Inhibition of graft-versus-host disease by double-negative regulatory T cells

Pretransplant infusion of lymphocytes that express a single allogeneic MHC class I Ag has been shown to induce tolerance to skin and heart allografts that express the same alloantigens. In this study, we demonstrate that reconstitution of immunoincompetent mice with spleen cells from MHC class I L(d)-mismatched donors does not cause graft-vs-host disease (GVHD). Recipient mice become tolerant to skin allografts of lymphocyte donor origin while retaining immunity to third-party alloantigens. The mechanism involves donor-derived CD3(+)CD4(-)CD8(-) double-negative T regulatory (DN Treg) cells, which greatly increase and form the majority of T lymphocytes in the spleen of recipient mice. DN Treg cells isolated from tolerant recipient mice can suppress the proliferation of syngeneic antihost CD8(+) T cells in vitro. Furthermore, we demonstrate that DN Treg cells can be generated in vitro by stimulating them with MHC class I L(d)-mismatched lymphocytes. These in vitro generated L(d)-specific DN Treg cells are able to down-regulate the activity of antihost CD8(+) T cells in vitro by directly killing activated CD8(+) T cells. Moreover, infusing in vitro generated L(d)-mismatched DN Treg cells prevented the development of GVHD caused by allogeneic CD8(+) T cells. Together these data demonstrate that infusion of single MHC class I locus-mismatched lymphocytes may induce donor-specific transplantation tolerance through activation of DN Treg cells, which can suppress antihost CD8(+) T cells and prevent the development of GVHD. This finding indicates that using single class I locus-mismatched grafts may be a viable alternative to using fully matched grafts in bone marrow transplantation.     

Roles of deletion and regulation in creating mixed chimerism and allograft tolerance using a nonlymphoablative irradiation-free protocol

The induction of mixed chimerism (MC) is a powerful and effective means to achieve transplantation tolerance in rodent models. Host conditioning with irradiation or cytotoxic drugs has been used in many protocols for chimeric induction across allogeneic barriers. The deletion of alloreactive T cell clones has been described as the main mechanism responsible for the induction of a stable MC. In this study, we demonstrate that a stable MC and skin allograft tolerance can be established across MHC barriers by a noncytotoxic, irradiation-free approach using costimulation blockade plus rapamycin treatment. By using an adoptive transfer model of skin allograft and using specific Vbeta TCR probes, we demonstrated that deletion of donor-reactive cytopathic T cell clones is indeed profound in tolerant hosts. Nonetheless, the challenge of tolerant mixed chimeras with 5 million mononuclear leukocytes (MNL) from naive syngeneic mice was neither able to abolish the stable MC nor to trigger skin allograft rejection, a hallmark of peripheral, not central tolerance. Furthermore, in an adoptive transfer model, MNLs harvested from tolerant hosts significantly inhibited the capacity of naive MNLs to reject same donor, but not third-party, skin allografts. Moreover, when we transplanted skin allografts from stable tolerant chimeras onto syngeneic immune-incompetent mice, graft-infiltrating T cells migrated from the graft site, expanded in the new host, and protected allografts from acute rejection by naive syngeneic MNLs. In this model, both deletional and immunoregulatory mechanisms are active during the induction and/or maintenance of allograft tolerance through creation of MC using a potentially clinically applicable regimen.     

Differential expression of Smad7 transcripts identifies the CD4+CD45RChigh regulatory T cells that mediate type V collagen-induced tolerance to lung allografts

Regulatory T cells (Tregs) induced by oral tolerance may suppress immunity by production of TGF-beta that could also enhance Treg activity. However, all cells that are phenotypically Tregs in rats (CD4(+)CD45RC(high)-RC(high)) may not have regulatory function. Because Smad7 expression in T cells is associated with inflammation and autoimmunity, then lack of Smad7 may identify those cells that function as Tregs. We reported that feeding type V collagen (col(V)) to WKY rats (RT1(l)) induces oral tolerance to lung allografts (F344-RT1(lvl)) by T cells that produce TGF-beta. The purpose of the current study was to identify the Tregs that mediate col(V)-induced tolerance, and determine Smad7 expression in these cells. RC(high) cells from tolerant rats were unresponsive to allogeneic stimulation and abrogated rejection after adoptive transfer. In contrast, CD4(+)CD45RC(low) (RC(low)) cells from tolerant rats and RC(high) or RC(low) cells from normal rats or untreated allograft recipients proliferated vigorously in response to donor Ags, and did not suppress rejection after adoptive transfer. TGF-beta enhanced proliferation in response to col(V) presented to tolerant RC(high), but not other cells. In contrast to other cells, only RC(high) cells from tolerant rats did not express Smad7. Collectively, these data show that the Tregs that mediate col(V)-induced tolerance to lung allografts do not express SMAD7 and, therefore, are permissive to TGF-beta-mediated signaling.     

3H-uridine incorporation by small lymphocytes of tolerant rats: relationship to T and B lymphocytes.

Tissue tolerance was induced in neonatal rats by the intravenous injection of bone marrow cells from adult allogeneic rat donors. After 6 to 8 weeks, lymphoid cells from rats in which tolerance had been induced were tested for mixed lymphocyte reactivity (MLR), 3H-uridine uptake, and the relationship of uridine incorporation to B and T lymphocytes. Lymph node (LN) and spleen (SPL) cells from the adult inoculated rats showed no reactivity in the MLR or normal lymphocyte transfer reaction (NLTRx), indicating that the animals were tolerant. After in vitro exposure to 3H-uridine, an abundance of small lymphocytes (SL) from these same tolerant rats were heavily labeled, in contrast to nontolerant controls, where relatively few SL were heavily labeled. In order to determine whether the heavily uridine-labeled cells were T cells or B cells, lymphoid cells from the LN and SPL of tolerant animals were exposed to either rabbit anti-AKR brain serum or rabbit anti-rat Ig conjugated with ferritin. The results showed that the heavily uridine-labeled SL of the tolerant rats were mainly Ig-positive cells.     

Induction of permanent mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 x 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 x 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE-) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vbeta11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 x 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).     

In vitro induction of immunological tolerance

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.     

Generation of H-2-reactive T cell lines that bear the 5936 idiotype(s)

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.     

Allo-I-J determinants participate in maintenance of neonatal H-2 tolerance

To evaluate the role of IJ antigens in maintenance of the tolerant state in adult H-2 tolerant mice, we have attempted to abolish tolerance by injecting monoclonal antibodies (mab) specific for host, donor, or third party IJ antigens into adult H-2 tolerant mice. Abolition of tolerance was evidenced by the rejection of fresh test skin grafts bearing the tolerated antigens. Whole H-2 tolerant mice treated with anti-IJ mab specific for donor (allo) IJ antigens rejected their test skin grafts, indicating that tolerance had been abolished. When two other types of tolerant mice were tested, we found that mice tolerant of class II antigens alone, but not mice tolerant of an IJ thru D disparity, were susceptible to the anti-donor IJ mab treatment. In addition, adult tolerant mice were unaffected by treatment with either anti-host or anti-third party IJ mab. When tested in vitro, lymphoid cells from tolerant mice, the tolerance of which was abolished by anti-IJ mab, remained unresponsive to the tolerogen, just as untreated (control) tolerant mice, in several in vitro assays (e.g., mixed lymphocyte reaction, cytotoxic T cell precursor frequency and bulk cell-mediated lysis without growth factor). Mice treated with antidonor IJ mab, however, unlike mice treated with anti-host or third party IJ mab, were capable of generating tolerogen-specific T cells in the absence of exogenous growth factor. Thus in the strain combinations we used, adult mice tolerant of either the entire H-2 region or of the class II major histocompatibility complex region alone are susceptible to abolition of the tolerant state by treatment with anti-donor IJ mab. Coincidentally, lymphoid cells from these mice generate sufficient endogenous T helper activity to activate the tolerogen-specific cytotoxic T cells. We suspect that these latter cells may be responsible for rejection of grafts bearing the tolerated antigens.     

Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

Active Suppression of the Allogeneic Histocompatibility Reactions During the Metamorphosis of the Clawed Toad Xenopus

Metamorphosis is a privileged period for the induction of tolerance to allografts. Transfers of lymphocytes from metamorphosing Xenopus into isogenic adults prevented the rejection of a skin graft differing from the adult host by minor histocompatibility antigens. This implies that active suppression is involved at one step of the induction of tolerance to the self-antigens that differentiate at the time of metamorphosis. The reciprocal experiments of preventing tolerance induction by transfer of normal adult cells into metamorphosing animals failed. However, passive transfer of anti-graft immunity in tolerant animals was partially observed, provided that a transfer of primed cells was done simultaneously with the challenging graft. Thus, memory cells are not as sensitive to the suppression as are the cells that respond in a first set reaction.     

Functional activity of T and B lymphocytes of mice undergoing spontaneous loss of tolerance]

The functional activity of splenocytes and thymocytes of mice tolerant to sheep red blood cells was investigated one and four weeks after tolerance induction. The tolerance was achieved by cyclophosphamide. The immunocompetence of thymocytes was fully reversed in lfour week time. The functional activity of T and B lymphocytes of the spleen was also partially recovered four weeks after tolerance induction. Preliminary thymectomy weakened but did not prevent completely the immunocompetence of T cells of the spleen from being recovered. No Tsuppressants were found in the thymus or spleen of the tolerant animals.     

CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance

Transplantation tolerance is induced reliably in experimental animals following intrathymic inoculation with the relevant donor strain Ags; however, the immunological mechanisms responsible for the induction and maintenance of the tolerant state remain unknown. We investigated these mechanisms using TCR transgenic mice (TS1) that carry T cells specific for an immunodominant, MHC class II-restricted peptide (S1) of the influenza PR8 hemagglutinin (HA) molecule. We demonstrated that TS1 mice reject skin grafts that have transgene-encoded HA molecules (HA104) as their sole antigenic disparity and that intrathymic but not i.v. inoculation of TS1 mice with S1 peptide induces tolerance to HA-expressing skin grafts. Intrathymic peptide inoculation was associated with a dose-dependent reduction in T cells bearing high levels of TCR specific for HA. However, this reduction was both incomplete and transient, with a full recovery of S1-specific thymocytes by 4 wk. Peptide inoculation into the thymus also resulted in the generation of immunoregulatory T cells (CD4+CD25+) that migrated to the peripheral lymphoid organs. Adoptive transfer experiments using FACS sorted CD4+CD25- and CD4+CD25+ T cells from tolerant mice revealed that the former but not the latter maintain the capacity to induce rejection of HA bearing skin allografts in syngeneic hosts. Our results suggest that both clonal frequency reduction in the thymus and immunoregulatory T cells exported from the thymus are critical to transplantation tolerance induced by intrathymic Ag inoculation.     

Evidence for involvement of clonal anergy in MHC class I and class II disparate skin allograft tolerance after the termination of intrathymic clonal deletion

Mechanisms of cyclophosphamide (CP)-induced tolerance to class I (D) and class II (IE) alloantigens were studied. Transplantation tolerance across H-2D plus IE Ag-barriers has been achieved when B10.Thy-1.1 (Kb,IAb,IE-,Db; Thy-1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) mice (5R; kb,IAb,IEb,Dd; Thy-1.2) and treated i.p. with 200 mg/kg of CP 2 days later. The tolerant state in the early and the late stage was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor Ag. In the tolerant B10.Thy-1.1 mice treated with 5R cells 28 days earlier and followed by CP, intrathymic clonal deletion of V beta 11+ T cells reactive to IE-encoded antigens was observed in association with intrathymic mixed chimerism. 5R skin survived, however, even after the clonal deletion of V beta 11+ T cells terminated by 180 days after tolerance induction. V beta 11+ T cells, which reappeared in the periphery of the recipient B10.Thy-1.1 mice bearing 5R skin at this stage, were not capable of proliferating in response to receptor cross-linking with V beta 11-specific mAb. Furthermore, the CTL activity against class I (Dd) alloantigens of spleen cells from these tolerant mice was restored by the addition of IL-2 to MLC. Thus, our experiments provide direct evidence that tolerance to both class I (Dd) and class II (IEb) alloantigens by clonal allergy occurs during the termination of intrathymic clonal deletion. These results clearly show practical hierarchy of the mechanisms of transplantation tolerance.     

Development of either split tolerance or robust tolerance along with humoral tolerance to donor and third-party alloantigens in nonmyeloablative mixed chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.     

Isolated islet transplantation in experimental diabetes.

Pancreatic islets have been isolated from the exocrine pancreas of inbred rats by the collagenase digestion method. Transplantation of isolated islets into the portal venous system of streptozotocin diabetic recipients resulted in complete abrogation of the diabetic state as measured by non-fasting serum glucose level, 24 h urinary output, rate of weight gain and glucose tolerance test. Transplantation to other sites resulted in less than optimal survival and function of islets. Allogeneic islets, transplanted across weak histocompatibility barriers, can survive and function for prolonged periods of time when transplanted recipients are immunosuppressed with antilymphocyte serum (ALS). Recipients of allogeneic islets, after a period of immunosuppression with ALS, become permanently tolerant to the allografted islets and to subsequent skin grafts from similar allogeneic donors. Allografted islets are able to prevent the occurrence of diabetic renal and ophthalmic changes that occur in control diabetic animals which had not undergone transplantation.     

Tolerance induction between two different strains of parental mice prevents graft-versus-host disease in haploidentical hematopoietic stem cell transplantation to F1 mice

Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) has been employed worldwide in recent years and led to favorable outcome in a group of patients who do not have human leukocyte antigen (HLA)-matched donors. However, the high incidence of severe graft-versus-host disease (GVHD) is a major problem for Haplo-HSCT. In the current study, we performed a proof of concept mouse study to test whether induction of allogeneic tolerance between two different parental strains was able to attenuate GVHD in Haplo-HSCT to the F1 mice. We induced alloantigen tolerance in C3H mice (H-2k) using ultraviolet B (UVB) irradiated immature dendritic cells (iDCs) derived from the cultures of Balb/c bone marrow cells. Then, we performed Haplo-HSCT using tolerant C3H mice as donors to F1 mice (C3H × Balb/c). The results demonstrated that this approach markedly reduced GVHD-associated death and significantly prolonged the survival of recipient mice in contrast to the groups with donors (C3H mice) that received infusion of non-UVB-irradiated DCs. Further studies showed that there were enhanced Tregs in the tolerant mice and alloantigen-specific T cell response was skewed to more IL-10-producing T cells, suggesting that these regulatory T cells might have contributed to the attenuation of GVHD. This study suggests that it is a feasible approach to preventing GVHD in Haplo-HSCT in children by pre-induction of alloantigen tolerance between the two parents. This concept may also lead to more opportunities in cell-based immunotherapy for GVHD post Haplo-HSCT.     

Cellular aspects of tolerance. II. Unresponsiveness of B cells

The responsiveness of bone marrow cells from tolerant donors was examined by reconstitution of lethally irradiated tolerogen-free recipients. In these animals, stem cells from tolerant donors gave rise to immunologically competent antigen sensitive B cells. The antibody produced by these cells could be detected by a sensitive plaque assay in liquid and by antigen elimination. The antibody was not demonstrable by an assay which only detected plaque forming antibody which was highly avid or was formed in large quantity per cell. In lethally irradiated animals, partially purified B cells from a tolerant animal could not cooperate with T cells from normal donors to reconstitute immunological responsiveness to immunogenic doses of the tolerance inducing antigen. We concluded that antigen sensitive B cells in the bone marrow become unresponsive following administration of tolerogenic forms of antigen. Responsiveness of the reconstituted recipient animals was due to the differentiation of donor stem cells and subsequent antibody production by their descendants. Earlier contradictory findings could be unified in terms of these observations and conclusions.     

Role for thymic and splenic regulatory CD4+ T cells induced by donor dendritic cells in allograft tolerance by LF15-0195 treatment

A 20-day treatment with LF15-0195, a deoxyspergualine analogue, induced allograft tolerance in a fully MHC-mismatched heart allograft model in the rat. Long-term allografts displayed minimal cell infiltration with no signs of chronic rejection. CD4+ spleen T cells from tolerant LF15-0195-treated recipients were able to suppress in vitro proliferation of allogeneic CD4+ T cells and to transfer tolerance to second syngeneic recipients, demonstrating dominant suppression by regulatory cells. A significant increase in the percentage of CD4+CD25+ T cells was observed in the thymus and spleen from tolerant LF15-0195-treated recipient. In vitro direct stimulation with donor APCs demonstrated that CD4+ regulatory T cells proliferated weakly and expressed low levels of IFN-gamma, IL-10, and IL-2. CD4+CD25+ cell depletion increased IL-2 production by CD4+CD25- thymic cells, but not splenic cells. Moreover, tolerance was transferable with splenic and thymic CD4+CD25+ cells, but also in 50% of cases with splenic CD4+CD25- cells, demonstrating that CD25 can be a marker for regulatory cells in the thymus, but not in the periphery. In addition, we presented evidences that donor APCs were required to induce tolerance and to expand regulatory CD4+ T cells. This study demonstrates that LF15-0195 treatment induces donor APCs to expand powerful regulatory CD4+CD25+/- T cells present in both the central and peripheral compartments.