Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.     

IL-4 deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of MHC class II disparate skin grafts

BACKGROUND: Acute rejection of MHC class II-disparate bm12 skin grafts by C57BL/6 recipient mice is characterized by massive graft infiltration by eosinophils, together with increased intragraft amounts of IL-4 and IL-5 mRNA. IL-5 blockade prevents the intragraft eosinophil infiltration and prolongs the survival of skin allografts. As the differentiation of T cell precursors into Th2 cells is largely driven by IL-4, we investigated the role of IL-4 in MHC class II-disparate allograft rejection. METHODS: We performed skin grafts from MHC class II incompatible bm12 mice into wild-type C57BL/6 mice (IL-4) or C57BL/6 IL-4 deficient mice (IL-4). Graft survival, in vitro T cell reactivity, and histology were compared. RESULTS: We observed that 50% of IL-4 mice rapidly rejected their bm12 allograft, whereas the other 50% retained their graft 60 days after transplantation. Histological examination of bm12 allografts retained by IL-4 mice showed a normal appearance with no inflammatory infiltrate and no eosinophils. Among IL-4 mice that acutely rejected their bm12 skin graft, we observed a dense polymorphonuclear infiltrate. The depletion of neutrophils significantly prolonged bm12 graft survival. CONCLUSIONS: Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.     

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.     

Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process.

BACKGROUND: Transient depletion of CD4+ T cells in cardiac allograft recipients prolongs allograft survival; however, grafts exhibit signs of chronic rejection characterized by collagen deposition and neointima development. Although it is believed that Th1 cells promote acute graft rejection, the role of these cells in chronic rejection remains unclear. Hence, our study evaluated whether Th1 cells are associated with the development of chronic cardiac allograft rejection. METHODS: Splenocytes obtained from C57BL/6 recipients bearing BALB/c hearts with signs of chronic rejection were adoptively transferred into C57BL/6 SCID cardiac allograft recipients. As a measure of Th1 function, interferon-y production was determined after restimulation of recipient splenocytes with donor alloantigens. RESULTS: Transfer of splenocytes in SCID allograft recipients resulted in accelerated chronic rejection in the majority of mice. Characterization of these cells before transfer revealed hyporesponsive Th1 function. However, donor-specific proliferative responses and precursor interleukin-2 producing helper and cytotoxic T lymphocyte frequencies were comparable to that of naive splenocytes. Further, splenocytes obtained from SCID recipients with advanced signs of chronic rejection remained deficient in Th1 function, suggesting that Th1 are not involved in this disease process. This possibility was further supported by the development of chronic rejection in IL-12 knockout recipients. Finally, when splenocytes used for adoptive transfer retained Th1 function, transfer of these cells into SCID recipients resulted in acute allograft rejection. CONCLUSIONS: We have established a model in which the mediators of chronic rejection may be further explored. In this system, the absence rather than the presence of donor-reactive Th1 is associated with chronic rejection. These data indicate that Th1-independent effector mechanisms are responsible for chronic rejection in this model.     

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.     

Acceptance of related and unrelated cardiac allografts in neonatally tolerized mice is cardio-specific and transferable by regulatory CD4+ T cells.

BACKGROUND: Non-donor-specific cardiac allograft acceptance is induced in C3H/He (C3H; H-2k) recipients injected as neonates with allogeneic BALB/c (BALB; H-2d) fetal liver cells (FLC). This occurs despite intact reactivity to donor-type and third-party alloantigens in in vitro assays and skin transplants. To investigate a role for regulatory T cells, we performed adoptive transfer studies and specifically assessed CD4+ and CD4- T cells. METHODS: Three cell populations (splenocytes, CD4+, CD4-) derived from neonatally-treated mice with accepted C57BL/6 (B6; H-2b) third-party cardiac grafts were adoptively transferred into sub-lethally-irradiated C3H mice. Reconstituted mice were challenged with B6 cardiac grafts, B6 skin grafts, or unrelated cardiac grafts. Separated cells were assessed in vitro. RESULTS: B6, BALB, and NZW (H-2z) graft acceptance was transferred by unfractionated splenocytes. CD4+ cells transferred B6 graft acceptance (85% survival > 100 days). CD4- cells, unfractionated cells from naive or only irradiated mice, and unfractionated cells from neonatally-treated non-transplanted C3H mice rejected grafts within 35 days. No inoculum induced skin graft acceptance. Co-cultured assays confirmed the suppressive function of CD4+ cells in vitro. CONCLUSIONS: Cardiac allograft acceptance in our model is regulated by CD4+ cells. The regulatory cell population is induced by the cardiac graft itself and mediates in vivo cardiac graft acceptance in a tissue-specific but not donor-strain-specific manner.     

CD4(+) T-cell-mediated mechanisms of corneal allograft rejection: role of Fas-induced apoptosis

BACKGROUND: The role of CD4(+) T cells as effector cells in corneal allograft rejection is poorly understood. We investigated the role of CD4(+) T cells as helper cells in the generation of allospecific effector macrophages in corneal graft rejection and the role of CD4(+) T cells as apoptosis-inducing effector cells. METHODS: Corneal allografts were transplanted to CD4 knockout, FasL-deficient, and macrophage-depleted hosts. An Annexin-V binding assay was used to evaluate the susceptibility of corneal cells to both Fas-dependent and CD4 T-cell-mediated apoptosis in vitro. RESULTS: Macrophages were essential for graft rejection, but not as effector cells. Anti-BALB/c CD4(+) T cells from immunized C57BL/6 mice induced apoptosis of BALB/c corneal epithelial and endothelial cells. However, anti-BALB/c CD4(+) T cells from FasL-deficient gld/gld mice did not induce apoptosis of BALB/c corneal endothelial cells. Moreover, gld/gld mice had a reduced capacity to reject BALB/c corneal allografts. Although the initial results suggested a role for Fas-induced apoptosis in corneal graft rejection, additional experiments indicated otherwise. The incidence and tempo of immune rejection of Fas-deficient lpr/lpr corneal allografts were no different than those for corneal grafts from Fas-bearing C57BL/6 donors. Moreover, CD4(+) T-cell-mediated apoptosis of corneal cells could not be blocked with either Fas-Fc fusion protein or anti-FasL blocking antibody. CONCLUSIONS: The results suggest that CD4(+) T cells function directly as effector cells and not as helper cells in the rejection of corneal allografts. Although the corneal endothelium is highly susceptible to Fas-induced apoptosis, this is apparently not the primary mechanism of CD4(+) T-cell-dependent rejection.     

Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bml pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days -1, 0, 1, 2 with grafting on day 0) with MST +/- S.D. of 71 +/- 29 and 44 +/- 36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bml mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to < 5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with > 20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (< 15%) and allografts survived for > 80 days. The GK5 allografts survived significantly longer than those from non-transgenic bml controls (MST 83 +/- 32 days, compared with 30 days, P < 0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.     

Production of nitric oxide during graft rejection is regulated by the Th1/Th2 balance, the arginase activity, and L-arginine metabolism

BACKGROUND: Production of nitric oxide (NO) by graft infiltrating macrophages has been proposed as an important effector mechanism of allograft rejection. Although high levels of NO are generated during allograft rejection, undetectable or only limited amounts of NO were found in rejected skin xenografts. METHODS: BALB/c mice were grafted with skin transplants from syngeneic, allogeneic or xenogeneic (rat) donors. The production of NO, cytokines and arginase in the grafts was determined by spectrophotometry, enzyme-linked immunosorbent assay, or polymerase chain reaction. Effects of depletion of CD4+ cells, neutralization of interleukin (IL)-4 or application of arginase inhibitors N(omega)-hydroxy-L-arginine (L-NOHA) and L-valine on production of NO in rejected xenografts were evaluated. RESULTS: Rejection of rat skin xenografts, on the contrary to rejection of allografts, was associated with a local high production of Th2 cytokines IL-4 and IL-10, overexpression of arginase genes, strongly enhanced arginase activity and attenuated NO generation in the graft. The supernatants obtained after cultivation of skin xenograft (but not allograft or syngeneic graft) explants contained a high arginase activity and strongly suppressed NO production by activated macrophages. This suppression was completely inhibited by L-NOHA or was overcome by an excess of exogenous L-arginine, a substrate for NO synthesis. Cocultivation of xenograft explants that did not produce NO with arginase inhibitors L-NOHA or L-valine restored NO generation in the graft. CONCLUSION: The results suggest that upregulation of arginase activity by Th2 cytokines during xenograft rejection limits the bioavailability of L-arginine for the inducible NO synthase and thus attenuates generation of NO by the graft-infiltrating macrophages.     

Acute Xenograft Rejection Mediated by Antibodies Produced Independently of TH1/TH2 Cytokine Profiles

There is substantial support for the hypothesis that T(H)1 cytokine responses are critical for the normal elaboration of allograft rejection. Recent studies by Wang et al. (1) underscore the importance of T(H)2 responses in xenograft rejection and revealed that T(H)1 cytokines, IL-12 and interferon-gamma (IFN-gamma), can negatively regulate the development of humoral responses necessary for xenograft rejection. Their exceptional studies prompted us to test whether the ability of allografts to elicit cellular rejection and xenografts to induce humoral rejection also result from the differential ability to induce T(H)1 and T(H)2 responses. We compared the kinetics of antibody and cytokine (IFN-gamma and IL-4) production in C57BL/6 mice following allograft transplantation with BALB/c hearts and in C57BL/6 and BALB/c mice following transplantation with Lewis rat hearts. We also compared the ability of BALB/c mice, deficient in the ability to produce IL-4 or IFN-gamma, to reject xenografts and produce xenoantibodies. We observed that T(H)1/T(H)2 cytokine production minimally affected the kinetics of graft rejection but regulated the magnitude of IgG subclass production. Anti-graft IgM played a critical role in initiating acute antibody-mediated xenograft rejection, and the production antigraft IgM was unaffected by IL-4 or IFN-gamma deficiency. In contrast to the report by Wang et al. (1), we conclude that antibody-mediated xenograft rejection in the concordant Lewis rat heart-to-C57BL/6 mouse xenotransplantation model is dependent on anti-IgM production but independent of T(H) cytokine profiles.     

Interleukin-9 promotes eosinophilic rejection of mouse heart allografts

BACKGROUND: Eosinophils participate in allograft rejection when donor-reactive helper T lymphocytes are T-helper type 2 (Th2)-biased. Whereas the involvement of interleukin (IL)-4 and IL-5 in these forms of rejection is well established, the role of IL-9, another Th2-type cytokine promoting eosinophilia, has not been determined. METHODS: We first used real-time polymerase chain reaction to quantify IL-9 mRNA in rejected allografts in a mouse model of fully mismatched heart transplantation in which recipients were devoid of CD8 T cells and developed a Th2 alloimmune response. We then compared allograft survival in wild-type versus IL-9-deficient mice depleted of CD8 T cells. Finally, we compared the fate of major histocompatibility complex class II-mismatched cardiac transplants from wild-type versus IL-9 transgenic donors to determine the influence of IL-9 overexpression within the graft. RESULTS: The Th2 alloimmune response in CD8-deficient mice was associated with the accumulation of IL-9 mRNA in the rejected graft. In IL-9-deficient recipients depleted of CD8 T cells, eosinophil infiltration of heart allografts did not develop, but rejection still occurred. In the major histocompatibility complex class II disparate model, heart allografts from IL-9 transgenic donors were acutely rejected, whereas grafts from wild-type donors did not develop rejection. Acute rejection of IL-9 transgenic hearts was associated with massive eosinophil infiltration and prevented by neutralization of either IL-4 or IL-5. CONCLUSION: IL-9 is critically involved in heart transplant eosinophilia in conjunction with IL-4 and IL-5.     

Induction of indefinite survival of fully mismatched cardiac allografts and generation of regulatory cells by sarpogrelate hydrochloride

BACKGROUND: At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. CONCLUSION: SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.     

Targeted gene disruption of the heat shock protein 72 gene (hsp70.1) in the donor tissue is associated with a prolonged rejection-free survival in the murine skin allograft model

It is known that the expression level of the heat shock protein (HSP) is elevated in allograft tissues, but the specific role of the HSP in the acute rejection has not been elucidated. This study aims to determine how and when the HSP72 molecule works immunologically in the process of acute allograft rejection from a skin graft model in which HSP72 (hsp70.1 gene) knock out (KO) mice were adopted either as a donor or as a recipient. In experiment I, tail skin was grafted from either the HSP72 KO C57BL/6 mice or wild-type C57BL/6 mice--as the allograft control--onto the trunk of B10BR mice. The grafts were observed for any signs of rejection until the 14th day after the graft. The survival of the grafted skin from the two donor groups was analyzed by a log-rank method. The grafted skin was observed for the degree of rejection using light microscopy. In addition, the degree of apoptosis was assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). A mixed lymphocyte reaction (MLR) was observed with HSP72 KO C57BL/6 splenocytes as the stimulator and B10BR mice lymphocytes as the responder cells. The cytokine levels, such as interleukin-2 (IL-2) and interferon-gamma, were measured from an MLR supernatant using enzyme-linked immunosorbent assay (ELISA). In experiment II, the tail skin was grafted in the opposite direction from the B10BR mice to the HSP72 KO or wild-type C57BL/6 mice, and the grafts were observed for any signs of rejection, as defined in experiment I. The absence of HSP72 expression was observed in the HSP72 KO mice lymphocytes after heat stress (42 degrees C) using Western blot analysis. In experiment I, the survival of the skin grafts from the HSP72 KO C57BL/6 mice was 12+/-1.3 days (median+/-S.E.), which was significantly longer than that from the allograft control donor (9+/-0.6 days; p=0.03). This coincided with the microscopic finding of the graft tissues. The MLR response of the CD4+ lymphocytes stimulated by HSP72 KO C57BL/6 splenocytes was lower, and the interferon-gamma concentration from the MLR supernatant was also lower. On Day 9, 3.7+/-1.1(mean+/-S.D.) TUNEL-positive cells per unit high-power field (HPF) were observed from the HSP72 KO C57BL/6 skin, which was significantly lower than that from the control skin (8.7+/-2.1 cells/HPF; p=0.045). In contrast, in experiment II, there was no difference in the survival of the skin graft either from the B10BR to HSP72 KO C57BL/6 mice or from the B10BR to the wild-type C57BL/6 mice. In summary, the survival of the skin grafts from the HSP72 KO C57BL/6 mice was prolonged, and the degree of rejection was lower than that from the allograft control. This means that the presence of HSP72 in the donor tissue is related to the up-regulation of acute rejection from the recipient immune system. The HSP72 KO mice as the donor were shown to have decreased level of antigen presentation to the recipient CD4+ lymphocytes. Therefore, the HSP72 molecule from the donor cells is believed to be related to the induction of the recipient immune reaction via alloantigen presentation.     

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.     

Characterization and manipulation of T cell immunity to skin grafts expressing a transgenic minor antigen.

BACKGROUND: Minor histocompatibility antigens play a significant role in allograft rejection when donor and recipient are matched at MHC loci. An improved understanding of T cell immunity directed toward a model minor antigen may provide new approaches for preventing graft rejection. METHODS: C57BL/6 (B6) recipient mice were engrafted with skin from B6 beta-galactosidase transgenic (beta-gal tg) donors and the induced T cell immune responses were characterized by cytokine ELISA spot assay. beta-gal-specific immunity was manipulated prior to transplant through preinjection with beta-gal in complete Freund's adjuvant (CFA) or through preinjection with soluble beta-gal i.v. RESULTS: B6 mice rejected beta-gal tg skin by day 25. Rejection was associated with a low frequency of predominantly CD8+, interferon-gamma-producing T cells capable of directly recognizing both beta-gal tg cells and an immunodominant major histocompatibility complex I-restricted peptide derived from the beta-gal protein. Rejection of multiple minor antigen disparate skin and major histocompatibility complex-disparate skin occurred significantly faster, and was associated with a 10- to 30-fold higher frequency of alloreactive T cells, than rejection of beta-gal tg skin. Prepriming of recipients with beta-gal in complete Freund's adjuvant resulted in an increased frequency of beta-gal-specific T cells and accelerated rejection of beta-gal tg skin. Intravenous injection of soluble beta-gal-induced graft tolerance and a lack of detectable beta-gal-specific immunity. CONCLUSIONS: The findings reveal that transgenically expressed beta-gal behaves as a minor transplantation antigen and that manipulation of the beta-gal-specific T cell repertoire can dramatically affect rejection of beta-gal tg skin grafts. The work provides the foundation for mechanistic studies of tolerogenesis to minor antigenic determinants.     

Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c and spontaneously-diabetic NOD recipient mice

BACKGROUND: The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. METHODS: The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. RESULTS: Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. CONCLUSIONS: These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.     

Comparison of successful and unsuccessful islet/Sertoli cell cotransplant grafts in streptozotocin-induced diabetic mice

Sertoli cells (SC) protect islet allografts from immune destruction in diabetic rodents. In this study, we examined the difference between successful and rejected islet/SC cografts in order to further improve this procedure for optimal extension of islet allograft survival. We cotransplanted 500 BALB/c islets with 1-8 million BALB/c SC under the kidney capsule of diabetic BALB/c, C3H-HeJ, and C57BL/6 mice. Cotransplantation of islets with up to 8 million SC was not detrimental to long-term islet graft function in syngeneic mice. However, large numbers of SC were detrimental to islet graft survival in allogeneic mice with the optimal dose for cotransplantation of 4 or 1 million SC in C3H-HeJ or C57BL/6 mice, respectively. Examination of successful grafts, from euglycemic recipients, revealed the presence of SC arranged in tubule structures with islets surrounding these tubules. Cellular infiltrate in successful grafts revealed CD4 T cells and macrophages along the periphery and within the grafts, and very few CD8 T cells. Conversely, examination of unsuccessful grafts, harvested from hyperglycemic recipients at the time of rejection, revealed the presence of SC arranged randomly with islets adjacent to the Sertoli cells, when present, and massive CD4 and CD8 T cell as well as macrophage cell infiltration. Prolongation of islet allograft survival appeared to be a function of SC transplant mass and recipient genetic background. A consequence of long-term graft acceptance is the formation of SC tubule structures, which may be an additional requirement for optimal protection of islet allografts.     

Tumor necrosis factor, macrophage colony-stimulating factor, and interleukin 1 production within sponge matrix allografts.

Neither the presence nor the specific role of secretory cytokines in in vivo allograft rejection has been extensively studied. We quantitated the levels of colony-stimulating factors, tumor necrosis factor, and interleukin 1 within the rejecting allograft. BALB/c (H-2d) mice were implanted with polyurethane sponges containing either allogeneic C57BL/6 (H-2b) or syngeneic splenocytes, or splenocyte-free media. At various days postgrafting, the sponges were harvested, and the cells infiltrating the grafts were analyzed for specific antidonor cytolytic activity, while IL-1, TNF, and CSF levels were measured in the graft exudate fluid. Allogeneic grafts had significantly higher concentrations of CSF, TNF, and IL-1 than syngeneic of splenocyte-free grafts. A specific radioimmunoassay revealed that macrophage colony-stimulating factor (M-CSF) is the primary CSF produced in the grafts. Peak TNF levels preceded peak M-CSF and IL-1 levels, which coincided with the initial appearance of allospecific cytotoxic T lymphocytes. Maximal CTL activity was seen on day 13, when the levels of these cytokines had already begun to fall. Specific bioassays for multi-CSF (IL-3), granulocyte CSF, granulocyte-macrophage CSF, IL-2, and IL-4 failed to detect these cytokines in the sponge fluid at any time. We hypothesize that TNF, M-CSF, and IL-1 probably play regulatory roles in the immunologic events at the site of allograft challenge.     

Pretransplant sensitization with major histocompatibility complex class I+ class II- hepatocytes leads to accelerated skin graft rejection.

The immunogenicity of major histocompatibility complex (MHC) class I+ class II- hepatocytes is controversial. We studied the effect of pretransplant donor-specific sensitization with either purified hepatocytes (HC) or splenocytes (Spl) on subsequent skin allograft survival. Five million Percoll-purified DBA HC or 10 x 10(6) DBA Spl were injected into C57BL/6 recipients either intraperitoneally (ip) or into a sponge matrix allograft. Twelve days later, sensitized mice received a DBA skin graft. On the same day, allogeneic (DBA) and syngeneic (BL/6) skin grafts were placed on naive BL/6 mice. In naive BL/6 mice, allogeneic skin graft survival was 7.8 +/- 0.5 days (n = 4), and syngeneic survival was indefinite (n = 5). Skin graft survival (mean +/- SD in days) in recipients sensitized with hepatocytes ip was 6.0 +/- 1.2 days (n = 5) compared with 5.6 +/- 0.5 days in recipients sensitized with splenocytes ip. Similarly, graft survival in recipients that received hepatocytes into a sponge matrix allograft was 5.67 +/- 1 days (n = 6) compared with 5.2 +/- 1.1 days (n = 8) in those that received splenocytes into the sponge. There was no difference in graft survival between mice sensitized with HC vs Spl, nor between mice injected ip vs with the sponge. All sensitized mice experienced accelerated graft rejection compared with naive controls (P less than 0.000). These results demonstrate that purified MHC class I+, class II- murine HCs are immunogenic in vivo. Sensitization with donor-specific HCs led to accelerated rejection of subsequent skin grafts, similar to the accelerated rejection seen after sensitization with MHC class I+ and class II+ splenocytes.