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.     

Xenoreactive CD4+ memory T cells resist inhibition by anti-CD44 mAb and reject islet grafts via a Th2-dependent pathway

Peng YZ, Chen JB, Shao W, Wang FY, Dai HL, Cheng PP, Xia JJ, Wang F, Huang R, Zhu Q, Qi Z. Xenoreactive CD4⁺ memory T cells resist inhibition by anti‐CD44 mAb and reject islet grafts via a Th2‐dependent pathway. Xenotransplantation 2011; 18: 252–261. © 2011 John Wiley & Sons A/S. Abstract: Background: Memory T cells are a significant barrier to the induction of transplant tolerance. Our previous study demonstrated that multiple applications of anti‐CD44 monoclonal antibody (mAb) could significantly inhibit CD4⁺ memory T cells from mediating rejection of cardiac allografts. Now, we sought to explore the effect and mechanism of anti‐CD44 mAb on the rejection of islet allografts and xenografts mediated by CD4⁺ memory T cells. Methods: In this study, we first engrafted skin grafts of C57BL/6 (B6) mice or Dark Agouti (DA) rats onto BALB/c mice to induce donor‐reactive memory T cells. We adoptively transferred purified CD4⁺ memory T cells to BALB/c origin nude mice and then transplanted islet allografts and xenografts to produce the Allo‐Tx and Xeno‐Tx models, respectively. We subsequently administered multiple anti‐CD44 mAb and observed changes in the survival times of the islet grafts. Results: In the Allo‐Tx model, the mean survival time (MST) of the grafts was 7.7 days in the isotype group, and 20.3 days in the anti‐CD44 group. In the Xeno‐Tx model, the MST of the grafts was 7.2 days in the isotype group and 8.2 days in the anti‐CD44 group. Compared with the isotype group, CD4⁺ T cells on the grafts in the anti‐CD44 group were significantly decreased in both the Allo‐Tx and Xeno‐Tx models, but the proportion of CD4⁺ memory T cells in the spleens and draining lymph nodes of the recipient nude mice in the anti‐CD44 group was significantly decreased in the Allo‐Tx model, while it was increased in the Xeno‐Tx model. The production of donor‐specific IgG antibody in the anti‐CD44 group did not vary in the Allo‐Tx model, while it was markedly elevated in the Xeno‐Tx model. Furthermore, the expression of interferon gamma in the anti‐CD44 group was markedly decreased in both the Allo‐Tx and Xeno‐Tx models, while the expression of IL‐4 in the anti‐CD44 group was significantly increased only in the Xeno‐Tx model. Conclusion: Multiple applications of the anti‐CD44 mAb could significantly inhibit donor‐reactive CD4⁺ memory T cells from rejecting grafts via a Th1‐dependent pathway, but xenoreactive CD4⁺ memory T cells can avoid the effects of anti‐CD44 mAb to reject islet xenografts via a Th2‐dependent pathway.     

Rejection of grafts with no H-2 disparity in TAP1 mutant mice: CD4 T cells are important effector cells and self H-2b class I molecules are target

Our previous results showed that TAP1 mutant mice rejected heart and skin grafts from donors with no H-2 disparity that express normal density of MHC class I molecules at the cell surface. During rejection, CD4 cells were predominant and essentially, no CD8 cells were found infiltrating the grafts. We hypothesized that TAP1 mutant mice, which developed and matured in an MHC class I-deficient environment, may have selected a repertoire of T cells with distinct reactivity to self class I molecules. The rejection of grafts with no H-2 disparity could be mediated by CD4+ T cells reactive to wild type H-2b class I molecules, or derived peptides, in the context of self-APC. Accordingly, we observed that transplanted TAP1 mutant mice presented a significant amplification of the proliferative T cell response to H-2Kb peptides, indicating that the stimulus with the graft was sufficient to induce peripheral expansion of these T cell repertoires. Therefore, the response to H-2Kb molecules could be a relevant pathway of activating T cells and triggering rejection of grafts expressing normal levels of these class I molecules. To test our hypothesis, we investigate the effect of pre-transplantation H-2Kb peptide-immunization on TAP1 mutant, which were then transplanted with C57BL/6 skin grafts (H-2b). Mice were immunized with a pool of five peptides derived from the polymorphic region of Kb alpha chain, before tail skin grafting. To study the role of CD4+ T cells in the rejection of C57BL/6 skin grafts, mice were in vivo depleted with an anti-CD4 monoclonal antibody GK1.5, and transplant evolution was observed. Sensitization of TAP1 mutant mice with H-2Kb peptides accelerated the rejection of skin grafts. Immunized mice rejected grafts with a MST of 13 days, compared to 16 days for the non-immunized mice (P=0.0089). The significant acceleration of graft rejection, induced by immunization with H-2Kb peptides, indicates that these peptides are capable of mobilizing effector T-cells that participate in rejection. These results support our hypothesis that class I molecules may be a target in the rejection of grafts with no MHC disparity. Depletion of CD4 T-cells resulted in a significant delay in rejection compared with the untreated control group. The MST of skin grafts in the controls was 16 days, whereas CD4-depleted recipients rejected skin grafts with a MST of 41 days (P=0.025). Moreover, some animals did not show macroscopic signs of rejection up to > 100 days posttransplantation. The contribution of CD4+ T cells to skin graft rejection, in our model, may reflect the occurrence of the presentation of H-2b peptides during graft rejection, in the context of self-APC. In conclusion, our results demonstrate an important role for H-2b molecules and CD4 T cells in the rejection of C57BL/6 grafts by TAP1 mutant mice. The low expression of MHC-I molecules on TAP1-/- mice may be determinant in the selection of a T cell repertoire strongly reactive to self MHC class I molecules which probably escapes the control of peripheral regulatory mechanisms.     

Transient Lymphopenia Breaks Costimulatory Blockade‐Based Peripheral Tolerance and Initiates Cardiac Allograft Rejection

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long‐term graft acceptance. After vascularized MHC‐mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti‐CD4 plus anti‐CD8 mAb. Long‐term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44^high effector/memory T cells in lymphatic organs and strong recovery of donor‐reactive T cell responses, indicating lymphopenia‐induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4⁺ Foxp³⁺) cell and B cell numbers as well as donor‐specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor‐reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.     

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.     

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

Endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts within 12–24 h posttransplant in mice and are activated to proliferate and produce IFN‐γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti‐LFA‐1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG‐treated recipients on day 7 posttransplant, allografts from anti‐LFA‐1 mAb‐treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor‐reactive CD8 T cells producing IFN‐γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti‐LFA‐1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG‐3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.     

Rapamycin Interferes With Postdepletion Regulatory T Cell Homeostasis and Enhances DSA Formation Corrected by CTLA4‐Ig

Previously, we demonstrated that alemtuzumab induction with rapamycin as sole maintenance therapy is associated with an increased incidence of humoral rejection in human kidney transplant patients. To investigate the role of rapamycin in posttransplant humoral responses after T cell depletion, fully MHC mismatched hearts were transplanted into hCD52Tg mice, followed by alemtuzumab treatment with or without a short course of rapamycin. While untreated hCD52Tg recipients acutely rejected B6 hearts (n = 12), hCD52Tg recipients treated with alemtuzumab alone or in conjunction with rapamycin showed a lack of acute rejection (MST > 100). However, additional rapamycin showed a reduced beating quality over time and increased incidence of vasculopathy. Furthermore, rapamycin supplementation showed an increased serum donor‐specific antibodies (DSA) level compared to alemtuzumab alone at postoperation days 50 and 100. Surprisingly, additional rapamycin treatment significantly reduced CD4⁺CD25⁺FoxP3⁺ T reg cell numbers during treatment. On the contrary, ICOS⁺PD‐1⁺CD4 follicular helper T cells in the lymph nodes were significantly increased. Interestingly, CTLA4‐Ig supplementation in conjunction with rapamycin corrected rapamycin‐induced accelerated posttransplant humoral response by directly modulating Tfh cells but not Treg cells. This suggests that rapamycin after T cell depletion could affect Treg cells leading to an increase of Tfh cells and DSA production that can be reversed by CTLA4‐Ig.     

CD4+ T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferon-gamma

BACKGROUND: T cells responding through the indirect pathway can induce allograft rejection, but mechanisms of rejection are not known. Interferon-y (IFN-gamma) may be an important mediator of rejection under these circumstances. METHODS: We transferred CD4+ T cells from IFN-gamma-deficient (IFN-gamma-/-) mice into SCID recipients of MHC II-deficient (MHC II-/-) skin grafts. Under these conditions, rejection can only occur via the indirect pathway and cannot be mediated by T-cell production of IFN-gamma. RESULTS: IFN-gamma-/- CD4+ T cells rejected MHC II -/- skin grafts. Flow cytometry revealed only CD4+ T cells in the recipients. Cytokine enzyme-linked immunosorbent spot assays confirmed only indirect recognition with an associated expansion of an alloreactive population of IL-2-, IL-4-, and IL-5-secreting T cells. CONCLUSION: CD4+ T cells recognizing alloantigens via the indirect pathway can mediate skin graft rejection in the absence of IFN-gamma.     

An immunosufficient murine model for the study of human islets

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti‐CD45RB. To assess whether anti‐CD45RB‐induced transplant tolerance requires B cells, B6 recipients received additional anti‐CD20 or B6μMT?/? mice were used. For some anti‐CD45RB‐treated B6μMT?/? mice, additional anti‐CD25 mAb was applied at the early or late stage post‐transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti‐CD45RB/anti‐CD20‐treated Foxp3‐GFP B6 mice. The results showed that anti‐CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti‐CD20, and 88.9% of xenografts accepted in anti‐CD45RB‐treated μMT?/? mice. These μMT?/? mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti‐CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post‐transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.     

The high‐risk corneal regraft model: a justification for tissue matching in humans

Models of high‐risk corneal graft rejection involve neovascularization induced via innate immune responses, e.g., suture‐mediated trauma. We describe a model of high‐risk corneal graft rejection using corneal graft donor‐recipient pairing based on a single‐antigen disparity. Donor corneas from transgenic mice on B10.BR (H‐2^k) background, in which hen‐egg lysozyme (HEL) as a membrane‐bound antigen (mHEL) was expressed under the major histocompatibility complex (MHC) Class I promoter (KLK‐mHEL, H‐2^k), were transplanted into wild type B10.BR recipient mice. Unmanipulated wild type recipient mice rejected KLK‐mHEL grafts (39%) slowly over 50–60 days. Graft rejection incidence was maximized (100%) and tempo accelerated (27 days) by priming with HEL‐pulsed syngeneic dendritic cells and less so by increasing T‐cell precursor frequency. Rejection also reached maximum levels (100%) and tempo (3–8 days) when mice which had rejected a first graft (‘rejectors’) were regrafted, and was associated with induction of HEL‐specific memory T cells. In contrast, ‘acceptors’ rejected a second graft at rates and tempo similar to naïve mice. These data reveal the importance of (i) donor MHC antigens as alloantigens for indirect recognition, (ii) alloantigen‐specific memory in high‐risk graft rejection involving regrafts, and (iii) suggest a role for tissue matching in human corneal graft to avoid sensitization to donor MHC antigens.     

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice

Abstract: Background: We have studied cellular components required for xenograft survival mediated by anti‐CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4⁺ but not CD8⁺ cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade‐induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Methods: Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jα18^null, CD1^null, or beta2 microglobulin (β2M)^null NK 1.1 depleted, or perforin^null. Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti‐CD154 mAb treatments. Additional groups received mAb or cells only. Results: We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jα18 component of the T‐cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jα18^null and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jα18^null recipients. These data imply a role for Jα18⁺ NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1^null mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1⁺ cells adversely affects rat skin graft survival. An additional assay in β2M^null mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin‐secreting NK cells. Conclusions: We conclude that MHC class I⁺ CD1⁺ Jα18⁺ NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti‐CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.     

Combination of antibodies inhibits accelerated rejection mediated by memory T cells in xenoantigen-primed mice

Wang F, Xia J, Chen J, Peng Y, Cheng P, Ekberg H, Wang X, Qi Z. Combination of antibodies inhibits accelerated rejection mediated by memory T cells in xenoantigen‐primed mice. Xenotransplantation 2010; 17: 460–468. © 2010 John Wiley & Sons A/S. Abstract: Background: Donor‐reactive memory T cells are known to accelerate allograft rejection; in our previous study, we reported that combined monoclonal antibodies (mAbs) could prolong islet allograft survival in alloantigen‐primed mice. In this study, we examine the effects of donor‐reactive memory T cells on the xenograft survival and methods to prolong the islet graft survival. Methods: To collect donor‐reactive T cells, we performed full‐thickness rat skin xenografting on BALB/c mice and isolated the T cells from the mice after 6–8 weeks. These cells were then adoptively transferred to syngenic mice 1 day before rat‐to‐mouse islet transplantation. Three experimental groups were established in the adoptive transfer model: recipient mice treated with isotype mAbs (isotype group); mice treated with anti‐CD40L mAb (anti‐CD40L group); and mice treated with anti‐CD40L, anti‐OX40L, and anti‐CD122 mAbs (3‐combined group). Results: Lewis rat islet xenografts transplanted in naïve mice showed a mean survival time (MST) of 12.8 days, while the graft rejection was accelerated if the recipient mice were treated with adoptively transferred donor‐reactive T cells (MST, 8.67 days). Treatment with anti‐CD40L mb could not reverse the accelerated rejection (MST, 9.3 days). However, when anti‐CD40L mb was combined with anti‐OX40L and anti‐CD122 mAbs, there was a considerable increase in the MST, which was 72.2 days. Compared to the isotype group, the 3‐combined group had significantly lesser proportion of memory T cells and greater proportion of regulatory T cells (Tregs) in the spleen. Meanwhile, in the 3‐combined group, the production of anti‐rat antibodies was markedly inhibited. Conclusion: Treatment with a combination of antibodies could significantly reverse the accelerated rejection mediated by donor‐reactive memory T cells by inhibiting cellular and humoral immune responses.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts

Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.     

Development of autoimmunity after skin graft rejection via an indirect alloresponse

BACKGROUND: T cell allorecognition occurs through direct contact with donor peptide: MHC complexes on graft cells and through indirect recognition of donor-derived determinants expressed by recipient MHC molecules. As both indirect allorecognition and autoantigen recognition are self-restricted, we hypothesized that chronic activation of indirectly primed T cells might result in determinant spreading to involve autoantigens, analogous to that which occurs during chronic autoimmune diseases. METHODS: We placed C57BL/6 MHC II knockout (B6 II-/-) skin grafts onto BALB/c SCID mice reconstituted with wild-type (WT) CD4+ T cells. Under these conditions the CD4+ cells could not recognize any antigen on the graft, but could respond through the indirect pathway. CD4+ cell-mediated rejection of WT B6 skin was studied to determine if autoreactivity was induced after direct allorecognition. Recall immune responses against donor- and self-stimulator cells were determined by ELISPOT and animals were tested for their ability to reject second isografts. RESULTS: WT allografts were rejected by day 14 although B6 II-/- grafts underwent delayed rejection over 4-5 weeks. CD4+ cells reisolated from the recipients of the MHC II-/- grafts, but not from the recipients of WT grafts, vigorously produced interferon-gamma and interleukin-2 in response to self, BALB/c stimulators. These autoreactive CD4+ T cells mediated rejection of a second isogenic BALB/c skin graft, demonstrating that the autoimmune response was pathogenic. CONCLUSION: Autoreactivity can develop after transplant rejection via the indirect pathway. Although the direct alloresponse is likely to be the driving force in acute graft rejection, posttransplantation induced autoimmune responses may be important elements of delayed or chronic rejection.     

Regulatory T Cells Are Critical to Tolerance Induction in Presensitized Mouse Transplant Recipients Through Targeting Memory T Cells

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40⁺CD44^hieffector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti‐OX40L monoclonal antibody (mAb) (α‐OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, α‐OX40L was added to the combined treatment protocol of LF15–0195 (LF) and anti‐CD45RB (α‐CD45RB) mAb—a protocol that induced heart allograft tolerance in non‐presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor‐specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs). Of note, CD25⁺ T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor‐primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.     

Anti‐huCD20 Antibody Therapy for Antibody‐Mediated Rejection of Renal Allografts in a Mouse Model

We have reported that B6.CCR5^?/? mice reject renal allografts with high serum donor‐specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody‐mediated rejection (AMR). B6.huCD20/CCR5^?/? mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti‐huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long‐term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti‐huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti‐huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.

     

Cellular pathways for rejection of class-I-MHC--disparate skin and tumor allografts

We have investigated the relative roles of the Lyt-2+ and L3T4+ T lymphocyte subsets in rejection of class-I-MHC-antigen-disparate skin and tumor allografts. To deplete T cells in vivo, rat anti-Lyt-2 or anti-L3T4 monoclonal antibodies (mAb) were administered to adult-thymectomized (ATX) recipient mice prior to transplantation. BALB/c (H-2d) recipient mice rejected the Ia- Sarcoma I (Sa1) (H-2a) tissue culture-derived tumor after depletion of the L3T4+ T cell subset in vivo. In contrast, depletion of the Lyt-2+ T cell subset permitted lethal tumor growth in all recipient mice. To determine the role of particular T cell subsets in rejection of Ld class-I-MHC-antigen-disparate allografts, BALB/c skin was transplanted to BALB/c-H-2dm2 recipient mice. Skin grafts were rejected by control mice with a mean survival time (MST) of 14.5 days. The MST of skin grafts for mice treated with anti-L3T4 mAb was 16.6 days. In contrast, administration of anti-Lyt-2 mAb alone (MST = greater than 47 days) or together with anti-L3T4 mAb (MST = greater than 50 days) caused prolonged or indefinite graft survival in all recipient mice. Depletion of specific T cell subsets was confirmed by flow cytometric analysis and by analysis of T cell function in vitro. These results suggest that Lyt-2+ T lymphocytes are essential for rejection of class-I-MHC-disparate allografts; indirect presentation of alloantigen to L3T4+ T cells may not be necessary for rejection.     

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.