ANTIGEN IMMUNOSUPPRESSION BY KILLER LANGERHANS CELLS

朗格汉斯细胞(LC)通常向T细胞传递激活信号。我们假设，通过基因修饰过表达CD95L(Fas配体)的LC，被称为“杀手”LC，将通过抗原特异性的相互作用向T细胞传递凋亡信号。为了验证这一点，我们将CD95L基因导入我们的LC系XS106(来源于A/J小鼠)，并选择了一个稳定表达CD95L的克隆(XS10-6-CD95L)。当用卵清蛋白(OVA)冲击时，这种杀伤性LC克隆在体外通过抗原特异性和CD95L依赖的机制触发OVA反应性CD4+T细胞的凋亡。致敏前后注射到A/J小鼠体内的卵子脉冲杀菌剂LC可抑制对DNFB的耳肿胀反应。重要的是，OVA脉冲杀手LC抑制OVA反应，但不抑制对无关抗原HEL的反应，而HEL脉冲杀手LC仅抑制HEL反应，建立抗原特异性。我们将在新的假设下定义机制，即杀手LC通过触发识别相应抗原的假定效应T细胞的凋亡来抑制不同的免疫反应。具体地说，我们将使用五个已建立的动物模型来研究杀手LC的影响：1)迟发型超敏反应：我们将在致敏前后注射OVA脉冲的杀手LC，以研究CD4+效应T细胞和记忆T细胞的影响、效应细胞的命运(过继转移D011.10转基因小鼠的OVA反应性、幼稚的CD4+T细胞)，以及杀手LC与T细胞进行细胞毒相互作用的关键时间(药物诱导的自杀系统)。2)接触性过敏症。我们将在致敏前或致敏后注射DNFB脉冲杀伤LC，以研究CD8+效应T细胞对Th2样调节性T细胞的影响、杀伤LC与CD8+T细胞的相互作用以及抗原特异性。3)Th2偏向免疫反应。小鼠将被OVA吸收的“贴片”皮下致敏，以产生OVA特异性IgE和IgG1抗体和特应性皮炎样皮肤病变。我们将注射OVA脉冲杀伤LC，以研究其对偏向Th2的效应T细胞和辅助T细胞的影响以及对皮损的治疗效果。4)实验性自身免疫性心肌炎。小鼠将被心肌肌球蛋白(CM)致敏，以产生自身免疫性心肌炎。我们将研究注射CM脉冲杀伤LC对识别组织特异性自身抗原的CD4+致病T细胞的影响，致病T细胞的去向，以及治疗的有效性和安全性。5)皮肤移植排斥反应。我们将研究杀手LC和“杀手LC杂交体”对同种反应性的CD4+和CD8+T细胞的影响，这两种细胞通常通过“直接”和“间接”途径被激活。这些研究将为建立一种治疗炎症性皮肤病的全新免疫抑制疗法奠定基础，该疗法旨在选择性地消除识别致病抗原(如半抗原、过敏原、自身抗原和同种异体抗原)的效应器T细胞。

Langerhans cells (LC) ordinarily deliver activation signals to T cells. We hypothesized that LC genetically modified to over-express CD95L (Fas ligand) termed "killer" LC, would deliver apoptotic signals to T cells upon antigen-specific interaction. To test this, we introduced CD95L cDNA into our LC line XS106 (derived from A/J mice) and selected a stable clone (XS 10-6-CD95L) that expressed abundant surface CD95L. This killer LC clone, when pulsed with ovalbumin (OVA), triggered apoptosis of OVA-reactive CD4+ T cells in vitro by an antigen-specific and CD95L-dependent mechanism. OVA-pulsed killer LC, when injected into A/J mice before or after sensitization, suppressed ear swelling responses to DNFB. Importantly, OVA-pulsed killer LC suppressed OVA responses, but not responses to the irrelevant antigen HEL, whereas HEL- pulsed killer LC inhibited only the HEL responses, establishing antigen- specificity. We will define mechanisms, under the new hypothesis that killer LC suppress diverse immunological responses by triggering apoptosis of putative effector T cells that recognize respective antigens. Specifically, we will study the impact of killer LC using five-established animal models: 1) Delayed type hypersensitivity: We will inject OVA- pulsed killer LC before or after sensitization to study the impact of CD4+ effect T cells and memory T cells, the fate of effector cells (adoptive transfer of OVA-reactive, naive CD4+ T cells from the D011.10 transgenic mice), and the critical timing for cytotoxic interaction of killer LC with T cells (drug-inducible suicide system). 2) Contact hypersensitivity. We will inject DNFB-pulsed killer LC before or after sensitization to study the impact of CD8+ effector T cells and on Th2-like regulatory T cells, killer LC interaction with CD8+ T cells and antigen- specificity. 3) Th2-biased immune responses. Mice will be sensitized epicutaneously with an OVA-absorbed "patch" to produce OVA-specific IgE and IgG1 antibodies and atopic dermatitis-like skin lesions. We will inject OVA-pulsed killer LC to study the impact on Th2-biased effector and helper T cells and "therapeutic" efficacy for skin lesions. 4) Experimental autoimmune myocarditis. Mice will be sensitized with cardiac myosin (CM) to produce autoimmune myocarditis. We will inject CM-pulsed killer LC to study the impact on CD4+ pathogenic T cells that recognize tissue-specific autoantigen, the fate of pathogenic T cells, and therapeutic efficacy and safety. 5) Skin graft rejection. We will study the impact of killer LC and "killer LC hybrids" on allo-reactive CD4+ and CD8+ T cells, which are ordinary activated via "direct" and "indirect" pathways. These studies will form the framework for establishing an entirely new immunosuppressive therapy for inflammatory skin diseases, the therapy designed to eliminate selectively the effector T cells that recognize pathogenic antigens (e.g., haptens, allergens, autoantigens, and alloantigens).