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

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

A novel tool for B-cell tolerance research: characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique

In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.     

T cell subsets required for in vivo and in vitro responses to single and multiple minor histocompatibility antigens.

The requirement for helper T cells in the in vivo and in vitro T cell response to a diverse panel of minor histocompatibility antigens in mice was investigated. Target H antigens included: (a) multiple antigens distinguishing H-2-matched, inbred strains, and (b) single H antigens, including H-4, H-3, and the male-specific (H-Y) antigen. The involvement of helper T cells in skin allograft rejection and CTL priming was evaluated by pretreating graft recipients with anti-CD4 antibody. Anti-CD4 treatment had no effect on rejection of H-3- and H-4-incompatible skin grafts, but slowed rejection of male and BALB.B skin grafts. Comparable pretreatment with anti-CD4 antibody in vivo eliminated the priming of H-4-specific CTL, multiple B10.BR anti-CBA/J CTL, and all but a minor C57BL/6 anti-BALB.B CTL population. However, CTL specific for the two classes of H antigens, single and multiple minor H antigens, differed in their in vitro requirements for CD4+ helper T cells: (a) multiple antigen-specific CTL required CD4+ helper T cells for optimal expansion, and (b) CTL specific for single H antigens expanded in the absence of helper T cells. The CTL specific for all tested H antigens were CD8+ T cells. These results suggest that CD4+ helper T cells are not always required for effective skin allograft rejection or CTL expansion in vitro; the requirement for helper T cells is apparently dependent upon the identity of the stimulatory minor H antigen(s). This variable dependency contrasts with the evident requirement for helper T cells in the in vivo priming of CTL specific for minor H antigens.     

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from na?ve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.     

Induction of specific unresponsiveness (tolerance) to skin allografts by intrathymic donor-specific splenocyte injection in antilymphocyte serum-treated mice.

Although chronic immunosuppression has been extremely successful in clinical organ transplantation, it is associated with severe complications such as opportunistic infections, spontaneous neoplasms, drug toxicities, metabolic complications, and the inability to control rejection. We therefore have investigated the ability of allogeneic donor lymphoid cells to produce specific tolerance following intrathymic (IT) injection into allograft recipients. Groups of B6AF1 mice received ALS on days -1 and +2 relative to C3H/He skin grafts on day 0; experimental groups received 1, 5, or 10 x 10(7) syngeneic (B6AF1) or allogeneic (C3H) spleen cells (SPCs) by IT injection on day +7. IT injection of C3H splenocytes significantly prolonged allograft survival at all cell doses tested when compared with ALS controls. The best survival was obtained following IT injection of 5 x 10(7) C3H cells (median survival time [MST] = 132 days; ALS controls = 21.5 days), with 8 of 13 skin grafts surviving longer than 100 days. IT injection of syngeneic splenocytes or third-party DBA/2 splenocytes did not prolong allograft survival beyond that observed in ALS controls. C3H spleen cells injected IT into ALS treated mice on day 0 relative to grafting of C3H skin also produced significant allograft survival (1, 5, or 10 x 10(7) SPCs = MSTs of 75, 47, and 35, respectively) but the results were inferior to those obtained by 5 x 10(7) SPCs IT on day +7. Spleen cells (1 or 5 x 10(7)) injected intraperitoneally or intravenously prolonged allograft survival beyond that seen in ALS controls but were inferior to IT injection at all doses and times studied. Bone marrow, thymocytes, or lymph node cells (5 x 10(7) cells) were substituted for SPCs for IT injection. IT injection of BM, LN or thymocytes all significantly prolonged graft survival over ALS controls. However none of these cell types was as effective as IT splenocytes. Eight B6Af1 recipients of IT splenocytes bearing C3H skin grafts for > 100 days received a second C3H skin graft as well as a simultaneous third-party B10.AKM skin graft. All rejected third-party grafts in normal first-set fashion. Three tolerated both 1st and 2nd C3H grafts without any sign of rejection; 1 rejected the 2nd C3H graft while tolerating the 1st graft; and 4 rejected the 2nd C3H graft in an attenuated fashion but also rejected the 1st graft at the same pace.(ABSTRACT TRUNCATED AT 400 WORDS)     

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

Mechanism of portal venous tolerant long-term MHC Class I L(d)-specific skin graft survival in transgenic 2CF1 mice

BACKGROUND: Administration of alloantigen via the portal vein (PV) in non-transgenic animals has been shown to promote immunologic tolerance and enhance transplant allograft survival. The underlying mechanisms remain unclear. In 2C x dm2 F1 (2CF1) transgenic mice, the monoclonal antibody, 1B2, identifies specific 2C TCR transgenic CD8+ T cells that are cytotoxic against Class I MHC L(d). In these mice, the specific response by these cells to L(d+) skin grafts after PV administration of L(d+) antigen was determined. MATERIALS AND METHODS: Saline (control) or allogeneic C57BL/6 x BALB/c F1 (CB6F1) spleen cells (25 x 10(6)), which differ from 2CF1 only at L(d), were injected PV into 2CF1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these 2CF1 mice. Skin graft rejection was defined as >50% loss of the graft. Parallel experiments were performed in non-transgenic littermates [B6F1 (C57BL/6 x dm2)]. FACS analysis of 2CF1 peripheral blood for 1B2+, CD4+, and CD8+ T cells was performed 2 days before PV injection (9 days prior to skin grafting), 5 days after PV injection (2 days prior to skin grafting), and 7, 14, 21, 28, and 60 days after skin grafting. FACS analysis of nai;ve, saline control, and CB6F1 PV-treated 2CF1 thymocytes was also performed. Responsiveness of saline (control)-treated and PV-treated 2CF1 splenocytes was measured by in vitro cytotoxic T lymphocyte (CTL). RESULTS: All CB6F1 skin grafts were rejected in <14 days by PV saline controls. However, a single PV injection of donor L(d+) CB6F1 cells was sufficient to induce indefinite CB6F1 (L(d+)) skin allograft survival in 100% of non-transgenic B6F1 and transgenic 2CF1 (anti-L(d)) TCR transgenic recipients. FACS analysis of 1B2+ T cells demonstrated that PV injection of donor antigen followed by a CB6F1 skin graft led to a 70% decrease in peripheral donor-reactive 1B2+ CD8+ T cells by day 7, while central thymocytes were unchanged. CTL of 2CF1 splenocytes following PV CB6F1 demonstrated that they were hyporesponsive to L(d) compared to saline-treated 2CF1 splenocytes. Despite recovery of peripheral CD8+ T cells to near normal levels by 60 days post-transplantation, skin graft survival persisted indefinitely. CONCLUSIONS: Administration of specific PV antigen results in exquisite long-term L(d+) skin allograft acceptance. This tolerance induction is related to a significant peripheral deletion of donor-reactive 1B2+ CD8+ transgenic T cells and anergy of the residual T cells.     

Mechanism of allorecognition and skin graft rejection in CD28 and CD40 ligand double-deficient mice

BACKGROUND: It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice. METHODS: Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+ and CD8+ T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay. RESULTS: BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+ T cells. In vitro studies indicated that CD8+ T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice. CONCLUSION: In the absence of CD28 and CD40 costimulatory signals, CD8+ T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Adoptive transfer of transplantation tolerance mediated by CD4+CD25+ and CD8+CD28- regulatory T cells induced by anti-donor-specific T-cell vaccination

Previous studies have shown that vaccinating rodents with anti-donor-specific T cells significantly prolonged allograft survival; however, the putative mechanism of the tolerance remains unclear. In this study, we used the model of heterotopic heart transplantation between the C57BL/6 donor mice and BALB/c recipient mice vaccinated with anti-donor (C57BL/6) or anti-third party (C3H)-specific T cells to determine whether T cells prolong survival of mouse heart allografts and which cells were involved in induction of allograft tolerance. We observed that the mean survival time (MST) of C57BL/6 heart grafts in BALB/c mice vaccinated with anti-C57BL/6 specific T cells (43.1 +/- 4.7 days) was prolonged from that in untreated BALB/c mice (9.5 +/- 1.1 days) or BALB/c mice receiving anti-C3H-specific T cells (10.4 +/- 1.9 days). These results suggested that alloantigen-specific T-cell vaccination significantly prolonged cardiac allograft survival. The CD4+CD25+ or CD8+CD28- T cells purified from splenocytes of BALB/c mice vaccinated with anti-donor-specific T cells proliferated markedly in response to irradiated anti-C57BL/6-specific T cells in vitro. Adoptive transfer of these CD4+CD25+ or CD8+CD28- T cells to na?ve syngenic mice significantly prolonged the survival of heart allografts. These data suggested that anti-donor-specific T-cell vaccination induced development of CD4+CD25+ or CD8+CD28- regulatory T cells, which in turn mediated allogeneic-specific tolerance.     

A differential requirement for CD8+ donor cells in the augmentation of allograft survival by posttransplantation administration of donor spleen cells and donor bone marrow cells

BACKGROUND: Peritransplant treatment with antithymocyte serum (ATS) and posttransplantation administration of donor bone marrow or donor splenocytes results in extended skin allograft survival. In this study, we examined the molecular basis of the tolerance promoting effect of donor bone marrow (BMC) cells and splenocytes with emphasis on the role of CD8 expression on the donor cells. METHODS: (C57BL/6J x A/J)F1 mice were treated on days -1 and +2 with ATS relative to transplantation with C3H/HeJ skin. On day +7, they were infused with CD8+ BMC, CD8- BMC, CD8+ splenocytes, or CD8- splenocyte donor subpopulations isolated by magnetic or fluorescence-based sorting. In additional experiments, B10.D2(R107) mice were treated in the same manner with C57BL/6 skin and BMC or splenocytes from C57BL/6 mice in which the CD8alpha gene had been inactivated. RESULTS: CD8+ donor bone marrow cells induced operational tolerance (defined as graft acceptance in the absence of chronic immunosuppression) in skin graft recipients at a dose that was reduced by 250-fold relative to unfractionated bone marrow cells (1.0x10(5) cells per recipient, median survival time (MST)=41 days vs. 2.5x10(7) cells per recipient, MST=49 days, P=0.40). Similarly, donor bone marrow cells from CD8 knockout mice did not promote graft acceptance (MST=98 days vs. animals not treated with bone marrow cells, MST=70 days, P=0.16). In contrast, the extension of graft survival by donor splenocytes did not require the presence of CD8+ donor cells because splenocytes depleted of CD8+ cells extended graft survival (MST=55 days) as well as unsorted splenocytes (44 days, P=0.2), and splenocytes from CD8 knockout animals (MST=145 days) extended graft survival at least as well as unsorted splenocytes (MST=74 days, P=0.4) CONCLUSIONS: These results suggest that the prolongation of graft survival by donor bone marrow is dependent on the presence of the CD8 molecule, whereas prolongation by donor splenocytes is not. Therefore, we suggest that the prolongation of graft survival by these cell types occurs via distinct molecular mechanisms probably mediated by different cell types.     

Anergic T cells generated in vitro suppress rejection response to islet allografts

BACKGROUND: Induction of antigen-specific unresponsiveness to grafts is the ultimate goal for organ transplantation. It has been shown that anergic T cells generated in vivo can be transferred as suppressor cells. Anergic cells generated in vitro have never been successfully used to prevent allograft rejection in vivo. We examined whether anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway can suppress allograft rejection in vivo. METHODS: Anergic T cells were generated in vitro by the addition of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) to primary mixed lymphocyte reaction (MLR) consisting of C57BL/6 (B6) splenocytes as responder and irradiated BALB/c splenocytes as stimulator. We tested the ability of these cells to respond to various stimuli and to suppress alloreactive T-cell responses in vitro. For in vivo studies, 4x10(7) anergic cells were injected intravenously immediately after transplantation of BALB/c islets under the renal subcapsular space of streptozotocin-induced diabetic and 2.5-Gy X-irradiated B6 mice. RESULTS: Anergic cells treated with both mAbs in the primary MLR did not proliferate in secondary MLR against BALB/c and third-party C3H/He stimulators. The cells also failed to respond to immobilized anti-CD3 mAb, although they proliferated in response to concanavalin A or phorbol myristate acetate + ionomycin. The anergic state was reversed by the addition of exogenous IL-2. Furthermore, these cells suppressed the proliferation of naive B6 T cells against either the same (BALB/c) or third-party (C3H/He) stimulator cells. In in vivo studies, irradiated B6 mice rejected BALB/c islet allografts acutely with a mean survival time of 27.0+/-8.3 days, whereas two of six animals injected with the anergic cells accepted the allografts indefinitely (>100 days) with a mean survival time of 52.0+/-38.2 days. CONCLUSIONS: Anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway suppress islet allograft rejection after adoptive transfer. This procedure might be clinically useful for promoting allograft survival.     

The role of B7 ligands (CD80 and CD86) in CD152-mediated allograft tolerance: a crosscheck hypothesis

BACKGROUND: The regulatory mechanism by which the B7 ligands (CD80 and CD86) direct the CD28/CD152 costimulatory pathways is unclear. This study investigated the role of CD80 and CD86 in a CD152-mediated allograft tolerance model. METHODS: A low-responding cardiac transplant model (BALB/c-->B10.A) with possible long-term acceptance was used. Immunocytochemical and flow cytometric analyses of the graft-infiltrating cells were conducted to characterize this transplant model. The influence of anti-CD80 and anti-CD86 treatments on the proliferation and interleukin (IL)-2 productions of the tolerated splenocytes (SC) was analyzed. The role of CD80 and CD86 in the induction and maintenance of the graft acceptance in this transplant model were also tested. RESULTS: B10.A mice could accept the BALA/c cardiac allografts (11/22), and an anti-CD152 antibody blocked the graft acceptance (10/10). Immunocytochemical and flow cytometric analyses showed that CD152+ cells were predominant among the CD4+ cells infiltrating the 100-day grafts of the B10.A recipients (B10.A-100). Either anti-CD80 or anti-CD86 treatment significantly enhanced polyclonal proliferation and IL-2 production of the B10.A-100 SC. Blockade of either CD80 or CD86 prohibited the tolerance transmitted by adoptive transfer, and anti-CD80 or anti-CD86 plus skin grafting undermined the established allograft tolerance. CONCLUSIONS: Both CD80 and CD86 were essential for the induction and maintenance of the CD152-mediated allograft tolerance.     

Induction of operational tolerance and generation of regulatory cells after intratracheal delivery of alloantigen combined with nondepleting anti-CD4 monoclonal antibody

BACKGROUND: We previously showed that intratracheal delivery of alloantigen induced prolonged survival of fully allogeneic cardiac grafts in mice. Here, this treatment protocol was combined with nondepleting anti-CD4 monoclonal antibody (mAb) to induce operational tolerance. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of whole splenocytes from C57BL/10 (H-2b) mice or a 15-mer Kb peptide, with or without intraperitoneal administration of nondepleting anti-CD4 mAb (YTS177). Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. In addition, some naive CBA mice underwent adoptive transfer of splenocytes from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time, 12 days). Mice given intratracheal delivery of whole splenocytes or Kb peptide demonstrated prolonged graft survival (median survival time, 84 and 76 days, respectively). Concurrent administration of YTS177 and intratracheal delivery of splenocytes or Kb peptide resulted in indefinite graft survival. Mice with long-surviving C57BL/10 cardiac grafts showed acceptance of skin grafts from C57BL/10 mice but not BALB/c mice, demonstrating that operational tolerance had been induced. Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of splenocytes or Kb peptide plus YTS177 induced indefinite survival of cardiac grafts in secondary recipients, indicating that regulatory cells had been generated. CONCLUSION: In a murine model, intratracheal delivery of donor splenocytes or Kb peptide combined with YTS177 induced operational tolerance and generated regulatory cells.     

Induction of indefinite survival of fully allogeneic cardiac grafts and generation of regulatory cells by intratracheal delivery of alloantigens under blockade of the CD40 pathway

BACKGROUND: The authors previously showed that intratracheal delivery (ITD) of donor splenocytes induced prolonged survival of fully allogeneic cardiac grafts in mice. In this study, this treatment protocol was combined with blockade of the CD40 pathway in an attempt to induce operational tolerance. METHODS: CBA mice were given donor splenocytes (1x107) or Kb peptide (100 microg) by ITD with or without antibody specific for mouse CD40 ligand (MR1, 200 microg) 7 days before transplantation of a C57BL/10 heart. Also, splenocyte (5 x 107) from primary recipient CBA mice given ITD of donor splenocytes or Kb peptide plus MR1 were adoptively transferred into naive CBA secondary recipients 7 days after the pretreatment and C57BL/10 hearts were transplanted into those recipients the same day. RESULTS: ITD of donor splenocytes and Kb peptide induced prolonged survival of cardiac grafts (median survival time [MST], 74 and 56 days, respectively), whereas naive control mice and mice pretreated with syngeneic splenocytes had acute graft rejection (MST in both groups, 7 days). When MR1 was included, all grafts survived indefinitely (>200 days), but mice pretreated with MR1 alone had graft rejection (MST, 54 days). Mice bearing cardiac grafts had acceptance of skin grafts from C57BL/10 but not BALB/c mice, demonstrating that operational tolerance was induced. Secondary recipients given adoptive transfer of splenocytes from primary recipients of the combined treatment had acceptance of C57BL/10 grafts, suggesting that regulatory cells were generated within 7 days of pretreatment. CONCLUSIONS: ITD of donor splenocytes or Kb peptide under blockade of the CD40 pathway induced operational tolerance and generated regulatory cells.     

Mediation of skin allograft rejection in scid mice by CD4+ and CD8+ T cells.

We analyzed the role of CD4+ and CD8+ T cells in H-2-disparate skin allograft rejection in the mutant mouse strain C.B-17/Icr scid with severe combined immunodeficiency. On the day of skin allografting, scid mice were adoptively transferred with negatively selected CD4+ or CD8+ splenocytes from normal unsensitized C.B-17/Icr mice. These populations were obtained using a double-mAb--plus--complement elimination protocol using anti-CD4 or anti-CD8 mAb that resulted in no detectable CD4+ or CD8+ cells by FACS and negligible numbers of cytolytic T lymphocytes by limiting dilution analysis in anti-CD8 treated populations. Spleen cells were removed from grafted mice at the time of rejection and were tested in vitro for antidonor reactivity in several assays: mixed lymphocyte culture, cell-mediated lympholysis, and LDA for CTL and for IL-2-producing HTL. The presence of Thy 1.2+, CD4+, or CD8+ cells was determined by FACS. All control C.B-17 mice and scid mice adoptively transferred with nondepleted CD4+, and CD8+ cells rejected skin allografts with similar mean survival times (15.6 +/- 1.5, 18.8 +/- 3.4, 18.0 +/- 5.4, respectively), whereas control scid mice retain skin allografts indefinitely (all greater than 100 days). C.B-17 syngeneic grafts survived indefinitely in all groups. At the time of rejection, splenocytes from scid mice receiving CD4+ cells had negligible donor-specific cytotoxicity in CML and negligible numbers of CTL by LDA, but demonstrated a good proliferative response in MLC and IL-2-producing cells by LDA (frequency = 1/1764). There were no detectable CD8+ cells present by FACS analysis. Conversely, splenocytes from scid mice adoptively transferred with CD8+ cells had strong donor-specific cytotoxicity in CML (58.8% +/- 16.1%) and CTL by LDA (frequency = 1/3448), but no significant proliferation was detected in MLC. There were no detectable CD4+ cells by FACS, but there were small numbers of IL-2-producing cells by LDA (frequency = 1/10,204). These data demonstrate that CD4+ cells adoptively transferred into scid mice are capable of mediating skin allograft rejection in the absence of any detectable CD8+ cells or significant functional cytolytic activity. The adoptive transfer of CD8+ cells also results in skin allograft rejection in the absence of detectable CD4+ cells. The detection of small numbers of IL-2 secreting cells in these mice may indicate that CD(8+)-mediated allograft rejection in this model is dependent on IL-2-secreting CD8+ cells.     

CD4+ T cells are critical for corneal, but not skin, allograft rejection

BACKGROUND: The relative contribution of CD4+ or CD8+ T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. METHODS: We compared the ability of CD4- knockout (KO), CD8- KO, and normal CD4+/CD8+ mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. RESULTS: Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8- KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 +/- 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 +/- 1 days), although most CD4- KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4- KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4- KO mice that accepted such grafts or that were not grafted. CONCLUSIONS: This study indicates that mice lacking CD4+ T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4+ T cells, the likely mechanism for rejection appears to involve the generation of CD8+ CTLs.     

CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

BACKGROUND: Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. METHODS: Neonatal BALB/c mice were injected with 108 live CAF, splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using 51Cr release assays of A/J or third-party targets. RESULTS: We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 to CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4+/CD25+ cells, but not CD4+/CD25- cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2. Moreover, when added back into cultures, purified CD4+/CD25+ cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. CONCLUSION: These data indicate that CD8 anergy is associated with the state of tolerance, and that CD4+CD25+ cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.     

A novel model of allograft rejection: immune reconstitution of Rag-1 recipients with 2C transgenic T-cell receptor lymphocytes

The complexity of allorejection (cell activation, homing, and effector function) makes detailed studies difficult. We have developed a model of allograft rejection using purified monoclonal alloreactive effector cells. Immunodeficient C57Bl/6-Rag-1 (H-2(b)) recipients of Balb/c (H-2(d)) islet or skin grafts were reconstituted via adoptive transfer of splenocytes from 2C transgenic mice containing CD8+ cytotoxic effector cells directed against L(d). Recipients were assessed for engraftment, activation and homing of effector cells, and ability to reject grafts. Both unpurified 2C splenocytes and purified 2C/CD8+ cells durably reconstitute immunodeficient mice. Naive 2C effector cells reject skin grafts, but not islet allografts. However, when effector cells are primed prior to reconstitution, islet allografts are rejected. Using this model, blockade of adhesion molecules LFA-1 and alpha4-integrin delayed infiltration of islet allografts and prolonged allograft survival. This model of allorejection may be useful to study the activation and homing of allospecific cells in vivo.