were preimmunized with WF skin grafts and subsequently received WF orthotopic corneal grafts rejected 100% of these corneas at an accelerated rate (MST = 9.7 days, P less than .02). We then employed a previously described technique of using latex beads to induce migration of Langerhans cells into the central cornea of the donor graft prior to transplantation. The presence of Langerhans cells in the donor cornea resulted in a higher incidence of rejection (96%) and an accelerated rate (MST = 11.8 days, P less than .02) when compared to untreated allografts. These rats also had a higher level of CTL activity and marked DTH responses. These data show that rejection of orthotopic allogeneic corneas is accompanied by the development of systemic alloimmunity as measured by CTL activity. However, these fully allogeneic corneas can be rejected in the absence of DTH responses. Langerhans cells have a dramatic effect on graft survival and are necessary for induction of DTH responsiveness in the host.     

Differential expression of helper versus effector activity in mice rendered neonatally tolerant of class II MHC antigens

Since tolerogen-specific helper activity is present in MLR-positive class II MHC tolerant mice, a loss of helper activity is unlikely to be responsible for the maintenance of tolerance in these mice. An alternative hypothesis, that effector cell function is selectively down-regulated, has been examined with lymphocytes from MLR-positive class-II MHC tolerant mice on both the A strain and the B10 background. The results demonstrate that lymphocytes from A-strain-tolerant mice were unable to generate tolerogen-specific effector cells in any of the assays tested (CML with or without exogenous growth factor and DTH following in vivo priming or local adoptive transfer), even though these mice possess tolerogen-responsive T helper cells. In contrast, a majority of MLR-positive tolerant mice on the B10 background generated measurable tolerogen-specific cytotoxic activity in the absence of exogenous growth factor, and all the mice examined generated substantial cytotoxic activity in the presence of exogenous growth factor. However, in a local adoptive transfer reaction, lymphocytes from these mice failed to display DTH. It is concluded that tolerance is maintained by selective impairment of class II specific effector functions and that regulation of DTH rather than CTL activity may be central to maintenance of in vivo tolerance to class II MHC antigens.     

Immune mechanisms in organ allograft rejection. VI. Delayed-type hypersensitivity and lymphotoxin in experimental renal allograft rejection

The cellular requirements for renal allograft rejection have been reassessed in a rat adoptive transfer model, preceding studies having shown that transplanted kidneys may be rejected in the absence of cytotoxic T cells or specific antibody. Unilaterally nephrectomized, sublethally irradiated (780 rads) LEW recipients of renal allografts from irradiated WF donors, were selectively reconstituted with spleen cells from sensitized syngeneic donors and subjected to delayed nephrectomy of the residual native kidney 3 days posttransplantation. In some experiments the reconstituting inocula were depleted of SIg+ cells (anti-Ig column) or additionally depleted of cytotoxic T cells and their precursors reactive with monoclonal OX8 (rosette depletion). Depleting the reconstituting inocula of SIg+ cells as well as cells reactive with monoclonal OX8 failed (n = 4) to alter the tempo of rejection, as demonstrated by a mean serum creatinine +/- SD on day 8 of 5.4 +/- 3.8 vs. 6.4 +/- 4.2 in recipients (n = 8) reconstituted with unfractionated inocula. These data support a link between DTH and graft rejection, so additional studies were performed to characterize rat lymphotoxin (LT), one of the potential mediators of DTH-induced tissue injury, and to demonstrate the presence of LT in rejecting rat renal allografts. Rat LT, generated in vitro by stimulating spleen cells from specifically sensitized rats with keyhole limpet hemocyanin (100 micrograms m/ml), was shown on gel filtration to have an MW of approximately 50,000. In-vitro-generated rat LT was shown to be heat stable (70 degrees C for 15 min) and soluble in 40% (NH4)2SO4. Rat LT eluted as a single peak on DEAE anion exchange chromatography (0-0.15 M, NaCl osmotic gradient), supporting the existence of but a single molecular form. LT was isolated from rejecting renal allografts on day 6 after renal transplant but undetected (less than 1 unit) in residual native kidneys. This study, therefore, provides substantial support for a link between DTH and renal allograft rejection. Lymphotoxin, one of the potential mediators of tissue injury in this model system, has been partially characterized and demonstrated to be present in rejecting rat renal allografts.     

The role of T4+ and Ly-2+ cells in skin graft rejection in the mouse

The role of the different T cell subsets (T4+ or Ly-2+) cells in the rejection of murine skin grafts was examined using the in vivo injection of monoclonal antibodies. The rat antimouse monoclonal antibodies used were shown to be active in vivo and to successfully deplete T4+ or Ly-2+ cells. H-2 mutant strains were used so that class I or class II MHC antigens could be studied in isolation; non-H-2 antigenic differences were also examined. The results were clear: (A) for class I antigens, Ly-2+ but not T4+ cells caused rejection; (B) for class II antigens, T4+ and Ly-2+ cells were both separately involved (additive studies indicated the unlikely involvement of Ly-2+T4+ cells); (C) for non-H-2 antigens, T4+ and not Ly-2+ cells were involved. In sensitized mice only anti-T4 antibodies caused any prolongation--for a class II difference. Our results relate strictly to phenotype and we have not measured cytotoxic or delayed-type hypersensitivity (DTH) (or IL-2 secretion) of cells in the graft causing rejection; nonetheless for class II antigens, anti-T4 treated mice had impaired in vivo DTH responses and the mediator of the DTH response to major histocompatibility complex (MHC) antigens is T4+; for class I antigens, DTH responses were not easily measured but mice with depleted DTH response were still able to reject grafts with a class I antigenic difference. The studies show that Ly-2+ cells, alone, can lead to graft rejection (for class I antigens) and T4+ cells alone (for non-H-2 antigens) can do the same. For class II antigens, T4+ cells are likely to influence the generation of Ly-2+ effector cells, although the precise mechanism is not clear. The studies therefore show that there is no special cell surface phenotype associated with graft rejection; rather the antigens presented dictate which cells are involved. How these cells function in the graft has yet to be demonstrated.     

Demonstration of local immunosuppression with methylprednisolone in the sponge matrix allograft model

This study evaluated the effect of locally delivered methylprednisolone on the systemic and local immune response in the sponge matrix allograft model. Polyurethane sponges were coated with peritoneal exudate cells from DBA/2 (H-2d) or C57Bl/6 mice (H-2b) and placed subcutaneously in C57BL/6 recipients 24 hr later. Each mouse received both a syngeneic and an allogeneic sponge graft. Local immunosuppression was effected by placement on day 0 of cellulose/matrix pellets containing a preparation of increasing quantity of controlled release MP or by daily intrasponge injection of MP. Local immunosuppression was demonstrated by decreased cytotoxicity on day 12 in the allogeneic sponge receiving MP directly, as compared with the groups that received MP in the opposite syngeneic sponge or no MP. This effect was noted at a specific MP dose (0.5 mg/kg/day). Absolute numbers of precursor cytolytic cells and mature cytolytic cells (determined by limiting dilution analyses) infiltrating the allografts were also decreased in the sponges receiving MP directly, relative to the groups receiving MP in the opposite syngeneic sponge. Maintenance of systemic (in contrast to local) immunity was also demonstrated. Animals treated with local MP into a simultaneously placed syngeneic sponge or in whom no MP was delivered became sensitized to a synchronous DBA/2 sponge and rejected a subsequent DBA/2 skin graft in second-set fashion. Conversely, animals that received local MP into their synchronous DBA/2 sponge rejected a subsequent DBA/2 skin graft or a third-party graft with first-set kinetics. The presence of local MP at the initial graft site prevented the animals from becoming sensitized to the presented alloantigen but did not keep the animal from developing a rejection response to a third-party skin graft with first-set kinetics. It appears that delivery of MP locally to the site of antigen is an effective method to modulate alloreactivity. In addition, the sponge matrix allograft appears to be a useful model for studying local immunosuppression.