Cytokine gene expression by alloactivated cells in SCID mice

OBJECTIVES: The severe combined immune deficient (SCID) mouse provides a neutral environment to study human immune responses. We therefore tested human gene expression of Interleukin (IL) 2, 4 and 10, interferon gamma (IFNgamma); transforming growth factor beta 1 (TGFbeta1); and CD40 ligand (CD40L) in splenic extracts of SCID mice after engraftment of PBLs from two persons (direct MLR) or one person plus allopeptides (indirect MLR) in the presence or absence of cyclosporin A (CsA) or FK506. METHODS: Cytokine gene expression was detected by RT and quantitative (for IFN-gamma, TGFbeta1 and CD40L) PCR. All cells, allopeptides, CsA (25 mg/kg/day for 7 days) or FK 506 (0.5 mg/kg/day for 7 days) were administered intraperitoneally (IP). RESULTS: In both direct and indirect MLR the numbers of SCID mice expressing the human cytokine genes varied between 33% for IL4 and 100% for IL10, IFN-gamma, TGFbeta1, and CD40L. There was significant interpersonal variation in levels of gene expression. Concomitant CsA or FK506 administration for 7 days did not abrogate early or late (1 week after discontinuation of CsA or FK506) cytokine gene expression in either the direct or indirect MLR, but paradoxically enhanced levels of IFN-gamma, TGFbeta1 and CD40L gene expression in some experiments. CONCLUSIONS: The results explain late rejection after rapid calcineurin inhibitor withdrawal or reduction, and illustrate the potential use of SCID mice as a surrogate model to study graft outcome by determination levels of gene expression and sensitivity to immunosuppressive agents in the in vivo alloresponse.     

Natural antibodies do not inhibit xenogeneic transplantation of human PBL in lethally irradiated mice

Abstract: Attempts to transfer human peripheral blood lymphocytes (hu-PBL) into lethally irradiated mice resulted in limited engraftment in recipients lacking natural antibodies (nAb) and could not be achieved in immunologically normal mice. It has been proposed that nAb with antihuman specificity play a major role in the rejection of the hu-PBL graft. In the present study we demonstrate that, following intensification of the conditioning protocol (thymectomy, supralethal dose of TBI, and radioprotection with bone marrow for donors with severe combined immune deficiency (SCID), transplants of 50 to 70 × 10⁶ hu-PBL were successfully engrafted in BALB/c, CBA/J and C3H/HeJ mice—regardless of the initial high levels of nAb. The percentage of human CD45⁺ cells in peritoneal lavage was not statistically different from that obtained in congenitally immune-deficient corresponding strains (SCID and CBA/N) lacking natural antibodies. Significant differences in engraftment of hu-PBL, between different human donors, were related neither to the nAb content (r = 0.29) nor to the ABO(H) blood group. The transfer of serum with high level of nAb into SCID and CBA/N mice or incubation of hu-PBL in such a serum prior to implantation, did not impede the engraftment and did not decrease the production of human immunoglobulins. These data demonstrate that the presence of nAb in supralethally irradiated normal mice does not inhibit the engraftment of hu-PBL, emphasizing the role of cellular mediated mechanisms in xenograft rejection.     

CD40/CD40L和B7-1/CD28在小鼠叶酸性肾病中的作用

目的研究CD40／CD40L、B7—1／CD28在肾小管间质浸润的免疫活性细胞、损伤的肾组织固有细胞上的表达以及其可能的作用。方法一次性腹腔注射叶酸制作CD1小时鼠叶酸性肾病模型。在第1、2、3、7、14、21天分别处死动物，取其右肾进行免疫组织化学染色；取左肾提取蛋白进行蛋白印迹分析；采血检测BUN、Scr。以抗B7—1功能性单克隆抗体（B7-1mAb）联合叶酸应用，观察21d后，采血行BUN、Scr检测，病理图像分析肾病变区域及保护率。结果小鼠在给予叶酸后第1天，CD40、B7-1在肾小管上皮表达上调，并持续至第21天。通过蛋白印迹半定量检测，CD40在各时间点实验组肾组织表达量均增加5倍以上（P均〈0．01），在间质区域浸润的免疫活性细胞上可见CD28和CD40L的表达。经B7-lmAb干预性治疗，小鼠死亡率从47．83％下降到11．01％。P〈0．01；BUN、Scr水平明显降低；肾组织保护率从7．45％上升至66．51％。结论在小鼠叶酸性肾病中．肾组织CD40／CD40L和B7-1／CD28的表达上调并参与了肾小管上皮细胞损伤、免疫活性细胞浸润和肾小管间质纤维化的过程。B7-1mAb干预治疗能减轻上述的肾损害，为肾间质纤维化的特异性免疫治疗提供新的途径。     

Aggressive skin allograft rejection in CD28-/- mice independent of the CD40/CD40L costimulatory pathway.

CD28-/- mice have been utilized to study the role of B7/CD28 and B7-CTLA4 interactions. There is evidence that CTLA4 ligation may be critical for tolerance induction. The aim of the current study is to further investigate rejection responses of CD28-/- mice and to define the role of B7-CTLA4 interactions in the absence of the CD40 and CD28 pathways. Balb/c skin allografts were transplanted onto C57BL/6 (B6) wild type or CD28-/- mice treated with anti-CD40L, CTLA4-Ig, or combination blockade. To investigate the cellular mechanism of rejection in CD28-/- recipients, mice were treated with anti-CD4 or anti-CD8 antibodies prior to treatment with costimulation blockade. The fluoroscein dye CFSE was utilized to study T cell expansion in vivo. Surprisingly, treatment of B6 CD28-/- mice with CTLA4-Ig alone (MST 12d), anti-CD40L alone (MST 13d), or combined blockade (MST 13d) had no effect on allograft survival compared to untreated B6 CD28 mice (MST 11d). CD28-/- recipients depleted of CD4+ cells and treated with CTLA4-Ig, anti-CD40L, or combination blockade also did not have prolonged survival compared with untreated mice (MST 10d). In contrast, CD28-/- recipients depleted of CD8+ cells had markedly prolonged allograft survival when treated with either anti-CD40L alone (MST 49d) or with combination blockade (MST 57d). Studies utilizing CFSE demonstrated that CD28-/- CD8+ T cells are not defective in in vivo proliferation responses compared with wild type CD8 cells. Thus, CD28-/- CD8+ T cells are responsible for aggressive rejection responses of CD28-/- mice independent of the CD40 pathway. In addition, CD40L blockade does not result in CD4+ T cell tolerance in CD28 recipients, despite an intact B7-CTLA4 pathway.     

Complex nature of the human antisperm antibody response in SCID mice

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely‐combined immunodeficient (SCID) mice in concentrations of 2.5–4.0 × 10⁷ cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8⁺ cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme‐linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with ‘naïve’ human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8⁺ immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from ‘naïve’ or pre‐sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre‐primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.     

Blockade of the PD-1/PD-1L pathway reverses the protective effect of anti-CD40L therapy in a rat to mouse concordant islet xenotransplantation model

BACKGROUND: We have previously demonstrated that costimulatory blockade with anti-CD40L monoclonal antibody (mAb) prolongs the survival of non-vascularized concordant rat to mouse islet xenografts. Here, we examine whether signaling through the PD-1/PD-1L pathway is required for the anti-CD40L therapy to prolong concordant islet graft survival using a novel anti-murine PD-1 mAb (clone 4F10). METHODS: C57BL/6 mice received a cellular concordant islet xenograft under the left kidney capsule and four experimental groups were prepared. Group I: untreated control; group II: recipient mice were treated with three doses of 0.5 mg of anti-CD40L mAb (clone MR1) on days 0, 2 and 4; group III: mice were treated with 0.5 mg of anti-PD-1 (CD279) mAb (clone 4F10) every other day for 8 days; and finally group IV: mice received the combined treatment that consisted of anti-CD40L plus anti-PD-1 mAb. RESULTS: Concordant islet xenografts transplanted in control untreated mice showed a median survival time (MST) of 17 +/- 7.43 days, whereas anti-CD40L treatment led to a significant prolongation of graft survival (MST: 154 +/- 65.56, P < 0.0001). The administration of anti-PD-1 alone significantly accelerated graft rejection compared to non-treated controls (MST: 10 +/- 2.24 vs. MST: 17 +/- 7.43, P < 0.0004). Remarkably, the combined administration of anti-CD40L and anti-PD-1 reversed the protective effect obtained with anti-CD40L alone (anti-CD40L, MST: 154 +/- 65.56 vs. anti-CD40L plus anti-PD-1, MST: 10 +/- 7.72, P < 0.0002). CONCLUSION: Overall, our data indicate that the PD-1/PD-1L pathway is required for the achievement of prolonged graft survival in anti-CD40L-treated mice in a setting of rat to mouse concordant islet xenotransplantation.     

CD14-159C/T基因多态性对全血培养CD14表达的影响

目的探讨CD14基因启动子-159C/T基因多态性对全血培养CD14mRNA表达及可溶性CD14(sCD14)浓度的影响。方法采集118例健康献血员血标本,用全血细胞培养模型检测内毒素刺激前后CD14mRNA表达及sCD14浓度的变化。采用聚合酶链反应(PCR)及限制性内切酶HaeⅢ对PCR产物的消化作用检测CD14基因多态性。同时,对内毒素刺激后肿瘤坏死因子α(TNF-α)诱生水平进行了分析。结果118例健康献血员中,等位基因T和C的频率分别为60.2%和39.8%;40例是T等位基因纯合子(TT),62例为杂合子(TC),还有16例基因型为CC。基因型TT与TC白细胞中CD14mRNA的表达及上清液sCD14浓度均明显高于CC纯合子(P<0.05或0.01)。并且TT纯合子TNF-α诱生水平为(352±215)pg/ml,显著高于基因型TC及CC[(261±163)pg/ml及(198±122)pg/ml,P<0.05]。结论内毒素受体CD14-159C/T基因多态性对全血培养CD14的表达及释放产生显著影响,并与内毒素诱导TNF-α的反应性相关。     

Sensitization to Minor Antigens Is a Significant Barrier in Bone Marrow Transplantation and Is Prevented by CD154:CD40 Blockade

Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation rejection. The mechanism of sensitization to minor histocompatibility antigens (Mi‐HAg) has not been thoroughly explored. We used a mouse model of allosensitization to Mi‐HAg to study the Mi‐HAg sensitization barrier in bone marrow transplantation (BMT). AKR mice were sensitized with MHC congenic Mi‐HAg disparate B10.BR skin grafts. Adaptive humoral (B‐cells) and cellular (T cells) responses to Mi‐HAg are elicited. In subsequent BMT, only 20% of sensitized mice engrafted, while 100% of unsensitized mice did. In vivo cytotoxicity assays showed that Mi‐HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes significantly more rapidly than naïve AKR mice but less rapidly than MHC‐sensitized recipients. Sera from Mi‐HAg sensitized mice also reacted with cells from other mouse strains, suggesting that Mi‐HAg peptides were broadly shared between mouse strains. The production of anti‐donor‐Mi‐HAg antibodies was totally prevented in mice treated with anti‐CD154 during skin grafting, suggesting a critical role for the CD154:CD40 pathway in B‐cell reactivity to Mi‐HAg. Moreover, anti‐CD154 treatment promoted BM engraftment to 100% in recipients previously sensitized to donor Mi‐HAg. Taken together, Mi‐HAg sensitization poses a significant barrier in BMT and can be overcome with CD154:CD40 costimulatory blockade.     

RNA干扰技术阻断CD40/CD40L共刺激通路对小鼠移植心存活时间的影响

目的 探讨慢病毒介导RNA干扰(RNAi)技术阻断CD40/CD40L共刺激通路对小鼠移植心存活时间的影响.方法 以针对小鼠CD40基因的RNAi慢病毒载体在体外感染供者骨髓来源的树突状细胞(DC),制备低表达CD40的耐受性DC(Tol-DC).荧光实时定量聚合酶链反应及流式细胞术检测DC感染前后CD40 mRNA及DC表面抗原CD40、CD11c和MHCⅡ的表达.建立小鼠异位腹腔心脏移植模型.在小鼠异位心脏移植前7 d,经静脉给受者输注体外制备的低表达CD40的Tol-EC(慢病毒感染DC注射组),并设置单纯移植对照组和未感染DC注射组作为对照.观察各组移植心的存活时间,评定各组术后第7天移植心排斥反应的病理分级.结果 CD40-RNAi慢病毒载体在体外感染DC 48 h后,CD40 mRNA表达明显受到抑制,抑制率为80.9%;CD40表达明显下降,由(74.37±4.08)%降至(40.07±4.03)%(P＜0.05).单纯移植对照组、未感染DC注射组和慢病毒感染DC注射组移植心存活时间分别为:(8±2)d、(9±1)d和(14±4)d,慢病毒感染DC注射组移植心存活时间明显延长,并且移植心排斥反应病理分级显著降低,差异有统计学意义(P＜0.05).结论 阻断CD40/C40L共刺激通路可抑制异系T淋巴细胞的活化,从而抑制急性排斥反应,延长小鼠移植心的存活时间.     

Neuroblastoma-induced inhibition of dendritic cell IL-12 production via abrogation of CD40 expression

Background/Purpose

CD40 expression by dendritic cells (DCs) critically regulates their maturation/antitumor activity. CD40-CD40 ligand (CD40L) signaling stimulates DC-mediated IL-12 production/cytotoxicity. Recent studies suggest that neuroblastoma (NB)-derived gangliosides impair DC maturation, IL-12 secretion, and NK/T-cell activity. Neuroblastoma ganglioside-mediated abrogation of CD40 expression by DC and tumor-induced tolerance has not been studied. The purpose of this study is to determine if NB inhibits DC IL-12 production via CD40. The contributory role of the NB-derived ganglioside G_M3 in this process is also examined.

Methods

Dendritic cells were generated from bone marrow of mice injected with saline (control) or murine NB. Control DCs were matured with or without G_M3. Dendritic cells were cocultured with NB cells treated with or without a ganglioside synthesis inhibitor. Dendritic cell groups were analyzed for maturation/costimulatory markers. Control and tumor-derived DC were stimulated with CD40L or Staphylococcus aureus and studied for IL-12 expression.

Results

CD40 expression on DC generated from NB bearing mice decreased by 64% (P < .001). G_M3 down-regulated DC maturation and CD40 expression. Only CD40-dependent IL-12 production was abrogated (60%, P < .01) in DC derived from NB-bearing mice. Dendritic cell capacity to synthesize IL-12 remained intact.

Conclusions

Neuroblastoma-induced inhibition of DC function may result from ganglioside-mediated CD40 signaling deficiency. Strategies to bypass/augment CD40-CD40L signaling may improve current NB immunotherapies.     

血管紧张素Ⅱ诱导Toll样受体4在腹膜间皮细胞的表达及其对脂多糖诱导CD40表达的影响

目的 观察血管紧张素Ⅱ（AngⅡ）对原代培养大鼠腹膜间皮细胞（RPMC）Toll样受体4（TLR4）表达的影响及其在脂多糖（LPS）诱导的核转录因子κB （NF-κB）活化及CD40表达中的作用。 方法 分离及培养RPMC。用不同浓度AngⅡ（10－9、10－8、10－7、10－6 mol/L）刺激细胞及用10-7 mol/L AngⅡ刺激细胞不同时间（mRNA为1、2、4、8、12、24、48 h和蛋白为6、12、24、36、48 h），观察血管紧张素1型受体（AT1R）阻滞剂洛沙坦（10－5 mol/L）和血管紧张素2型受体（AT2R）阻滞剂PD123177（10-5 mol/L）对AngⅡ诱导TLR4表达的影响。将细胞随机分为下列4组：对照组、AngⅡ (10－7 mol/L)组、LPS(1 mg/L)组、AngⅡ (10－7 mol/L)+LPS(1 mg/L)组，观察AngⅡ对LPS诱导的NF-κB激活和CD40表达的影响。RT-PCR检测TLR4、CD40 mRNA表达；Western印迹检测TLR4、IκBα、磷酸化IκBα（p-IκBα）、NF-κB p65、磷酸化NF-κB（p-p65）蛋白表达；免疫荧光检测细胞NF-κB p65亚单位的表达及分布。 结果 (1)10－9、10－8、10－7、10－6 mol/L AngⅡ作用RPMC 12 h，TLR4 mRNA表达分别增加70.5%、89.5%、102.9%和121.9%；作用24 h TLR4蛋白表达分别增加12.1%、27.7%、51.2%和41.6%。AngⅡ（10－7 mol/L）作用RPMC不同时间，TLR4 mRNA表达高峰为8 h和12 h（P < 0.01），蛋白表达高峰为12 h和24 h（P < 0.01）。(2)洛沙坦阻断后，AngⅡ诱导的TLR4表达与未阻断组比较，下调33.5%（P < 0.05）。PD123177对AngⅡ诱导的TLR4表达无显著影响（P > 0.05）。(3) 与正常对照组比较，LPS作用60 min p-IκBα/IκBα、p-p65/p65表达分别上调362.6%（P < 0.01）和67.4%（P < 0.05）；作用4 h CD40 mRNA表达上调299.9%（P < 0.01）；与LPS组比较，AngⅡ预刺激24 h加LPS作用60 min，p-IκBα/IκBα、p-p65/p65表达分别上调49.1%（P < 0.01）和29.3%（P < 0.05）；作用4 h CD40 mRNA表达上调56.8%（P < 0.01）。(4)免疫荧光结果显示正常对照组与AngⅡ组细胞中，p65信号定位于细胞胞质；LPS作用60 min，p65信号从胞质进入胞核；AngⅡ+LPS组NF-κB p65胞核信号显著增强。 结论 AngⅡ呈浓度、时间依赖性诱导RPMC TLR4表达，并显著增强LPS诱导NF-κB激活及CD40的表达。提示腹膜组织局部产生的AngⅡ可能对LPS诱导的腹膜组织炎性反应具有放大作用。     

Transplant Acceptance Following Anti‐CD4 Versus Anti‐CD40L Therapy: Evidence for Differential Maintenance of Graft‐Reactive T Cells

Inductive therapy with anti‐CD4 or anti‐CD40L monoclonal antibodies (mAb) leads to long‐term allograft acceptance but the immune parameters responsible for graft maintenance are not well understood. This study employed an adoptive transfer system in which cells from mice bearing long‐term cardiac allografts following inductive anti‐CD4 or anti‐CD40L therapy were transferred into severe combined immunodeficiency (SCID) allograft recipients. SCID recipients of cells from anti‐CD4‐treated mice (anti‐CD4 cells) did not reject allografts while those receiving cells from anti‐CD40L‐treated mice (anti‐CD40L cells) did reject allografts. Carboxyfluorescein succinimidyl ester (CFSE) labeling of transferred cells revealed that this difference was not associated with differential proliferative capacities of these cells in SCID recipients. Like cells from naïve mice, anti‐CD40L cells mounted a Th1 response following transfer while anti‐CD4 cells mounted a dominant Th2 response. Early (day 10) T‐cell priming was detectable in both groups of primary allograft recipients but persisted to day 30 only in recipients treated with anti‐CD4 mAb. Thus, anti‐CD40L therapy appears to result in graft‐reactive T cells with a naïve phenotype while anti‐CD4 therapy allows progression to an altered state of differentiation. Additional data herein support the notion that anti‐CD40L mAb targets activated, but not memory, cells for removal or functional silencing.     

Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease

BACKGROUND: Interaction between CD40 and CD40 ligand (CD40L) is involved in both cognate and innate immune responses. Blockade of CD40-CD40L interactions reduces severity of renal injury in murine lupus nephritis and membranous nephropathy. We hypothesized that CD40-CD40L could contribute to renal injury in models that are not antibody-dependent, and that anti-CD40L could diminish inflammation and fibrosis in murine adriamycin nephropathy. METHODS: Male BALB/c mice were divided into three groups (N = 6 per group): (1). saline-treated, age-matched control; (2). adriamycin only; and (3). MR1 + adriamycin. In group 3, mice were treated with intraperitoneal injections of anti-CD40L antibody (clone MR1, 0.4 mg per mouse) after the onset of proteinuria at days 5, 7, 9, and 11 after adriamycin treatment. Animal subgroups were compared at 14 and 42 days after induction of adriamycin nephropathy. Functional and pathologic markers of disease severity, cellular components of interstitial inflammation, and the degree of CD40 expression were assessed. Relative cortical RNA expression of the chemokine monocyte-chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) was also compared between animal groups. RESULTS: CD40 was weakly expressed in tubules of normal mice but was expressed in tubules, interstitium, and glomeruli of mice with adriamycin nephropathy in a time-dependent manner. MR1 treatment resulted in a significant attenuation of the severity of adriamycin nephropathy at day 42 [e.g., glomerular sclerosis (%), group 3, 20.1 +/- 4.7 vs. group 2, 30.2 +/- 7.2, P < 0.001]. CD40L blockade significantly reduced tubulointerstitial injury as well [tubular diameter microm), group 3, 42.5 +/- 6.9 vs. group 2, 66.3 +/- 13.7, P < 0.001; and group 1, 37.3 +/- 5.7, P < 0.01; tubular cell height microm), group 3, 16.3 +/- 1.7 vs. group 2, 11 +/- 1.8, P < 0.01; and group 1, 18.2 +/- 1.9, P < 0.01; interstitial volume (%), group 3, 13.9 +/- 5.1 vs. group 2, 26.2 +/- 4.9, P < 0.001; and group 1, 1.3 +/- 0.7, P < 0.001; proteinuria (mg/24 hours), group 3, 1.8 +/- 0.6 vs. group 2, 4.3 +/- 0.8, P < 0.001; and group 1, 0.7 +/- 0.2, P < 0.05; and creatinine clearance microL/min), group 3, 75 +/- 4 vs. group 2, 35 +/- 2, P < 0.001; and group 1, 82 +/- 4, P < 0.01] were also improved by MR1. MR1 treatment also resulted in a significant reduction in the number of cortical macrophages at both 14 and 42 days after adriamycin (P < 0.01). Cortical expression of MCP-1 and RANTES was significantly reduced by MR1 treatment at 42 days after adriamycin (P < 0.01 and P < 0.05, respectively). CONCLUSION: Blockade of CD40-CD40L interaction protects against renal structural and functional injury in this murine model of chronic proteinuric renal disease.     

CTLA-4Ig in Combination with Anti-CD40L Prolongs Xenograft Survival and Inhibits Anti-Gal Ab Production in GT-Ko Mice

The generation of GT-Ko mice has provided unique opportunities to study allograft and xenograft rejection in the context of anti-alpha1,3-Gal antibody (anti-Gal Ab) responses. In this study we used the allotransplantation model of C3H hearts into galactosyltransferase-deficient (GT-Ko) mice and the xenotransplantation model of baby Lewis rat hearts into GT-Ko mice to investigate the ability of CTLA-41g in combination with anti-CD40L mAb to control graft rejection and anti-Gal Ab production. Murine CTLA-41g or anti-CD40L monotherapy prolonged allograft survival, and the combination of these reagents was most immunosuppressive. However short-term treatment with murine cytotoxic T lymphocyte associated antigen-4 (muCTLA-41g) and/or CD40 ligand (CD154) monoclonal antibodies (anti-CD40L mAbs) was unable to induce indefinite allograft survival. CTLA-4-immunoglobulin fusion protein (CTLA-41g) or anti-CD40L monotherapy only marginally prolonged xenograft survival; the combination of human CTLA-41g and anti-CD40L significantly prolonged xenograft survival (74days), while the combination of murine CTLA-41g and anti-CD40L resulted in graft survival of >120days. CTLA-41g or anti-CD40L monotherapy or the combination of these agents inhibited the production of alloAbs, including anti-Gal Abs. CTLA-41g or anti-CD40L monotherapy partially controlled xenoAb and anti-Gal Ab production, while the combination was more effective. These observations corroborate our previous observations that humoral, including anti-Gal Ab, responses and rejection following allograft or concordant xenograft transplantation in GT-Ko mice are T-cell dependent and can be controlled by costimulation blockade.     

核转录因子-κB通路在CD40-CD154诱导血管内皮细胞活化中的作用

目的 探讨血管内皮细胞(VEC)内核转录因子-κB(NF-κB)通路在CD40-CD154相互作用诱导VEC活化中的作用.方法 应用重组人肿瘤坏死因子α(rhTNF-α)和重组人γ干扰素(rhIFN-γ)刺激人主动脉VEC,通过流式细胞仪(FACS)检测CD40和粘附因子表达水平;通过表达CD154的细胞株D1.1和VEC共同培养后,观察VEC粘附因子的表达水平;通过抗CD154单克隆抗体阻断CD40-CD154间的反应;应用NF-κB阻断剂BAY11-7082观察阻断NF-κB通路后对CD40-CD154诱导VEC活化的影响.结果 未活化的VEC膜表达低水平的CD40、CD54和CD106,但不表达CD62E,经白细胞介素刺激后可上调这些蛋白分子的表达水平.D1.1细胞株和VEC共同培养后可诱导VEC活化并上调CD62E、CD54和CD106的表达水平;抗CD154抗体可阻断CD40-CD154间的反应所诱导的VEC活化;NF-κB阻断剂可阻断rhTNF-α诱导的VEC活化以及CD40-CD154间的反应所诱导的VEC活化.结论 VEC中CD40和T淋巴细胞中CD154之间的相互作用在诱导VEC活化过程中起重要作用;CD40-CD154相互作用并通过NF-κB通路诱导VEC活化;NF-κB通路阻断剂可抑制VEC活化.     

A GM-CSF/CD40L producing cell augments anti-tumor T cell responses

BACKGROUND: Tumors evade T cell-mediated rejection despite the presence of tumor associated antigens (TAAs) and T cells specific for these TAAs in cancer patients. Therapeutic tumor vaccines are being developed to prevent this evasion. Previous reports revealed that anti-tumor T cell responses could be activated in mice when granulocyte macrophage-colony stimulating factor (GM-CSF) or CD40L are produced at tumor vaccine sites. We sought to test the hypothesis that production of GM-CSF and CD40L by a bystander cell line could induce an anti-tumor T cell response in an in vitro human model. MATERIALS AND METHODS: The K562 cell line was stably transfected with the human GM-CSF and CD40L genes. The effect of this cell line on T cell responses was tested in a human autologous mixed tumor cell/lymph node cell model using tissue from a series of cancer patients. RESULTS: There was no significant anti-tumor T cell response when human lymphocytes derived from tumor-draining lymph nodes were stimulated with autologous tumor cells in vitro. However, significant anti-tumor T cell responses were observed when bystander cells transfected with CD40L and GM-CSF were added to the cultures. CONCLUSIONS: A fully autologous human model consisting of tumor cells as stimulator cells and tumor-draining lymph nodes as responder cells can be used to test immunotherapeutic strategies. T cells in these lymph nodes are unresponsive to autologous tumor cells, but this lack of responsiveness can be reversed in the presence of GM-CSF and CD40L. These data provide a rationale for testing tumor cell vaccines incorporating GM-CSF- and CD40L-expressing bystanders in clinical trials.     

Adenovirus-mediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model

BACKGROUND: The interaction between CD40 ligand (CD40L) and CD40 on antigen presenting cells is essential for the initiation of antigen-specific T-cell responses, whereas CD40L stimulation of CD40+ tumor cells can induce cellular apoptosis. We investigated the anti-tumor effects induced by CD40L gene transfer into the mouse prostate adenocarcinoma cell line TRAMP-C2, both in vitro and in vivo. METHODS: TRAMP-C2 cells were transduced with an adenoviral vector encoding CD40L (AdCD40L). The induced expression of co-stimulatory molecules and cell viability was analyzed. AdCD40L-transduced TRAMP-C2 cells were used in prophylactic vaccination studies, while therapeutic studies were performed using peritumoral injections of AdCD40L. RESULTS: AdCD40L yielded reduced TRAMP-C2 cell viability and induced apoptosis in vitro. Vaccination with CD40L-expressing TRAMP-C2 cells induced anti-tumor immunity and peritumoral AdCD40L injections induced tumor growth suppression. CONCLUSIONS: Our observations highlight the therapeutic potential of using AdCD40L as a monotherapy or in combination with conventional chemotherapy or novel therapies (e.g., oncolytic viruses). The use of AdCD40L offers an attractive option for future clinical trials.     

Analysis of the CD40 and CD28 pathways on alloimmune responses by CD4+ T cells in vivo

BACKGROUND: Blockade of the CD40 and CD28 pathways is a powerful strategy to inhibit CD4-mediated alloimmune responses. In this study, we examine the relative roles of the CD40 and CD28 pathways on CD4-mediated allograft rejection responses, and further characterize the role of these pathways on CD4+ T-cell activation, priming for cytokine production, and cell proliferation in response to alloantigen in vivo. METHODS: BALB/c skin allografts were transplanted onto C57BL/6 Rag 1-/- recipients reconstituted with CD4 cells from CD28-/- or CD40L-/- donors. The popliteal lymph node assay was used to study the role of these pathways on CD4-cell activation and priming in vivo. To investigate the role of CD40 and CD28 blockade on CD4-cell proliferation, the fluorescein dye carboxyfluorescein diacetate succinimidyl ester was used. We performed heterotopic cardiac transplantation using CD40-/- mice to evaluate the role of CD40 on donor versus recipient cells in CD4-mediated rejection. RESULTS: B6 Rag 1-/- recipients reconstituted with CD28-/- CD4+ T cells acutely rejected allografts (median survival time 15 days), whereas recipients reconstituted with CD40L-/- CD4+ T cells had significantly prolonged survival of BALB/c skin grafts (MST 71 days). CD40L blockade was equivalent to or inferior to CD28 blockade in inhibition of in vivo CD4-cell activation, priming for cytokine production, and proliferation responses to alloantigen. BALB/c recipients depleted of CD8 cells promptly rejected donor B6 CD40-/- cardiac allografts, whereas B6 CD40-/- recipients depleted of CD8 cells had significantly prolonged survival of BALB/c wild-type cardiac allografts. CONCLUSIONS: The CD40/CD40L pathway, but not the CD28/B7 pathway, is critical for CD4-mediated rejection responses, however, the responsible mechanisms remain unclear.