Induction of transplantation tolerance in rats by spleen allografts. I. Evidence that rats tolerant of spleen allografts contain two phenotypically distinct T suppressor cells

We have examined suppressor cell activity in transplantation tolerant (TT) rats bearing vascularized spleen allografts in several different donor-recipient combinations. More than 60% of WAG (RT-1u) and 65% of AGUS (RT-1l) spleen allografts were permanently accepted when transplanted to AGUS and PVG (RT-1c) rats, respectively. All (WAG X AGUS)F1 to AGUS and (AGUS X PVG)F1 to PVG spleen allografts survived indefinitely. Unseparated LNC, TDL, and whole T cell or W3/25+, OX8- T cell populations obtained from AGUS rats bearing (WAG X AGUS)F1 spleens exhibited reduced mixed lymphocyte reaction (MLR) responses to the spleen donor, and to some extent to BN(RT1n) third-party stimulators, but responded normally to PVG.A(RT1a) stimulators. Coculture experiments demonstrated that lymph node cells (LNC) and thoracic duct lymphocytes (TDL) of TT rats contain RT1 specific suppressor cells. Furthermore, T cells isolated from all donor-recipient combinations contained two phenotypically distinct suppressor cell populations: a radiosensitive W3/25+, OX8- (Th/i) and a relatively radioresistant W3/25-, OX8+ (Ts/c). These Ts may be responsible for the maintenance of TT.     

The role of T suppressor cells in the maintenance of spontaneously accepted orthotopic rat liver allografts.

Orthotopic liver allografts from BN donors to LEW recipients are spontaneously accepted, and the recipients develop donor-specific immunological unresponsiveness. This unresponsiveness may be mediated by suppressor T cells. Immunomagnetically purified splenic T cells from LEW rats bearing BN liver grafts were shown to adoptively transfer suppression of skin, heart, and kidney graft rejection in a donor-specific manner, prolonging the survival of BN but not third-party DA grafts. However, the suppressor T cells were sessile, being resident in the spleen but not present in thoracic duct lymph. The presence of a nonrecirculating suppressor T cell in rats spontaneously accepting liver transplants is strongly suggestive of an important function in the maintenance of donor-specific unresponsiveness, although the contribution of other possible mechanisms of unresponsiveness has not been investigated.     

The mechanism of the induction of immunologic unresponsiveness to rat cardiac allografts by recipient pretreatment with donor lymphocyte subsets

In continuation of our studies using UV-B-irradiated DST and donor leukocyte (DL) recipient pretreatment to induce specific unresponsiveness to organ allografts, we have examined the relative contributions of splenic lymphocyte populations and T lymphocyte subsets in the induction of immunologic unresponsiveness. Our data show that enriched populations of MHC class II-positive B lymphocytes and the W3/25+ T cell subset obtained from splenic leukocytes using immunoadsorbent columns in conjunction with mAbs led to indefinite graft survival (greater than 100 days) in the Lewis-to-ACI rat cardiac allograft model. In contrast, pretreatment with T lymphocytes or the Ox8+ T subset was relatively ineffective in prolonging cardiac allograft survival. In addition, third-party (W/F) W3/25+ T cell recipient pretreatment did not influence the survival of Lewis cardiac allografts in ACI recipients, thus confirming the specificity of pretreatment with the T cell subset in graft prolongation. Furthermore, we have examined the underlying mechanisms of donor-specific unresponsiveness induced by donor spleen cells, B lymphocytes, and W3/25+ T cells using adoptive transfer assays. Serial adoptive transfer studies demonstrated the presence of 0x8+ suppressor T cells in the spleens of unresponsive recipients bearing well-functioning cardiac allografts and of serum "suppressor factors" that have the capacity for specifically prolonging donor-type test graft survival in naive syngeneic rats. Our findings suggest that the induction of specific unresponsiveness in this model is dependent on a sequential collaboration between the appearance of donor-specific serum factor(s) (humoral phase) and donor-specific suppressor T cells (cellular phase). These results may be potentially useful in planning future strategies for the induction of unresponsiveness to clinical organ allografts by immunologic manipulation of the host with MHC class II-positive B cell and CD4+ T cell clones.     

Induction of transplantation tolerance in rats by spleen allografts. II. Evidence that W3/25+ T suppressor/inducer and OX8+ T suppressor/effector cells are required to mediate specific unresponsiveness

The results presented in this report demonstrate that T cells, isolated from AGUS rats bearing long-term (WAG X AGUS)F1 spleen allografts adoptively transferred to irradiated AGUS recipients could not mediate the rejection of WAG hearts but rejected PVG. A hearts in acute fashion. Further, unresponsive T cells were able to suppress the capacity of adoptively transferred (40 X 10(6) normal T cells to reject WAG but not PVG.A heart allografts. We also studied the role of W3/25+ and OX8+ T cells subsets in the maintenance of unresponsiveness. Isolated W3/25+ or OX8+ unresponsive T cells were not able to mediate acute rejection, but were less effective in prolonging WAG allograft survival than the unresponsive whole T cell population, suggesting that both W3/25+ Ts1 and OX8+ Ts2 subsets were required for effective suppression in vivo. When, however, unresponsive W3/25+ T cells were infused simultaneously with normal OX8+ T cells, they could produce indefinite survival of WAG heart allografts. These results indicate that the unresponsive state induced by (WAG X AGUS)F1 spleen allografts transplanted to AGUS rats is maintained by the interaction of W3/25+ T suppressor/inducer and OX8+ T suppressor/effector cells.     

Induction of suppressor cells by donor-specific blood transfusions and heart transplantation in rats

In the BN/Rij to WAG/Rij rat donor-host combination, a single injection of 1 ml of donor blood 7 days before transplantation leads to permanent acceptance of BN/Rij hearts. In this model of specific unresponsiveness, it was investigated whether suppressor cells were present in the steady-state phase at 5 to 6 weeks after transplantation. Thymocytes, spleen cells, peripheral blood lymphocytes, and lymph node cells from blood-conditioned recipients were adoptively transferred to WAG/Rij recipients irradiated with 450-rad X-rays. BN/Rij heart transplantation was performed after 14 days. It was found that suppressor cells were present in the spleen and thymus of unresponsive recipients but not in the peripheral blood or lymph nodes. Adoptive transfer of 25 x 10(6) spleen cells led to permanent survival of BN/Rij hearts in four of nine cases, whereas transfer of 25 x 10(6) thymocytes always resulted in permanent graft survival. Fractionation of suppressor spleen cells into T and B cell-enriched populations and macrophages revealed that the suppression was mediated by T cells.     

The course of pancreas allografts in rats conditioned by spleen allografts.

A new technique for transplanting duct-ligated rat pancreas grafts, rather similar to the technique for spleen grafting in rats, is presented. Inbred AGUS and WAG rats with a strong Ag-B incompatibility were used. Duct-ligated pancreas AGUS to AGUS isografts survived indefinitely in streptozotocin-induced diabetic hosts while WAG to AGUS allografts were quickly rejected. However, when WAG spleen and pancreas were transplanted en bloc to AGUS rats, endocrine pancreas graft function persisted for up to 6 weeks. This finding of a transient protection of pancreas allografts by donor-strain spleen allografts led to further experiments. AGUS recipients first received WAG spleen allografts which then were removed after 3 to 5 months, at which time WAG pancreas allografts were inserted. Sixty-eight per cent of these grafts survived and cured their hosts of streptozotocin-induced diabetes.     

Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients

Twenty patients with well-functioning kidney grafts from one-haplotype-mismatched related donors, were studied 1-10 years after transplantation (A). Another group of six patients were studied at various times after transplantation (B). The presence of donor-specific transplantation tolerance, using mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) tests was investigated, as well as the possible existence of cells with suppressive activity. All recipients were transfused prior to transplantation and treated with conventional immunosuppression. The patients in group A showed MLC reactivity against donor and third-party cells, indicating a allogeneic response capacity. The CML activity against the donor was low, however, and remained low also following removal of adherent cells. The CML activity toward third-party cells was within the normal range of unmatched individuals. In group B, two of six recipients and high postoperative CML activity against the donor. Both recipients showed clinical signs of rejection. In the remaining four recipients, the antidonor CML reactivity one week after transplantation was lower than the preoperative level. The decrease was even more pronounced at 12 months, although the reactivity against third-party cells was unaltered. The CML reactivity from unrelated fourth-party individuals toward donors was suppressed when cells from recipients with long-term functioning kidneys were added to the cell cultures. The results suggest the presence of a donor-specific cellular suppressor mechanism underlying the donor-specific CML unresponsiveness in recipients with long-term-functioning kidney allografts.     

Induction of donor-specific tolerance to rat cardiac allografts by intrathymic inoculation of bone marrow.

BACKGROUND. Induction of donor-specific tolerance to tissue or organ allografts can readily be achieved by administration of allogeneic bone marrow to neonatal rodents; however, in adult recipients induction of transplantation tolerance by this strategy generally requires intensive cytoablative conditioning. Described here is a novel method of promoting transplantation tolerance that involves inoculation of donor bone marrow into the thymus of transiently immunosuppressed adult recipients. METHODS. Prospective Wistar-Furth recipients were inoculated with allogeneic Lewis bone marrow cells (BMCs) either intrathymically or intravenously in conjunction with a single dose of antilymphocyte serum 2 to 3 weeks before receiving donor-strain cardiac allografts. Recipients were monitored for graft survival and examined for presence of hematopoietic chimerism. RESULTS. Intrathymic but not intravenous inoculation of donor BMCs led to permanent survival of donor-strain cardiac allografts, whereas third-party Dark agouti cardiac allografts were rejected promptly. Persistence of donor chimerism was demonstrated in the thymus of Wistar-Furth recipients of intrathymic Lewis BMCs for as long as 3 weeks after BMC inoculation. CONCLUSIONS. Intrathymic inoculation of BMCs concurrently with a single dose of antilymphocyte serum induces donor-specific unresponsiveness to rat cardiac allografts. The unresponsiveness may be the result of deletion or functional inactivation of alloreactive clones maturing in a thymus bearing donor alloantigen. Intrathymic inoculation of BMCs deserves further evaluation as a possible clinical strategy for the induction of transplantation tolerance.     

Characteristics and function of suppressor T lymphocytes in immunologically unresponsive rats following pretreatment with UV-B-irradiated donor leukocytes and peritransplant cyclosporine

Lewis rats pretreated with UV-B-irradiated donor leukocytes (UV-DL) and peritransplant cyclosporine (CsA...CsA, 20 mg/kg on days 0, +1, and +2) accepted W/F heart allografts permanently. This study of donor-specific immunologic unresponsiveness and its cellular mechanisms shows that the induction phase of unresponsiveness is partially mediated by W3/25+ T cells, while its maintenance is dependent on the presence of 0 x 8+ T cells. In vivo adoptive transfer of either splenocytes, T lymphocytes (T cells), or W3/25+ T cells from ungrafted, UV-DL-transfused rats into unmodified syngeneic Lewis rats that received test grafts 24 hr later led to significant prolongation of donor-specific graft survival. Adoptive transfer of 0 x 8+ cells did not influence donor-type (W/F) test graft rejection. Adoptive transfer of SpL, T cells and 0 x 8+ cells from UV-DL and CsA-treated recipients of W/F heart allografts at 20 or 180 days after transplantation led to significant donor-specific graft prolongation in naive syngeneic hosts, while adoptive transfer of W3/25+ cells, in this group, did not affect test graft survival. However, the adoptive transfer of SpL or of T cell subsets did not influence third-party (ACI) graft survival. In-vitro mixed lymphocyte reaction between thoracic duct lymphocytes obtained at various intervals following grafting from UV-DL and CsA treated Lewis recipients of W/F heart allografts and donor-type SpL resulted in significantly reduced reactivity by 78%, 75%, 69% (P less than 0.001) and 43% (P less than 0.02) compared with controls when responder TDL were obtained at 20, 50, 100, and 180 days after transplantation, respectively. In coculture studies, the MLR response to donor SpL was specifically suppressed by 60%, 57%, 46%, and 50% (P less than 0.01) at 20, 50, 100, and 180 days after transplantation, respectively, compared with controls. These data indicate that the induction of specific unresponsiveness to heart allografts in this model is mediated, in part, initially by the appearance in the host of specific W3/25+ cells either induced or recalled by UV-DL transfusion, and that a stable state of immunologic unresponsiveness is subsequently dependent on the presence of 0 x 8+ suppressor cells.     

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.     

In vitro analysis of T cell-mediated cytotoxicity displayed by rat heart allograft recipients rendered unresponsive by total-lymphoid irradiation and extracted donor antigen

The addition of 3M KCl-extracted donor antigen (HAg) to immunosuppressive therapy with 16 Gy total lymphoid irradiation produces a significantly higher fraction of Wistar-Furth (WFu) recipients displaying indefinite survival of heterotopic buffalo (BUF) heart allografts, namely 80 versus 20%. The experiments presented herein analyzed the direct activity as well as estimated the potential precursor numbers at 1 and 3 months in treated recipients. At 1 month post-TLI/HAg therapy, recipients showed reduced proliferative responses in mixed lymphocyte reactions (MLR) in a specific pattern toward donor but not third-party stimulators. Both TLI/Graft and TLI/HAg/Graft groups showed a higher frequency of BUF antigen-directed T-cytotoxic cells (fTc) than TLI-treated, but nontransplanted, WFu hosts. In addition, the TLI/HAg group alone displayed alloantigen-specific suppressor cells that suppressed the MLR proliferative responses of normal spleen T cells against donor, but not third-party, alloantigens. At 3 months postirradition, both TLI/Graft and TLI/HAg/Graft groups displayed variable MLR proliferative responses toward donor and third-party alloantigens. Whereas nontransplanted, TLI-treated WFu rats recovered their fTc to normal levels at 3 months, the TLI and TLI/HAg treated recipients bearing functional heart allografts demonstrated significantly decreased splenic fTc. These results show that reduced numbers of cytotoxic cell precursors may afford more reliable indices of prolonged heart allograft survival than MLR responses. The observations suggest that TLI/HAg transplant hosts display both reduced cytotoxic precursors and activated suppressor elements.     

Suppressor cell induction in donor-specific transfused mouse heart recipients

Administration of donor-specific blood presenting major or minor foreign histocompatibility antigens to the mouse recipient improved heart allograft survival when a single transfusion of 0.25 ml was given to the recipient prior to transplantation. Multiple transfusions did not prolong allograft survival, which suggested a presensitization effect. In addition, when the single transfusion volume was reduced to 0.025 ml, no significant effect in prolongation of graft survival was observed. Thus the amount of blood transfused seemed to be a critical factor in achieving the transfusion effect. Splenic suppressor cells after transfusion and transplantation (as determined by adoptive transfer) were present during the stable maintenance phase of graft survival in transfused recipients with long-term surviving heart allografts. Also, an intact spleen was required to achieve improved allograft survival in mice transfused with donor-specific blood. Thus a mechanism for the favorable effect of blood transfusion may be the generation of splenic suppressor cells in response to transfusion followed by transplantation.     

Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients.

The study was designed to compare second heart and skin grafts and in vitro assays as a means of assessing peripheral tolerance in C57BL/6 mice. Vascularized heterotopic BALB/c hearts were placed in C57BL/6 recipients treated with anti-CD4, GK1.5 (1 mg total per 20 g mouse i.p. on days 0, 1, 2, 3). Those mice in which hearts survived for >60 days were challenged with donor and third-party (CBA) skin grafts or with second heart grafts, of donor or third-party origin, attached to the carotid artery and jugular vein. In vitro alloreactivity was assessed by mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) using recipient spleen cells. Parenchymal damage, cellular infiltration and vascular disease were assessed from the histology of long-term allografts and isografts. Allografts in untreated recipients were rapidly rejected while isografts survived > 100 days. Primary allografts in anti-CD4 treated recipients also survived > 100 days, as did donor strain secondary heart transplants given at >60 days after the first graft. Third-party hearts were rapidly rejected, as were donor and third-party skin grafts placed on recipients with long-term allografts. These recipients showed low MLR response to both donor and third-party stimulators and donor-specific suppression of CML at 60 days post graft. Long-surviving heart allografts all showed evidence of parenchymal damage and vascular intimal thickening. Thus in the BALB/c to C57BL/6 donor-recipient strain combination, hearts, but not skin grafts, could be used to demonstrate peripheral tolerance, which seemed to be both organ and major histocompatibility complex (MHC) specific. Despite long survival, BALB/c hearts all showed evidence of parenchymal damage and vascular intimal thickening, a sign of chronic rejection.     

Posttransplant infusion of donor-specific blood induces immunological unresponsiveness in rat hepatic allografts

BACKGROUND: We previously reported that pretransplant donor-specific blood transfusion (DST) induces CD45RC-CD4+ T cells, Th2-like effector cells, and prolongs rat hepatic allograft survival. Our study investigated the effects of posttransplant DST on rat hepatic allograft survival. METHODS: Three days after transplantation, LEW (RT1(1)) recipient rats with ACI (RT1a) livers were injected i.v. with freshly heparinized donor-specific blood. The time kinetics of CD45RC-CD4+ and CD45RC+CD4+ T cell subsets in hepatic infiltrates were examined. RESULTS: Posttransplant DST significantly prolonged rat hepatic allograft survival. Interferon (IFN)-gamma, interleukin (IL)-12, and IL-18 mRNA levels in hepatic allografts of untreated recipients were significantly greater than in recipients treated with posttransplant DST. However, hepatic allografts of recipients treated with posttransplant DST showed significantly higher IL-4, IL-10, and transforming growth factor (TGF)-beta mRNA levels than untreated recipients. The ratio of CD45RC-CD4+ T cells to CD45RC+CD4+ T cells was significantly higher in hepatic allografts treated with posttransplant DST than in untreated animals. Immunostaining with anti-rat dendritic cell (OX-62) monoclonal antibody revealed that OX-62+ cells were distributed to the splenic red pulp of animals treated with posttransplant DST and to the splenic white pulp in untreated animals. Most OX62+ cells isolated from the spleen of recipients treated with posttransplant DST expressed donor RT1Ba class II major histocompatibility complex antigens, suggesting that OX-62+ cells were of donor origin. CONCLUSION: Posttransplant DST was associated with persistent infiltration of CD45RC-CD4+ T cells, Th2-like effector cells, in rat hepatic allografts, causing immunologic unresponsiveness and establishment of microchimerism in the spleen.     

Suppressor T cells in rats with prolonged cardiac allograft survival after treatment with cyclosporine

DA rats treated with cyclosporine for 2 weeks after being grafted with an RT1-incompatible PVG heart graft did not reject the graft and developed a state of specific unresponsiveness to graft antigens. The cellular mechanisms maintaining this state of unresponsiveness were studied by testing the capacity of lymphocytes from these animals to effect or inhibit graft rejection in irradiated grafted hosts. Whole lymph node and spleen cell populations, and the T cell subpopulation separated from the latter, failed to restore the rejection of PVG hearts in irradiated DA recipients but restored third-party Wistar-Furth (W/F) rejection. Both whole spleen cells and the splenic T cell subpopulation had the capacity to suppress the ability of normal DA lymphocytes to cause graft rejection. Suppression was not dependent upon a state of chimerism in grafted cyclosporine -treated animals, and was not associated with any measurable alterations in the proportion of cytotoxic/suppressor T cells in lymphoid tissues. These studies show that the state of specific unresponsiveness that follows the treatment of heart grafted rats with cyclosporine is dependent, in part, upon active suppression that is induced or mediated by T lymphocytes. Many features of the immune reactivity of cyclosporine -treated grafted rats support the hypothesis that the mechanism of specific suppression in these animals is akin to that of enhancement, rather than to that of transplantation tolerance induced in neonatal rats.     

Soluble antigen and cyclosporine-induced specific unresponsiveness in rats. Frequency of alloantigen-specific T cytotoxic cells in normal, sensitized, and unresponsive rats

The cellular mechanisms of unresponsiveness induced with a combined KCl-extracted donor antigen (HAg) and CsA regimen were dissected by limiting-dilution (LD) assay. While untreated Wistar-Furth (WFu, RT1u) rats reject Buffalo (BUF, RT1b) heart allografts within a mean survival time of 6.6 +/- 0.5 days, recipients treated with 3 cycles of CsA alone (-1,0,1; 7,8,9; 15,16,17) maintained BUF heart allografts up to an MST of 22.5 +/- 8.9 days. When CsA was combined with BUF HAg (-1), BUF heart survival was further prolonged up to an MST of 34.2 +/- 6.6 days, while third-party BN HAg was ineffective (MST of 21.5 +/- 2.1 days). On day 10 postgrafting, the frequency of T cytotoxic cells (fTc) within the splenic pan-T-cell population was 1:1437 +/- 301 in CsA and 1:1087 +/- 438 in CsA/HAg treated recipients. In contrast, on day 30 postgrafting, both CsA and CsA/HAg treated WFu rats bearing functional BUF hearts showed within their splenic pan-T-cell populations a profound decrease in fTc to 1:2966 +/- 824 with CsA alone and to 1:4946 +/- 938 with CsA/HAg treatment. In contrast, both untreated WFu rats who rejected BUF heart allografts and CsA-treated WFu recipients who had rejected their BUF heart allografts on day 20 displayed an increased fTc to 696 +/- 243 and to 1:1169, respectively, when examined at day 30 postgrafting. Additionally, both the W3/25+ and OX8+ T cell subsets specifically suppressed the proliferative response of normal WFu T cells against BUF and, to a lesser degree, third-party BN irradiated stimulators. Thus, CsA-treated animals develop a potent specific-suppressor mechanism that is augmented by pretreatment with donor soluble antigen. This suppressor activity may decrease the frequency of alloantigen-specific Tc cells and thereby prolong the survival of BUF heart allografts.     

The immunosuppressive action of suppressor cells from antigen-cyclosporine-treated hosts on renal allograft survival

Systemic adoptive transfer was employed to assess the immunosuppressive efficacy of antigen-specific suppressor T (Ts) cells purified from recipients treated with 3M KCl-extracted donor histocompatibility antigen (Ag) and cyclosporine (CsA). Suppressor cells were obtained from Wistar-Furth (WFu, RT-1u) hosts treated with a single i.v. injection of 5 mg 3M KCl-extracted donor Buffalo (Buf, RT-1b) antigen combined with a three-day course of CsA, a group that displays prolonged renal allograft survival (MST 23.2 +/- 10.2 days) compared with animals treated with CsA alone (MST 12.2 +/- 2.4 days). These noncytolytic, OX-8 phenotype, 800-rad-resistant/1500-rad-sensitive, nylon-wool-nonadherent and cyclophosphamide-sensitive suppressor T cells (1 X 10(6)) were adoptively transferred ten days after transplantation into virgin, secondary syngeneic hosts-thereby prolonging Buf graft survival from 7.2 to 17.5 days. The suppressor effect was immunologically specific; adoptive transfer did not prolong the survival of third-party Brown-Norway (BN) grafts (MST 10.4 +/- 3.1 days) compared with the nontreated control group (MST 11.0 +/- 2.9 days). The potency of Ts cells purified from Ag-CsA-treated hosts to transfer unresponsiveness into normal secondary WFu hosts (MST 17.5 +/- 8.0 days) was stronger than that of Ts cells from hosts treated with CsA only (MST 10.6 +/- 2.6 days). Moreover, in vitro stimulation of monoclonal-antibody-purified Ts cells by irradiated donor Buf spleen cells potentiated the in vivo induced suppressor activity, leading to an MST of 38.1 +/- 32.6 days; indeed 3 of 12 animals (25%) displayed permanent unresponsiveness. Furthermore, Ts cells from Ag-CsA-treated hosts displayed a synergistic effect with a three-day course of CsA administration into the secondary hosts (MST 24.2 +/- 8.0 days) compared with animals only treated with CsA (MST 12.2 +/- 2.4 days, P less than 0.001). Moreover, the combination of the Ag-CsA regimen with Ts cells administered one day after transplantation caused even greater prolongation of graft survival (MST 34.2 +/- 14.2 days) compared with Ag-CsA-treated hosts (MST 23.2 +/- 10.2 days, P less than 0.025). Thus adoptively transferred antigen-specific suppressor T cells may be explored to intensify the specific immunosuppressive effect of the Ag-CsA regimen to achieve long-term unresponsiveness.     

Analysis of primed donor-specific T cells in recipient mice bearing orthotopic corneal allografts

BACKGROUND: Orthotopic corneal allografts placed in normal eyes of mice are often not rejected, whereas grafts placed in high-risk (neovascularized) eyes are routinely destroyed. Because rejection of solid tissue allografts is usually mediated by donor-specific T cells, we wished to determine the extent to which donor-specific T cells become primed in mice bearing orthotopic corneal allografts in normal and "high-risk" eyes. METHODS AND RESULTS: Our data indicate corneal allografts placed in neovascularized eyes were rejected within 2 weeks, and lymph nodes draining these grafts contained primed donor-specific T cells that proliferated in vitro and displayed cytotoxic activity. By contrast, only 50% of corneal allografts placed in normal eyes experienced rejection. Lymphoid cells from all of these mice displayed donor-specific proliferative activity, irrespective of whether the graft was accepted or rejected. At no time were donor-specific cytotoxic T cells detected. Failure to detect primed cytotoxic T cells was not the result of anergy or deletion of unprimed donor-specific precursors of CTL. CONCLUSIONS: We conclude that primed donor-specific proliferative and cytotoxic T cells directed at MHC alloantigens correlate well with rejection of orthotopic corneal allografts in neovascularized high-risk eyes. However, rejection of cornea allografts in normal eyes does not correlate well with proliferative T cells, nor are donor MHC-specific cytotoxic T cells detected. The possibility is discussed that graft rejection in normal eyes is not mediated by T cells that recognize MHC alloantigens via the direct pathway, but via T cells that recognize donor alloantigens presented by recipient MHC molecules (indirect pathway).     

Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts

BACKGROUND: We investigated whether intrathymic inoculation of donor bone marrow at the time of transplantation induced long-term acceptance of lung allografts. METHODS: Four- to-six-week-old August Copenhagen Irish (ACI) and Wistar Furth (WF) rats were used as donors and recipients, respectively. After being inoculated intrathymically with either donor-specific (ACI) or third-party (F344) bone marrow (2.0 x 10(7) cells/lobe), the recipient (WF) animal received a left lung transplant from an ACI donor. A short course of tacrolimus (1 mg/kg per day for 5 days) was administered. Animals were sacrificed at timed intervals after transplantation, and rejection was graded on a scale of 0 (none) to 4 (severe). RESULTS: At 28 days, animals receiving donor-specific bone marrow have lower (p < 0.01) median rejection grade (MRG = 0.25; n = 6) than those receiving third-party bone marrow (MRG = 3; n = 6) and controls (no bone marrow; MRG = 2.5; n = 6). Animals receiving intrathymic donor bone marrow accepted lung allografts up to 380 days with minimal rejection (MRG = 2; n = 6). Long-term lung recipients also accepted a challenging donor-specific heart graft (n = 4) for more than 150 days. In mixed lymphocyte reaction assays, T lymphocytes of WF recipients that had received intrathymic bone marrow (from ACI donor) exhibited low response (similar to self antigens) to donor (ACI) cells, but reacted strongly (five times higher) to third-party (F344) cells. CONCLUSIONS: Intrathymic inoculation of donor bone marrow at the time of transplantation along with a short course of tacrolimus induces long-term acceptance of lung allografts in rats. This simple approach of tolerance induction may have clinical application.     

Examination of the mechanisms responsible for tolerance induction after intrathymic inoculation of allogeneic bone marrow.

OBJECTIVE: This study examined the immunologic mechanism(s) responsible for the induction of transplantation tolerance in rats pretreated with intrathymic inoculation of donor strain bone marrow. SUMMARY BACKGROUND DATA: Induction of unresponsiveness may involve deletion and/or inactivation of donor-reactive T-cell precursors maturing in a thymus harboring donor alloantigen or generation of regulatory/suppressor cells. It was reasoned that, if unresponsiveness is caused by deletion of alloreactive clones, the presence of additional thymic tissue devoid of donor alloantigen permits normal maturation of T-cells and, thus, prevents induction of tolerance. However, if unresponsiveness were primarily mediated by regulatory/suppressor cells, the presence of noninoculated thymic tissue should not affect the induction of tolerance. METHODS: Three strategies were used to define the cellular basis of cardiac and islet allograft survival in WF recipients of intrathymic LEW donor bone marrow as follows: (1) inoculation of bone marrow either into the native thymus and/or into an ectopic thymus, (2) limiting dilution analyses of the frequency of precursor cytotoxic T-lymphocytes (CTLp), and (3) adoptive transfer to syngeneic secondary hosts. RESULTS: Inoculation of bone marrow into only one lobe of the native thymus and/or into an ectopic thymus did not promote consistent survival of subsequent LEW cardiac allografts. Tolerant hosts displayed significant reductions in CTLp frequencies against donor alloantigens. Adoptive transfer of spleen cells from tolerant WF hosts harboring long-standing cardiac allografts led to permanent survival of LEW cardiac allografts in all secondary recipients. However, transfer of spleen cells from WF animals that received intrathymic LEW bone marrow (but no cardiac allograft) did not promote survival of LEW cardiac allografts in naive secondary hosts. CONCLUSIONS: These results indicate that the unresponsive state after intrathymic inoculation of bone marrow cells is primarily mediated by deletion and/or inactivation of donor-specific T-cell precursors maturing in a chimeric thymus. The demonstration by adoptive transfer studies of putative regulatory/suppressor cells suggested an important role for the persistence of donor alloantigen (supplied by a vascularized allograft) in the maintenance of the unresponsive state.