CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Complement depletion with cobra venom factor delays acute cell‐mediated rejection in pig‐to‐mouse corneal xenotransplantation

Oh JY, Kim MK, Lee HJ, Ko JH, Kim Y, Park CS, Kang HJ, Park CG, Kim SJ, Lee JH, Wee WR. Complement depletion with cobra venom factor delays acute cell‐mediated rejection in pig‐to‐mouse corneal xenotransplantation. Xenotransplantation 2010; 17: 140–146. © 2010 John Wiley & Sons A/S. Abstract: Background: We have demonstrated earlier that porcine corneal xenografts underwent an acute cell‐mediated rejection in mice despite the absence of T cells. In the present study, we investigated the effect of complement depletion by cobra venom factor (CVF) on the corneal xenograft rejection in a pig‐to‐mouse model. Methods: Porcine corneas were orthotopically transplanted into C57BL/6 (B6) and severe combined immunodeficiency (SCID) mice. For complement depletion, 25 μg of CVF (1 g/kg bodyweight) was injected intraperitoneally on the day before and 1, 3, 5, and 7 days after transplantation. Graft survival was clinically assessed by slit lamp biomicroscopy and the median survival time (MST) was calculated. The grafts were histologically evaluated serially after transplantation using antibodies against CD4, CD8, NK1.1, and F4/80. Results: The CVF treatment significantly prolonged the porcine corneal xenograft survival in both B6 (MST 9.4 vs. 15.5 days; P = 0.0011) and SCID mice (MST 16.4 vs. 20.5 days; P = 0.0474). Histologically, whereas macrophages and CD4⁺ T cells were progressively infiltrated into porcine corneal grafts in CVF‐untreated B6 mice, the infiltration by both cells was markedly delayed and decreased in the xenografts in CVF‐treated B6 mice. Likewise, macrophage infiltration, which was prominent in rejected porcine xenografts in SCID mice, was also reduced in CVF‐treated SCID mice. Conclusions: Our results suggest that complement depletion by CVF delayed, although did not prevent, an acute cell‐mediated rejection in a pig‐to‐mouse corneal xenotransplantation.     

Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

CD8 T Cells Can Reject Major Histocompatibility Complex Class I-Deficient Skin Allografts

Following transplantation, recipient T cells can recognize and respond to donor antigens expressed directly on donor cells, and can respond to donor-derived peptides that have been processed and presented in the context of recipient MHC through the indirect pathway. Indirectly primed CD4⁺ T cells have been well studied in transplantation, but little information is available regarding whether indirectly primed CD8⁺ T cells participate in rejection. To address this, we placed MHC class I-deficient D^bK^b knockout skin grafts onto allogeneic H-2 ^k SCID recipients followed by adoptive transfer of purified H-2 ^k CD8⁺ T cells. The MHC class I-deficient grafts were rejected and only CD8⁺ T cells were detectable in the recipient lymphoid organs and in the skin grafts. Immunohistochemical analysis showed that CD8⁺ T cells were found in close proximity to vascular endothelial cells and to recipient infiltrating macrophages, suggesting specific interactions. The data demonstrate that cross-primed polyclonal CD8⁺ T cells can function as active participants in the effector phase of rejection. The findings confirm and extend previous studies using a monoclonal TCR transgenic T cell and shed light on mechanisms of acute and chronic graft injury that are potentially relevant to human transplant recipients.     

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice

Abstract: Mouse CD4⁺ T cells efficiently develop in fetal pig thymus (FPTHY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8⁺ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of Vβ5.1/5.2⁺ and Vβ11⁺ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of Vβ6, Vβ7, and Vβ8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthyrnic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8⁺ cells can undergo normal functional and phenotypic maturation in FPTHY grafts. Thus, other explanations must be sought for the failure of CD8'cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THYLIV-grafted mice.     

Blockade of indirect recognition mediated by CD4+ T cells leads to prolonged cardiac xenograft survival

Abstract:  The T-cell response to xenografts is induced by direct and indirect recognition of xenoantigens. Although the importance of indirect recognition is well established in vitro, the contribution of this pathway to xenograft rejection in vivo remains to be fully elucidated. We herein investigated the direct contribution of indirect recognition to cardiac xenograft rejection in the rat-to-mouse (PVG.R8-to-C57BL/10) concordant model. Rat xenoantigens invoked a vigorous proliferative response in mouse T cells harvested from naïve or graft recipients at rejection. Indirect recognition predominated the response, as antibodies against mouse class II I-A^b, CD80, or CD86 molecules significantly (45 to 60%) blocked the proliferative response. Importantly, the blockade of indirect recognition in vivo by treating the graft recipients with a monoclonal antibody (mAb) against class II I-A^b molecule on days 0, 1, and 3 post-transplantation resulted in significant ( P  < 0.009) prolongation of cardiac xenograft survival (Mean Survival Time (MST) >94 ± 55 days vs. 7 ± 0.8 days for controls). In contrast, treatment of recipients with a mAb against mouse class I H-2K^b/D^b molecules did not significantly affect graft rejection (MST = 8 ± 1 days). These results demonstrate that indirect recognition mediated by CD4⁺ T cells plays a critical role in the rejection of cardiac grafts in the rat-to-mouse xenogeneic model.     

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor-Specific CD8+ Memory T Cells

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8⁺ T-cell responses in mice that lack the IFN-I receptor (IFN-IR^−/−). We found that IFN-IR^−/− mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR^−/− male skin was transplanted to syngeneic IFN-IR^−/− female mice. Quantitation of the male (H-Y)-specific CD8⁺ T-cell response in these mice revealed normal generation of donor-specific CD8⁺ effector T cells but fourfold reduction in CD8⁺ memory T cells. Memory CD8⁺ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR^−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8⁺ T cells into long-lived, functional memory T cells.     

Differential Roles of CD8+ and CD8− T Lymphocytes in Corneal Allograft Rejection in ''High-Risk'' Hosts

We examined the role of perforin and FasL in corneal allograft rejection mediated by CD8+ and CD8 T cells. BALB/c corneas were transplanted orthotopically into vascularized, 'high-risk' graft beds in C57BL/6 mice, perforin knockout mice and FasL-defective gld/gld mice. CD8+ and CD8 T cells were collected following graft rejection and adoptively transferred to SCID mice, which were then challenged with BALB/c corneal allografts. In every case, CD8 T cells could mediate graft rejection when adoptively transferred to SCID mice that received BALB/c corneal allografts. Although CD8+ T cells also mediated graft rejection, the tempo was slower. Moreover, CD8+ T cells collected FasL-defective donors that had rejected corneal allografts, mediated corneal allograft rejection in only 50% of the SCID mice that received the adoptively transferred cells. In some cases, CD8+ T-cell-mediated rejection occurred in the absence of delayed-type hypersensitivity and cytotoxic T-lymphocyte activity, but was associated with CD8+ T-cell-mediated apoptosis of BALB/c corneal cells in vitro. The results demonstrate the redundancy in immune mechanisms of corneal allograft rejection. Either CD8+ or CD8 T cells can produce corneal allograft rejection, however functional FasL is necessary for optimal rejection, even in a high-risk setting.     

CD4+ T-cell-independent rejection of corneal allografts

BACKGROUND: Several studies suggest that a significant number of corneal allografts undergo rejection in the absence of CD4 T cells. This study examined the role of CD4 T cell-independent mechanisms of corneal allograft rejection. METHODS: BALB/c corneal allografts were transplanted to C57BL/6 beige nude mice that received either CD8 or CD8 T cells from C57BL/6 CD4 knockout (KO) mice that had rejected BALB/c corneal allografts. Immune effector functions of CD8 or CD8 T cells from C57BL/6 CD4 KO mice were assessed using delayed-type hypersensitivity assays and Annexin V apoptosis assays respectively. RESULTS.: Both CD8 and CD8 T cells from CD4 KO corneal allograft rejector mice mediated corneal allograft rejection following adoptive transfer to nude mice. CD8 T cells, but not CD8 T cells, from CD4 KO mice adoptively transferred donor-specific DTH and induced apoptosis of BALB/c corneal endothelial cells in vitro. Apoptosis of BALB/c corneal endothelial cells was mediated by double negative (DN) T cells, as treatment of CD8 cells from CD4 KO mice with anti-Thy 1.2 plus complement abolished their effector function. CONCLUSION: The results support the proposition that CD4 T cell-independent rejection of corneal allografts can be mediated by either CD8 or CD8 T cells. The CD8 T cells represent a unique DN T cell population that might mediate rejection by either direct cytolysis or by inducing apoptosis of the donor corneal endothelium.     

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.     

Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice

INTRODUCTION: The purpose of the current study was to investigate the capacity of CD4+, CD8+, or non-T cells to independently initiate acute rejection of allogeneic hepatocytes using reconstituted SCID, CD4 or CD8 knockout (KO) recipient mice. METHODS: Allogeneic hepatocytes (FVB/N, H-2q) were transplanted into C57BL/6.SCID (H-2b), CD4 KO (H-2b), CD8 KO (H-2b), or beige/beige (H-2b) mice. SCID mice with functioning hepatocellular allografts subsequently received purified non-T cells (NTC), CD4+, or CD8+ splenocytes. Some mice were treated with anti-CD4, anti-CD8, and/or anti-nkl.1 mAb. Recipient mice were also assessed for donor-reactive delayed-type hypersensitivity (DTH) responses and donor-reactive alloantibody production. RESULTS: Median hepatocellular allograft survival time (MST) was 28 days in CD4+ reconstituted SCID mice and 14 days in CD8+ reconstituted SCID mice. SCID hosts reconstituted with NTC demonstrated indefinite hepatocellular allograft survival (>120 days). MST was 10 days in untreated beige/beige (NK cell deficient) mice. MST was 14 days in untreated, 35 days in anti-CD4 mAb treated, and 10 days in anti-nkl.1 mAb treated CD8 KO mice. MST was 10 days in untreated, 35 days in anti-CD8 mAb treated, and 7 days in anti-nk1.1 mAb treated CD4 KO mice. Donor-reactive DTH responses were not detected in reconstituted SCID mice, were minimal in CD4 KO mice, and were prominent in CD8 KO mice after rejection of allogeneic hepatocytes. Similarly, donor-reactive alloantibody, was not detected in CD4 KO hosts, but was readily detected in CD8 KO hosts. CONCLUSIONS: These studies show that both CD4+ and CD8+ T cells (but not host NTC) can independently initiate the rejection of allogeneic hepatocytes. While hepatocyte rejection by isolated CD4+ T cells is not surprising, rejection by CD8+ T cells (in the absence of CD4+ T cells) was unusual, and may explain the failure of "standard" immunosuppressive regimens to suppress acute rejection of allogeneic hepatocytes, as noted in prior studies. Furthermore, NK cells do not appear to be required for either CD4+ T cell or CD8+ T cell initiated hepatocyte rejection.     

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

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.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with rejection.     

Atorvastatin enhances humoral immune responses but does not alter renal injury in experimental crescentic glomerulonephritis

Aim:   Statins are widely used for their cholesterol-lowering effects and for prevention of cardiovascular disease. Evidence indicates that these drugs also have immunomodulatory and other non-lipid lowering effects, with studies suggesting benefit in some animal models of immune (particularly T helper (Th)1)-mediated inflammatory disease and their corresponding human disease counterparts. We sought to evaluate the immunomodulatory effects and therapeutic potential of atorvastatin in experimental crescentic glomerulonephritis, a Th1-predominant animal model of glomerulonephritis.
Methods:   Autologous phase, anti-glomerular basement membrane glomerulonephritis was induced in C57BL/6 mice by intravenous injection of sheep anti-mouse glomerular basement membrane globulin. Mice were administered atorvastatin (10 or 100 mg/kg) or control (phosphate-buffered saline) daily by oral gavage. Immune responses and renal injury were assessed after 21 days.
Results:   Compared with control-treated mice, treatment with atorvastatin did not alter renal injury (serum creatinine, proteinuria, glomerular crescent formation) or glomerular leukocytic infiltration (CD4⁺ T cells or macrophages). Atorvastatin resulted in a dose-related increase in circulating serum antibody to the disease-inducing antigen but no differences in antigen-stimulated splenocyte production of Th1/Th2 cytokines. At the higher dose, atorvastatin also led to a significant reduction in apoptosis of splenic CD4⁺ T lymphocytes.
Conclusion:   This study demonstrates that statins modulate humoral responses and alter splenic CD4⁺ T cell apoptosis. However, atorvastatin does not lead to significant changes in T helper cell polarization or renal injury in experimental crescentic glomerulonephritis.     

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

Aim:   To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4^- CD8⁺ TIL-clone derived from human renal cell carcinoma (RCC).
Methods:   Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8⁺ enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed.
Results:   A clone, T16-13, by day 94 numbering 1 × 10⁷ cells, was harvested and characterized as a CD4^- CD8⁺ clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 ± 0.7 LU₃₀/10⁶ cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (−0.1 LU₃₀/10⁶ cells).
Conclusions:   A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.