Islet transplantation in the discordant tilapia-to-mouse model: a novel application of alginate microencapsulation in the study of xenograft rejection

BACKGROUND: Tilapia islet xenograft rejection is characterized by infiltration with macrophages (Mphis), eosinophils (Ephis), and T lymphocytes. The presence of these cells indicates they contribute to rejection; therefore, an attempt was made to assess their role through host immunomodulation. METHODS: Tilapia islet cells were transplanted under the kidney capsule of streptozotocin diabetic Balb/c mice, which were then treated with one of several immunomodulatory regimes targeting Mphis, Ephis, or T cells. Mphis were depleted using either silica or liposome-entrapped Cl2MDP. Ephi migration was blocked using monoclonal antibodies (mAbs) targeting interleukin (IL)-4 or IL-5. T-cell function was altered with mAbs targeting CD3, CD4, or CD8. Finally, T helper (Th)1 and Th2 activity was altered by depleting essential Th1 or Th2 cytokines with mAbs or by promoting a Th1 response with the injection of exogenous IL-12. The effects of antibody-mediated immunomodulation on graft survival were initially screened by cotransplanting alginate-encapsulated, mAb-secreting hybridoma cells into the peritoneal cavity at the time of islet transplantation. Significant prolongation was then confirmed using purified antibodies injected at the time of islet transplantation. Rejected grafts were examined histologically, and immunohistochemistry was used to assess the cellular infiltrates for each of the treatment groups. RESULTS: Modulation of Mphis and Ephis alone did not significantly delay functional rejection of tilapia islet grafts (maximal mean graft survival time [mGST]=7.1+/-1.7 and 9.4+/-3.4, respectively) compared with untreated controls (mGST=8.2+/-1.0). Treatment of transplanted animals with antibodies against CD3 or CD4 significantly promoted graft survival (maximal mGST=16.3+/-5.8 and 34.0+/-11.6, respectively), whereas targeting CD8 and Th1 and Th2 cytokines showed no prolonging effect (maximal mGST=7.8+/-2.9 and 9.5+/-4.3, respectively). CONCLUSION: Our results indicate that rejection in the tilapia-to-mouse model follows a pattern similar to other models of discordant islet cell xenotransplantation.     

Local expression of IDO, either alone or in combination with CD40Ig, IL10 or CTLA4Ig, inhibits indirect xenorejection responses

Abstract: Background: To overcome cell‐mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3‐dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo. Methods: The PIEC stable lines expressing IDO, CD40Ig, and IL10 as single molecules were generated. In addition, PIEC lines expressing IDO with either CD40Ig, IL10 or CTLA4Ig were generated to produce cell lines expressing two molecules. BALB/c mice were primed with wild type PIEC, followed by harvesting splenocytes used as responder cells and PIEC expressing immunomodulatory molecules as stimulators, in proliferation and cytokine assays. In vivo effects of modified PIEC were examined by transplantation of PIEC lines expressing the immunomodulatory molecules under the renal capsule of naïve mice. PIEC grafts were harvested for histological evaluation at days 7 and 14. Results: Proliferation of primed BALB/c splenocytes was inhibited most significantly by IDO compared with control cells (49%, P = 0.02). In addition both Th1 (interferon‐gamma) and Th2 (IL4 and IL10) cytokines were markedly inhibited in vitro by IDO expression. IL10 expressing cells did not inhibit proliferation as potently (37%, P = 0.03) whilst CD40Ig lead to an increase in proliferative responses (59%, P = 0.02). Co‐expression of CD40Ig, IL10, and CTLA4Ig with IDO resulted in further modest reductions in proliferation compared with IDO expression alone. When transplanted under the renal capsule of BALB/c mice, those grafts expressing IDO demonstrated significantly lower levels of lymphocyte infiltration at days 7 and 14 than control grafts and those expressing CD40Ig, CTLA4Ig or IL10 alone. Grafts co‐expressing IDO and a second molecule were no better protected than those expressing IDO alone. Graft cell viability (PIECs) was reduced in some IDO expressing grafts suggesting high levels of IDO expression may inhibit PIEC viability, however, grafts co‐expressing IDO‐CTLA4Ig and IDO‐IL10 were not affected in this way. Conclusion: Indoleamine 2,3‐dioxygenase appears to be a potent molecule for protecting xenografts from cell‐mediated rejection responses activated via the indirect pathway. Co‐expression of IDO with both CTLA4Ig and IL10 warrants further investigation. Overall these findings support pursuing further studies, in larger animal models, to determine whether increased IDO activity within the graft itself can attenuate xenorejection responses.     

Antisense deoxyoligonucleotides or antibodies to murine MD-1 inhibit rejection of allogeneic and xenogeneic skin grafts in C3H mice

BACKGROUND: Altered expression of murine MD-1, a molecule controlling expression of members of the interleukin (IL)-1 receptor family of signaling proteins, regulates antigen-presenting cell-induced alloreactions. We investigated the effect of treatment with antisense deoxyoligonucleotides or antibodies to MD-1 in vivo on allogeneic and xenogeneic skin graft survival and the immune responses in transplanted mice. METHODS: C3H mice received C57BL/6 or Lewis rat skin grafts, followed by i.v. injections of anti-MD-1 antibody or antisense oligonucleotides or control reagents at 48-hr intervals. Survival was monitored. In separate studies, mice were sacrificed at 5-day intervals. Serum was analyzed for circulating MD-1 antigen, and peritoneal cells for surface expression of MD-1. The proliferative and cytolytic response of lymphocytes harvested from treated animals and restimulated in vitro with allo- or xenogeneic cells, and the cytokines produced, was measured. Graft histology was assessed at 11 days after transplantation. RESULTS: Treatment with anti-MD-1 oligonucleotides or antibodies suppressed rejection of both xeno- and allogeneic grafts, decreased induction of graft-specific cytotoxic T cells, increased production of type-2 cytokines (IL-4 and IL-10), and decreased production of type-1 cytokines (IL-2 and interferon-gamma). Serum levels of MD-1 were suppressed, as was expression of MD-1 on the surface of antigen-presenting cells. Grafts from MD-1-treated mice showed little lymphocyte infiltration, and no signs of graft necrosis. CONCLUSION: Our data suggest a critical in vivo role for MD-1 expression in regulating graft rejection, as well as in the concomitant sensitization of T cells and their cytokine production profile, which parallels the rejection response.     

IL-13 prolongs allograft survival: association with inhibition of macrophage cytokine activation

Th2 cytokines, especially IL-4 and IL-10, may facilitate transplant tolerance induction but the role of IL-13, another Th2 cytokine, is not known. This study examined the effects of rat recombinant IL-13 (rIL-13) on alloimmune responses. In vitro effects of rIL-13 were compared in mixed lymphocyte cultures (MLC) on rat lymphocytes cultured with PVG stimulator cells. DA rats grafted with fully allogeneic PVG neonatal heart grafts were treated with 40,000 units of rIL-13 for 10 days and graft survival monitored by ECG. Cytokine mRNA expression in the graft and lymphoid tissues was studied by RT-PCR and alloantibody levels assayed. rIL-13 had no effect on MLC, unlike rIL-4 which enhanced proliferation and induced Th2 and inhibited Th1 cytokines in MLC. rIL-13 inhibited IL-12p35, IL-12p40 and TNF-alpha mRNA induction in dendritic cell cultures. Treatment with rIL-13 prolonged fully allogeneic PVG neonatal heart graft survival to 18-21 (13-27) days (median (range)); compared to 12 (9-15) days in untreated normal rejection (p<0.05) and 14 (10-24) days in sham treated controls (p<0.05). RT-PCR studies on graft tissue identified reduced mRNA expression for the dendritic cell/macrophage molecules iNOS, TNF-alpha and IL-12 compared to normal rejection. rIL-13 treatment did not increase Th2 cytokines as compared to normal rejection, or the Th2 dependent IgG1 alloantibody response, while IL-4 did. These studies demonstrated that rIL-13 can prolong allograft survival associated with inhibition of IL-12, TNF-alpha and iNOS mRNA induction, and suggest IL-13 could modify graft rejection by inhibition of dendritic cell and/or macrophage function.     

Local secretion of a chimeric anti-CD4 antibody protects against graft rejection in the NOD mouse

BACKGROUND: Engineering a graft to secrete its own immunosuppressive antibodies may minimize the risks associated with current high dose systemic immunosuppression. METHODS AND RESULTS: A beta cell insulinoma cell line (NIT-1) was transfected with genes encoding a chimeric anti-CD4 antibody. The NIT-1 cells secreted functional chimeric anti-CD4 antibody that bound to the CD4 molecule on mouse thymocytes and inhibited in vitro proliferation of CD4+ve T cells. Both test and control transfected cell lines grew at a similar rate in immunodeficient mice. In immunocompetent NOD mice, NIT-1 cells are normally rejected by a cellular immune response against the SV40 T antigen. Although control transfected NIT-1 cells were rapidly rejected by NOD mice, anti-CD4 secreting NIT-1 cells grew significantly better and were able to form tumors at the site of injection. CONCLUSIONS: The local secretion of chimeric anti-CD4 antibody from transfected cells can contribute to graft survival in our transplantation model.     

CD8+ T cell subsets TC1 and TC2 cause different histopathologic forms of murine cardiac allograft rejection

BACKGROUND: CD4+ T cell effector function is sufficient to mediate allograft rejection, and it is suggested that CD8+ T cell-mediated effects are dependent on CD4+ T cell help. CD8+ T cells can be classified into at least two functional subsets: Tc1, producing high amounts of interferon (IFN)-gamma and Tc2, producing interleukin (IL)-4, -5, -10, and -13 and low levels of IFN-gamma. Because these subsets express different chemokine receptors, they may have different capabilities of migrating into grafts. Once in the graft, each subset may perform different effector functions dependent on the cytokines it produces. We asked whether allospecific CD8+ T cells, in the absence of CD4+ T cells, are capable of mediating rejection of a primarily vascularized allograft, and if Tcl and Tc2 cells differ in their ability to mediate rejection. METHODS: Hearts from H-2d mice were transplanted into H-2b RAG 1-/- recipients. Without manipulation, these fully mismatched allografts would survive indefinitely due to the absence of mature T and B cells. We adoptively transferred allo-(H-2d)-reactive Tcl or Tc2 cells from H-2b mice into each recipient. Grafts were harvested and analyzed on predefined timepoints, rejection was graded on a modified ISHLT scale. RESULTS: On day 7, grafts from Tc1- or Tc2-injected animals showed grade 1-2 parenchymal rejection with stable phenotype and comparable distribution of graft infiltrating CD8+ T cells. Adoptive transfer of IFN-gammahigh Tc1, but not of IFN-gammalow Tc2 cells was followed by the development of graft vasculitis, as well as graft arteriopathy. Adoptive transfer of IL-4high IL-5high Tc2, but not of IL-4low IL-5low Tc1 cells lead to extensive infiltration of eosinophils and formation of giant cells. CONCLUSIONS: Both Tc1 and Tc2 cells can mediate murine cardiac allograft rejection in the absence of CD4+ T cell help, although each subset elicits a different type of inflammatory response. In this model, cytokine secretion of either functional CD8+ T effector cell subset is an important effector mechanism in the process of allograft rejection: IFN-gammahigh Tc1 cells are important in early graft vasculitis, although IL-4high IL-5high Tc2 cells promote recruitment of secondary effectors like eosinophils.     

Anti‐Complement Component C5 mAb Synergizes with CTLA4Ig to Inhibit Alloreactive T cells and Prolong Cardiac Allograft Survival in Mice

While activation of serum complement mediates antibody‐initiated vascular allograft injury, increasing evidence indicates that complement also functions as a modulator of alloreactive T cells. We tested whether blockade of complement activation at the C5 convertase step affects T cell‐mediated cardiac allograft rejection in mice. The anti‐C5 mAb BB5.1, which prevents the formation of C5a and C5b, synergized with subtherapeutic doses of CTLA4Ig to significantly prolong the survival of C57BL/6 heart grafts that were transplanted into naive BALB/c recipients. Anti‐C5 mAb treatment limited the induction of donor‐specific IFNγ‐producing T cell alloimmunity without inducing Th2 or Th17 immunity in vivo and inhibited primed T cells from responding to donor antigens in secondary mixed lymphocyte responses. Additional administration of anti‐C5 mAb to the donor prior to graft recovery further prolonged graft survival and concomitantly reduced both the in vivo trafficking of primed T cells into the transplanted allograft and decreased expression of T cell chemoattractant chemokines within the graft. Together these results support the novel concept that C5 blockade can inhibit T cell‐mediated allograft rejection through multiple mechanisms, and suggest that C5 blockade may constitute a viable strategy to prevent and/or treat T cell‐mediated allograft rejection in humans.     

Suppressed splenocyte proliferation following a xenogeneic skin graft due to implanted biomaterials

BACKGROUND: Immune system responses to antigens in the context of biomaterials are poorly understood. Biomaterial implantation results in an inflammatory reaction, which is anticipated to alter the adaptive immune response, in the case presented here, to a skin xenograft. Our earlier work showed unexpectedly low splenocyte proliferation following a xenogeneic cell implant in tandem with a biomaterial in the form of a microcapsule. Here we explore whether that effect was due to the cells or the biomaterial, and attempt to dissect the mechanism of immune deviation. METHODS: We assessed the immune response of Balb/c mice to hamster skin grafts accompanied by one of three implants: encapsulated xenogeneic cells; free cells accompanied by the same encapsulation biomaterials; and the encapsulation biomaterials without cells. Cells were encapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer then embedded in an agarose gel. Splenocyte proliferation upon re-challenge in vitro, antibody titer in serum, and Th1/2 polarization (by cytokines in splenocyte challenge supernatants and antibody isotypes in serum) were measured. RESULTS: All skin grafts with encapsulation materials (even without cells) suppressed subsequent splenocyte proliferation at 10 days postimplant, although this effect disappeared by two months. In contrast, the antibody response was equal to or greater than that for a skin graft alone. Th1/2 polarization could not explain these observations because it did not correlate with the suppression of splenocyte proliferation. CONCLUSIONS: Implanted biomaterials caused nonspecific, transient suppression of splenocyte responses to hamster cells following hamster skin grafts, which is potentially important in the context of tissue engineering.     

Exhaustive differentiation of alloreactive CD8+ T cells: critical for determination of graft acceptance or rejection

BACKGROUND: The precise role that CD8+ T cells play in the rejection and acceptance of different types of allograft is unclear and has been shown to vary between donor-recipient combinations. METHODS: The response of adoptively transferred CD8+ T cells reactive to the donor alloantigen H2Kb was examined after transplantation of H2Kb liver, kidney, and heart grafts in mice. RESULTS: After transfer of 6 x 10(6) alloreactive CD8+ T cells to T-cell depleted syngeneic mice spontaneous long-term acceptance of liver grafts was observed, whereas kidney and heart grafts were acutely rejected. Within 5 days of liver transplantation, we found that the entire H2Kb-reactive T-cell pool was stimulated to proliferate and differentiate into memory or effector cells that were detectable within lymphoid tissues as well as the liver graft itself. However, despite the generation of effector or memory T cells, liver allografts were accepted, which correlated with the exhaustion or deletion of such cells. In contrast, although activation and proliferation of H2Kb-reactive CD8+ T cells was observed after transplantation of heart or kidney grafts, unactivated, H2Kb-reactive CD8+ T cells were still present in the spleen even long term. Interestingly, differences in the effector function of liver and kidney graft infiltrating donor-reactive CD8+ T cells were not detected after adoptive transfer into immunodeficient mice, despite a reduction in Th1-type cytokines within liver grafts. CONCLUSIONS: The rapid and extensive initial activation and differentiation of donor-reactive CD8+ T cells that occurs after liver transplantation leads to clonal exhaustion or deletion of the alloreactive CD8+ T-cell repertoire resulting in spontaneous tolerance induction.     

Down-regulation of interleukin-2 and interferon-gamma and maintenance of interleukin-4 and interleukin-10 production after administration of an anti-CD3 monoclonal antibody in mice.

BACKGROUND: Activating anti-CD3 monoclonal antibodies (mAbs), such as OKT3, are potent immunosuppressive agents that are widely used in clinical transplantation.We investigated whether the in vivo induction of T cell unresponsiveness contributes to the immunosuppressive properties of the anti-mouse-CD3 mAb 145-2C11. METHODS: After a single in vivo administration of 145-2C11 residual T cells were restimulated in vivo and in vitro to assess cytokine production. Mice were also transplanted with allogeneic skin 9 days after 145-2C11 administration to investigate whether the immunosuppressive properties of the antibody persist after the reexpression of the T cell receptor. RESULTS: Pretreatment with anti-CD3 mAbs caused a profound deficit in both interleukin- (IL) 2 and interferon- (IFN) y secretion upon restimulation in vivo, whereas IL-4 was only partially inhibited and IL-10 production was significantly increased. Purified T cells obtained from mice injected with anti-CD3 mAb also displayed deficient IL-2 and IFN-gamma production together with persisting IL-4 and IL-10 secretion. 145-2C11 had immunosuppressive properties that per sisted after the reexpression of the T cell receptor because mice transplanted with allogeneic skin 9 days after a single anti-CD3 mAb injection still had significantly prolonged graft survival (14.1+/-0.6 days vs. 10.7+/-0.4 days in controls, P<0.02). Blocking IL-4 and IL-10 by neutralizing mAbs further prolonged skin graft survival in mice injected with 145-2C11 (18.3+/-0.7 vs. 14.8+/-0.6 days, P<0.02). CONCLUSION: The in vivo administration of the 145-2C11 anti-CD3 mAb results in the selective inhibition of Thl-type cytokine secretion upon restimulation, which correlates with a state of immunosuppression. The persistent production of Th2-type cytokines does not contribute to the anti-CD3 mAb-mediated prolonged survival of skin allografts in our experimental model.     

Mechanisms of survival prolongation of murine cardiac allografts using the treatment of CTLA4-Ig and MR1

BACKGROUND: [corrected] The present study was undertaken to determine the role of costimulatory blockade in a murine cardiac transplant model. MATERIALS AND METHODS: We blocked the CD28/B7 and CD154/CD40 costimulatory pathways by transient administration of CTLA4-Ig and MR1 antibody to study the effects on allograft survival time, deviation of Th1 and Th2 cytokine secretion, and other mechanisms related to prolonged survival. RESULTS: Costimulatory blockade prolonged the mean survival time (MST) of cardiac allografts to 43 days for the treated group vs 8 days for the untreated group (P < .01). The costimulatory blockade down-regulated the expression of 2 Th1 cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and 2 Th2 cytokines (IL-4 and IL-10), reduced the numbers of graft-infiltrating CD4+ and CD8+ lymphocytes, and inhibited the expression of both perforin/GrB and FasL in allografts. CONCLUSIONS: Combined administration of CTLA4-Ig/MR1 inhibited acute rejection reactions in murine cardiac allografts, prolonging the survival of cardiac grafts through several mechanisms, including inhibition of Th1 and Th2 cytokine expression, graft infiltration by CD4+ and CD8+ T lymphocytes, and reduced both perforin/GrB and Fas-FasL.     

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens

BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed na?ve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or na?ve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either na?ve or tolerant hosts effected rejection. In contrast neither tolerant nor na?ve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.     

In vivo blockade of macrophage migration inhibitory factor prevents skin graft destruction after indirect allorecognition.

BACKGROUND: The effector mechanisms that ultimately destroy transplanted tissues are poorly understood. In particular, it is not clear how CD4+ T cells primed to donor-derived determinants expressed on recipient MHC molecules (the indirect pathway) can mediate graft destruction in the absence of cognate recognition of peptide: MHC on the graft cells themselves. Macrophage migration inhibitory factor (MIF) inhibits macrophage movement and is a proinflammatory and regulatory cytokine known to be essential for development of delayed-type hypersensitivity reactions. METHODS: To test whether MIF participates in graft destruction following indirect recognition, we studied rejection of MHC-II-deficient skin grafts placed on allogeneic SCID recipients adoptively transferred with na?ve CD4+ T cells, and the recipients were treated with neutralizing anti-MIF monoclonal antibody or isotype control IgG. In this model graft rejection can only occur indirectly as the graft cells lack MHC II for recognition by the recipient CD4+ T cells. RESULTS: We found that in vivo blockade of MIF inhibited indirect CD4+ cell-mediated skin graft destruction, and markedly reduced detectable macrophages within the grafts. The neutralizing anti-MIF antibody significantly inhibited alloreactive DTH but did not prevent T cell priming or interferon-gamma release by primed T cells. CONCLUSIONS: The results strongly implicate MIF as an active participant in skin graft destruction after indirect recognition and suggest that this effect is mediated through an inhibition of macrophage migration and/or function.     

Protective genes expressed in endothelial cells of second hamster heart transplants to rats carrying an accommodated first graft

Abstract: Accommodation refers to survival of a xenograft despite the presence of anti-donor organ antibodies and complement. We have recently shown that accommodation of a hamster heart transplanted to a rat receiving short-term cobra venom factor (CVF) and continuing cyclosporine A (CyA) therapy is associated with i) the expression in the endothelial cells (EC) and smooth muscle cells of the graft of a number of "protective" genes, ii) a prominent intragraft Th2 cytokine profile, and iii) the relatively heavy deposition of IgG2c antibodies on the EC of the graft. In contrast, rejecting grafts do not express the protective genes, have a Th1 cytokine profile, and apparently have lesser amounts of IgG2c. These findings are consistent with host factors (Th2 cytokines and IgG2c) contributing to xenograft accommodation. To test whether these host factors may predispose to the development of accommodation, we placed a second hamster heart into each of 12 rats carrying a surviving first heart; recipients were, at the time, receiving only CyA. Whereas first grafts transplanted to rats receiving only CyA survive for 3 to 4 days, 11 out of 12 second transplants survived more than 20 days, and the other survived for 7 days. Nine of the twelve were not rejected: of these, four were removed between day 35 and 132 for study, and the remainder are still beating at 35 to 52 days. The surviving second hearts we studied had accommodated in that the picture on immunopathology was the same as for surviving first hearts. We suggest that the Th2 cytokines and perhaps the IgG2c response are factors in allowing prolonged survival of the second grafts and, further, that these factors contribute to the expression in the EC and smooth muscle cells of the surviving second hearts of the protective genes.     

Induction of Donor-Specific Allograft Tolerance by Short-Term Treatment with LF15-0195 After Transplantation. Evidence for a Direct Effect on T-Cell Differentiation

A 20-day treatment with LF15-0195, a deoxyspergualine analog, induced long-term heart allograft survival in the rat without signs of chronic rejection. LF15-0195-treated recipients did not develop an anti-donor alloantibody response. Analysis of graft-infiltrating cells, IL10, TNFalpha, IFNgamma mRNA and iNOS protein expression in allografts, 5 days after transplantation, showed that they were markedly decreased in allografts from LF15-0195-treated recipients compared with allografts from untreated recipients. Surprisingly, spleen T cells from LF15-0195 recipients, 5days after grafting, were able to proliferate strongly in vitro, when stimulated with donor cells, but had reduced mRNA expression for IFNy compared with spleen T cells from untreated graft recipients. Furthermore, when T cells from naive animals were stimulated in vitro, using anti-CD3 and anti-CD28, LF15-0195 also increased T-cell proliferation in a dose-dependent fashion: however, these cells expressed less of the Th1 -related cytokines, IFNgamma and IL2, compared with untreated cells, suggesting that LF15-0195 could act on T-cell differentiation. In conclusion, we show here that a short-term treatment with LF15-0195 induced long-term allograft tolerance, decreasing the in situ anti-donor response, and we illustrate evidence for the development of regulatory mechanisms.     

Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model

Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0±1.8 days and xenografts in 6.5±1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4+ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.