Role of double-negative regulatory T cells in long-term cardiac xenograft survival

A novel subset of CD3(+)CD4(-)CD8(-) (double negative; DN) regulatory T cells has recently been shown to induce donor-specific skin allograft acceptance following donor lymphocyte infusion (DLI). In this study, we investigated the effect of DLI on rat to mouse cardiac xenotransplant survival and the ability of DN T cells to regulate xenoreactive T cells. B6 mice were given either DLI from Lewis rats, a short course of depleting anti-CD4 mAb, both DLI and anti-CD4 treatment together, or left untreated. DLI alone did not prolong graft survival when compared with untreated controls. Although anti-CD4-depleting mAb alone significantly prolonged graft survival, grafts were eventually rejected by all recipients. However, the combination of DLI and anti-CD4 treatment induced permanent cardiac xenograft survival. We demonstrate that recipients given both DLI and anti-CD4 treatment had a significant increase in the total number of DN T cells in their spleens when compared with all other treatment groups. Furthermore, DN T cells harvested from the spleens of DLI plus anti-CD4-treated mice could dose-dependently inhibit the proliferation of syngeneic antidonor T cells. Suppression mediated by these DN T cells was specific for antidonor T cells as T cells stimulated by third-party Ags were not suppressed. These results demonstrate for the first time that a combination of pretransplant DLI and anti-CD4-depleting mAb can induce permanent survival of rat to mouse cardiac xenografts and that DN T regulatory cells play an important role in preventing long-term concordant xenograft rejection through the specific suppression of antidonor T cells.     

Both infiltrating regulatory T cells and insufficient antigen presentation are involved in long-term cardiac xenograft survival

We have previously shown that pretransplant donor lymphocyte infusion (DLI) together with transient depletion of CD4(+) T cells could induce permanent rat-to-mouse heart graft survival, whereas depleting CD4(+) T cells alone failed to do so. In this study, we investigated the mechanism leading to long-term xenograft survival. We found that peripheral CD4(+) T cells from DLI/anti-CD4-treated mice could mount rat heart graft rejection after adoptive transfer into B6 CD4(-/-) mice. Infusing donor-Ag-loaded mature dendritic cells (DCs) could break long-term cardiac xenograft survival in DLI/anti-CD4-treated mice. Interestingly, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, we observed a significant increase in both the number and suppressive activity of alphabeta-TCR(+)CD3(+)CD4(-)CD8(-) double negative regulatory T cells and decrease in the numbers of CD4(+) and CD8(+) T cells in the xenografts of DLI/anti-CD4-treated mice. Moreover, there was a significant reduction in MHC class II-high DCs within the xenografts of DLI/anti-CD4-treated recipients. DCs isolated from the xenografts of anti-CD4- but not DLI/anti-CD4-treated recipients could stimulate CD4(+) T cell proliferation. Our data indicate that functional anti-donor T cells are present in the secondary lymphoid organs of the mice that permanently accepted cardiac xenografts. Their failure to reject xenografts is associated with an increase in double negative regulatory T cells as well as a reduction in Ag stimulation by DCs found within grafts. These findings suggest that local regulatory mechanisms need to be taken into account to control anti-xenograft T cell responses.     

Double-negative T cells, activated by xenoantigen, lyse autologous B and T cells using a perforin/granzyme-dependent, Fas-Fas ligand-independent pathway

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-beta, TNF-alpha, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 +/- 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.     

Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells

In this study, we investigated whether mesenchymal stem cells (MSC) had immunomodulatory properties in solid organ allotransplantation, using a semiallogeneic heart transplant mouse model, and studied the mechanism(s) underlying MSC tolerogenic effects. Either single (portal vein, day -7) or double (portal vein, day -7 and tail vein, day -1) pretransplant infusions of donor-derived B6C3 MSC in B6 recipients induced a profound T cell hyporesponsiveness and prolonged B6C3 cardiac allograft survival. The protolerogenic effect was abrogated when donor-derived MSC were injected together with B6C3 hematopoietic stem cells (HSC), suggesting that HSC negatively impact MSC immunomodulatory properties. Both the induction (pretransplant) and the maintenance phase (>100 days posttransplant) of donor-derived MSC-induced tolerance were associated with CD4(+)CD25(+)Foxp3(+) Treg expansion and impaired anti-donor Th1 activity. MSC-induced regulatory T cells (Treg) were donor-specific since adoptive transfer of splenocytes from tolerant mice prevented the rejection of fully MHC-mismatched donor-specific secondary allografts but not of third-party grafts. In addition, infusion of recipient-derived B6 MSC tolerized a semiallogeneic B6C3 cardiac allograft, but not a fully MHC-mismatched BALB/c graft, and expanded Treg. A double i.v. pretransplant infusion of recipient-derived MSC had the same tolerogenic effect as the combined intraportal/i.v. MSC infusions, which makes the tolerogenic protocol applicable in a clinical setting. In contrast, single MSC infusions given either peritransplant or 1 day after transplant were less effective. Altogether these findings indicate that MSC immunomodulatory properties require HSC removal, partial sharing of MHC Ags between the donor and the recipient and pretransplant infusion, and are associated with expansion of donor-specific Treg.     

Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

Pretransplant donor lymphocyte infusion (DLI) has been shown to enhance donor-specific allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored donor cell subsets after DLI in a murine MHC class I L^d-mismatched skin transplantation model. We found that donor B cells, but not DCs, are the major surviving donor APCs in recipients following DLI. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Furthermore, mice that had received donor B cells showed higher expression of Ly6A and CD62L on antigen-specific TCRαβ⁺CD3⁺CD4⁻CD8⁻NK1.1⁻ double negative (DN) regulatory T cells (Tregs). B cells presented alloantigen to DN Tregs and primed their proliferation in an antigen-specific fashion. Importantly, DN Tregs, activated by donor B cells, showed increased cytotoxicity toward anti-donor CD8⁺ T cells. These data demonstrate that donor B cells can enhance skin allograft survival, at least partially, by increasing recipient DN Treg-mediated killing of anti-donor CD8⁺ T cells. These findings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specific immune therapy to enhance allograft survival.     

Abrogation of antibody-mediated allograft rejection by regulatory CD4 T cells with indirect allospecificity

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.     

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.     

Achieving donor-specific hyporesponsiveness is associated with FOXP3+ regulatory T cell recruitment in human renal allograft infiltrates

Exploring new immunosuppressive strategies inducing donor-specific hyporesponsiveness is an important challenge in transplantation. For this purpose, a careful immune monitoring and graft histology assessment is mandatory. Here, we report the results of a pilot study conducted in twenty renal transplant recipients, analyzing the immunomodulatory effects of a protocol based on induction therapy with rabbit anti-thymocyte globulin low doses, sirolimus, and mofetil mycophenolate. Evolution of donor-specific cellular and humoral alloimmune response, peripheral blood lymphocyte subsets and apoptosis was evaluated. Six-month protocol biopsies were performed to assess histological lesions and presence of FOXP3+ regulatory T cells (Tregs) in interstitial infiltrates. After transplantation, there was an early and transient apoptotic effect, mainly within the CD8+ HLADR+ T cells, combined with a sustained enhancement of CD4+ CD25(+high) lymphocytes in peripheral blood. The incidence of acute rejection was 35%, all steroid sensitive. Importantly, only pretransplant donor-specific cellular alloreactivity could discriminate patients at risk to develop acute rejection. Two thirds of the patients became donor-specific hyporesponders at 6 and 24 mo, and the achievement of this immunologic state was not abrogated by prior acute rejection episodes. Remarkably, donor-specific hyporesponders had the better renal function and less chronic renal damage. Donor-specific hyporesponsiveness was inhibited by depleting CD4+ CD25(+high) T cells, which showed donor-Ag specificity. FOXP3+ CD4+ CD25(+high) Tregs both in peripheral blood and in renal infiltrates were higher in donor-specific hyporesponders than in nonhyporesponders, suggesting that the recruitment of Tregs in the allograft plays an important role for renal acceptance. In conclusion, reaching donor-specific hyporesponsiveness is feasible after renal transplantation and associated with Treg recruitment in the graft.     

Human CD4+ T cells mediate rejection of porcine xenografts

It has previously been demonstrated that xenograft rejection in rodents is dependent on CD4+ T cells. However, because of the lack of an appropriate in vivo model, little is known about the cellular basis of human T cell-mediated rejection of xenografts. In this study, we have evaluated the ability of human T cells to mediate rejection of porcine skin grafts in a novel in vivo experimental system using immunodeficient mice as recipients. Recombinase-activating gene-1-deficient mice (R-) lacking mature B and T cells were grafted with porcine skin and received human lymphocytes stimulated in vitro with irradiated porcine PBMC. Skin grafts on mice given either unseparated, activated human lymphocytes, or NK cell-depleted lymphocyte populations were rejected within 18 days after adoptive cell transfer. In contrast, skin grafts on mice given T cell-depleted human lymphocytes or saline showed no gross or histologic evidence of rejection up to 100 days after adoptive transfer. Purified CD4+ T cells were also able to mediate rejection of porcine skin grafts. These data suggest that human CD4+ T cells are sufficient to induce rejection of porcine xenografts. Thus, strategies directed toward CD4+ T cells may effectively prevent cellular rejection of porcine xenografts in humans.     

FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function

TCRalphabeta+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRgamma was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRgamma-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRgamma-sufficient DN T cells, FcRgamma-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRgamma, DN Treg cell clones also express higher levels of TCRbeta, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRgamma associates with the TCR complex and that both FcRgamma and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRgamma expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRgamma deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRgamma plays a role in mediating TCR signaling in DN Treg cells.     

T cell-activated macrophages are capable of both recognition and rejection of pancreatic islet xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4(+) T cells from BALB/c mice. Twelve days after CD4(+) T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4(+) T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4(+) T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4(+) T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4(+) T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.     

Antibody-mediated rejection of cardiac allografts in CCR5-deficient recipients

Rejected MHC-mismatched cardiac allografts in CCR5(-/-) recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5(-/-) recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5(-/-) (H-2(b)) recipients of A/J (H-2(a)) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-gamma, whereas CD4 T cells producing IL-4 were increased in CCR5(-/-) recipients. Numbers of donor-reactive CD8 T cells producing IFN-gamma were reduced 60% in CCR5(-/-) recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5(-/-) allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1(-/-) recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5(-/-) recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12-60, mean 21 days) in muMT(-/-)/CCR5(-/-) recipients. These results indicate that the donor-specific Ab produced in CCR5(-/-) heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.     

Distinct subsets of dendritic cells regulate the pattern of acute xenograft rejection and susceptibility to cyclosporine therapy

We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c(+)CD8alpha(+) splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c(+)CD8alpha(-) splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c(+)IL-12(+) splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8alpha(-) bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8alpha(+) bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 +/- 0.6 days to 14.2 +/- 0.8 days. CD8alpha(+) DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8alpha(+) DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.     

Alloantibodies prevent the induction of transplantation tolerance by enhancing alloreactive T cell priming

Circulating alloantibodies in transplant recipients are often associated with increased Ab-mediated as well as cellular rejection. We tested the hypothesis that alloantibodies facilitate cellular rejection by functioning as opsonins to enhance T cell activation using a BALB/c to C57BL/6 heart or skin transplant model. Long-term heart and skin survival induced with anti-CD154 alone or in combination with donor-specific transfusion (DST), respectively, was abrogated by the presence of anti-K(d) mAbs, and alloreactive T cell activation as well as acute rejection was observed. The prevention of graft acceptance in the skin model was dependent on anti-K(d) binding to and converting DST from tolerigenic to immunogenic. Adoptive transfer of CFSE-labeled TCR-transgenic T cells into B6 recipients treated with anti-CD154/DST revealed the ability of anti-K(d) to enhance the proliferation of anti-K(d)-specific T cells via the indirect pathway as well as of non-K(d)-reactive, recipient MHC-restricted CD4(+) and CD8(+) T cells. Thus, alloantibodies with restricted specificity are able to facilitate the indirect presentation as well as the cross-presentation of a larger repertoire of "linked" donor-derived Ags. These observations highlight the ability of alloantibodies to function not only in classical humoral rejection but also as opsonins that facilitate the CD40-CD154-independent activation of alloreactive T cells.     

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.     

CD4+ T regulatory cell induction and function in transplant recipients after CD154 blockade is TLR4 independent

Although the role of CD4(+) T regulatory cells (Treg) in transplantation tolerance has been established, putative mechanisms of Treg induction and function in vivo remain unclear. TLR4 signaling has been implicated in the regulation of CD4(+)CD25(+) Treg functions recently. In this study, we first examined the role of recipient TLR4 in the acquisition of operational CD4(+) Treg following CD154 blockade in a murine cardiac transplant model. Then, we determined whether TLR4 activation in allograft tolerant recipients would reverse alloimmune suppression mediated by CD4(+) Treg. We document that donor-specific immune tolerance was readily induced in TLR4-deficient recipients by a single dose of anti-CD154 mAb, similar to wild-type counterparts. The function and phenotype of CD4(+) Treg in both wild-type and TLR4 knockout long-term hosts was demonstrated by a series of depletion experiments examining their ability to suppress the rejection of secondary donor-type test skin grafts and to inhibit alloreactive CD8(+) T cell activation in vivo. Furthermore, TLR4 activation in tolerant recipients following exogenous LPS infusion in conjunction with donor-type skin graft challenge, failed to break Treg-mediated immune suppression. In conclusion, our data reveals a distinctive property of CD4(+) Treg in tolerant allograft recipients, whose induction and function are independent of TLR4 signaling.     

Specific B cell tolerance is induced by cyclosporin A plus donor-specific blood transfusion pretreatment: prolonged survival of MHC class I disparate cardiac allografts

Donor-specific blood transfusion (DST), designed to prolong allograft survival, sensitized recipients of the high-responder PVG-RT1u strain, resulting in accelerated rejection of MHC-class I mismatched (PVG-R8) allografts. Rejection was found to be mediated by anti-MHC class I (Aa) alloantibody. By pretreating recipients 4 wk before grafting with cyclosporin A (CsA) daily (x7), combined with once weekly (x4) DST, rejection was prevented. The investigation explores the mechanism for this induced unresponsiveness. CD4 T cells purified from the thoracic duct of CsA/DST-pretreated RT1u rats induced rejection when transferred to R8 heart-grafted RT1u athymic nude recipients, indicating that CD4 T cells were not tolerized by the pretreatment. To determine whether B cells were affected, nude recipients were pretreated, in the absence of T cells, with CsA/DST (or CsA/third party blood) 4 wk before grafting. The subsequent transfer of normal CD4 T cells induced acute rejection of R8 cardiac allografts in third party- but not DST-pretreated recipients; prolonged allograft survival was reversed by the cotransfer of B cells with the CD4 T cells. Graft survival correlated with reduced production of anti-MHC class I (Aa) cytotoxic alloantibody. The results indicated that the combined pretransplant treatment of CsA and DST induced tolerance in allospecific B cells independently of T cells. The resulting suppression of allospecific cytotoxic Ab correlated with the survival of MHC class I mismatched allografts. The induction of B cell tolerance by CsA has important implications for clinical transplantation.     

Immunoregulatory function of CD3+, CD4-, and CD8- T cells. Gamma delta T cell receptor-positive T cells from nude mice abrogate oral tolerance

Previous work has shown that abrogation of oral tolerance is mediated by T cells which are found in the CD3+, L3T4- (CD4-), and Lyt-2- (CD8-) subset (termed double-negative; DN) in mice. Inasmuch as it is known that athymic, nude (nu/nu) mice possess Thy 1+, CD4-, and CD8- T cells which also exhibit a functionally rearranged TCR gamma-chain, we investigated whether this subset of nude T cells contained functional immunoregulatory cells. In this report, we examined the phenotype and distribution of CD3+ T cells in the spleen and in the mesenteric and peripheral lymph nodes of BALB/c nu/nu mice in comparison with normal mice (+/+). In the spleens of nude mice, the predominant CD3+ T cell subpopulation was DN. Further, in mesenteric and peripheral lymph nodes, approximately one-third and one-half of the CD3+ T cells were double negative, respectively. In contrast, CD3+, DN T cells represent a small subpopulation in normal (+/+) mice. We next showed that functional regulatory T cells which possess the ability to abrogate oral tolerance were induced in nu/nu mice by Ag priming. BALB/c nude mice were immunized with SRBC, and the splenic CD3+, Vicia villosa-adherent cells were obtained by panning. Adoptive transfer of CD3+, V. villosa-adherent T cells to orally tolerant BALB/c mice restored responsiveness to SRBC, whereas V. villosa nonadherent cells were without effect. In other experiments, CD3+ T cells from the spleens of SRBC-primed mice were further enriched for the CD5+, DN phenotype and adoptive transfer of this subset completely abrogated oral tolerance to SRBC. To characterize the nature of the TCR expressed on these CD3+, DN T cells, we developed a rabbit antibody to a synthetic peptide (residues 209-218: Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) which was synthesized from a deduced sequence of the murine delta-gene. Immunoprecipitation of a cell membrane fraction from CD3+, DN T cells with anti-delta TCR antibody isolated a 45-kDa band. Furthermore, immunoprecipitation of these cells with anti-CD3 (145-2C11) revealed bands at 45 and 35 kDa (corresponding to delta- and gamma-chains, respectively). Taken together, these results are the first to show that gamma delta-TCR bearing CD3+, CD4-, and CD8- T cells are functional and reverse oral tolerance when adoptively transferred.