Directing T Cell Responses during Immune Reconstitution

该项目的目标是研究T细胞再生的生物学，并开发可用于T细胞枯竭宿主的治疗方法，以提高宿主的整体免疫能力，并在最小残留肿瘤疾病的情况下指导T细胞对肿瘤特异性抗原的反应。使用小鼠HY皮肤移植排斥模型，在T细胞耗尽后，我们已经表明胸腺去核小鼠通过外周扩张进行免疫重建无法排斥HY不同的皮肤移植物，而通过胸腺依赖途径重建的宿主正常排斥移植物。如果提供了足够数量的T细胞，在胸腺去切的小鼠中就能完成排斥反应，我们已经证明，这需要大约10%的正常T细胞库。在这些实验中，我们也证明了IL-7可以有效地增强胸腺非依赖性T细胞再生，并且IL-7治疗可以恢复在T细胞数量不足的胸腺化宿主中对HY异种移植物的排斥能力。从机制上讲，这似乎与IL-7相关，至少部分相关。通过使用bcl-2转基因T细胞疫苗抑制程序性细胞死亡（PCD）， bcl-2抑制程序性细胞死亡的能力也增强了该模型的免疫能力。因此，这些结果表明PCD可能在外周扩增过程中限制宿主的免疫能力，并提出了体内PCD调节可能作为增强胸腺缺陷TCD宿主免疫能力的有用方法的可能性。该模型还强调了IL-7在体内调节外周扩张过程中的关键作用。基于这些结果，我们假设内源性IL-7的产生可以通过在T细胞耗竭的情况下增强外周扩增过程来促进T细胞的稳态。为了验证这一假设，我们试图评估由于HIV感染而导致T细胞耗竭的儿童血清IL-7水平。我们观察到，在与HIV相关的CD4+耗竭的儿童中，血清IL-7水平和T细胞计数之间存在显著的负相关。统计上，血清IL-7水平似乎与CD4+和CD8+ T细胞计数有关，但与B细胞计数无关。目前的工作正在进行中，以确定是否类似的关系存在于成人T细胞耗竭和T细胞耗竭相关的细胞毒性治疗。这些研究可能有助于确定IL-7在维持T细胞稳态中的新作用。关于T细胞再生的胸腺依赖途径，我们目前正在研究年龄或疾病相关的胸腺功能障碍在人类T细胞耗竭后限制T细胞受体库多样性中的相对重要性。为了评估人类低水平胸腺功能随着时间的推移使T细胞受体（TCR）库多样化的能力，目前的工作重点是评估一系列接受高活性抗逆转录病毒治疗的儿童患者的TCR库多样性。我们假设，随着时间的推移，即使这些患者的胸腺功能不理想，也可能导致TCR曲目的多样化。为了测量TCR多样性的这种变化，我们开发了一种半定量方法，使用互补确定区3 （CDR3）大小分析连续稀释纯化CD4+ T细胞。有趣的是，在正常个体中使用这种方法的初步结果显示，T细胞受体库多样性的显著年龄相关变化。脐带血显示出多样化的储备，只有10e5个输入CD4细胞，而年轻人直到使用5 × 10e5个输入CD4细胞才显示出多样化的储备。老年人（40-50岁）直到使用1 × 10e6输入CD4+ T细胞才显示出多样化的功能库。这些结果表明，TCR库多样性的可测量下降发生在整个正常人类寿命中。对于接受高活性抗逆转录病毒治疗的儿童，我们观察到CD4+ T细胞计数<200细胞/mcl的儿童TCR库多样性显著降低。相比之下，CD4+ T细胞计数较高的儿童的初步结果显示TCR库多样性基本正常（例如bb0 - 400个细胞/mcl）。我们还使用这种方法比较了来自HIV感染患者的CD45RA+和CD45RO+ CD4+ T细胞的TCR库多样性。到目前为止，没有观察到剧目多样性的显著差异。在我们正在进行的临床试验中，未来的计划包括使用该技术来监测接受免疫恢复治疗的癌症患者TCR库多样性的变化。艾滋病相关100%

The objectives of this project are to study the biology of T cell regeneration and to develop therapeutic approaches which could be used in T cell depleted hosts to improve overall host immunocompetence and to direct T cell responses toward tumor-specific antigens in the setting of minimal residual neoplastic disease. Using a murine model of HY skin graft rejection, following T cell depletion, we have shown that thymectomized mice which have undergone immune reconstitution via peripheral expansion are unable to reject HY disparate skin grafts while hosts reconstituted via thymic-dependent pathways reject grafts normally. Rejection is accomplished in thymectomized mice if sufficient numbers of T cells are provided which we have shown requires approximately 10% of the normal T cell repertoire. In these experiments, we have also shown that IL-7 potently enhances thymic-independent T cell regeneration and that IL-7 therapy restores the capacity to reject HY disparate grafts in thymectomized hosts reconstituted with insufficient T cell numbers. Mechanistically, this appears to be related, at least in part, to IL-7?s ability to inhibit programmed cell death since inhibition of programmed cell death (PCD) by the use of bcl-2 transgenic T cell inocula also enhances immune competence in this model. Therefore, these results suggest that PCD may serve to limit host immune competence during peripheral expansion and raise the possibility that modulation of PCD in vivo may serve as a useful approach for enhancing immune competence in thymic-deficient TCD hosts. This model has also emphasized the critical role of IL-7 in modulating the process of peripheral expansion in vivo. Based upon these results, we have hypothesized that endogenous IL-7 production could contribute to T cell homeostasis by enhancing the process of peripheral expansion in the setting of T cell depletion. In order to test this hypothesis, we sought to evaluate serum IL-7 levels in children with T cell depletion due to HIV infection. We have observed a striking inverse correlation between serum IL-7 levels and T cell counts in children with HIV associated CD4+ depletion. Statistically, the serum IL-7 levels appear to be related to both CD4+ and CD8+ T cell counts, but not B cell counts. Current work is underway to address whether similar relationships exist in adults with T cell depletion and in T cell depletion related to cytotoxic therapy. These studies may serve to identify a new role for IL-7 in maintenance of T cell homeostasis.With regard to thymic-dependent pathways of T cell regeneration, we are currently investigating the relative importance of age- or disease-associated thymic dysfunction in limiting T cell receptor repertoire diversity following T cell depletion in humans. In order to evaluate the ability of low level thymic function to diversify the T cell receptor (TCR) repertoire over time in humans, current work is focused upon evaluation of TCR repertoire diversity in a series of pediatric patients treated with highly active antiretroviral therapy for HIV infection. We have hypothesized that even suboptimal thymopoiesis in this patient population may lead to diversification of the TCR repertoire over time. In an attempt to measure such changes in TCR diversity, we have developed a semi-quantitative approach using complementarity determining region 3 (CDR3) size analysis of serially diluted purified CD4+ T cells. Interestingly, preliminary results using this approach in normal individuals have shown significant age related changes in T cell receptor repertoire diversity. Whereas cord blood shows a diverse repertoire with as few as 10e5 input CD4 cells, young adults do not show a diverse repertoire until 5 x 10e5 input CD4 cells are utilized. Older adults (40-50 years) do not show a diverse repertoire until 1 x 10e6 input CD4+ T cells are utilized. These results suggest that measurable declines in TCR repertoire diversity occur throughout the normal human lifespan. With regard to children treated with highly active antiretroviral therapy, we have observed significantly reduced TCR repertoire diversity in children with CD4+ T cell counts <200 cells/mcl. In contrast, preliminary results in children with higher CD4+ T cell counts (e.g. >400 cells/mcl) reveal essentially normal TCR repertoire diversity. We have also used this approach to compare TCR repertoire diversity in CD45RA+ vs. CD45RO+ CD4+ T cells from patients with HIV infection. Thus far, no significant differences in repertoire diversity have been observed. Future plans involve the use of this technique to monitor changes in TCR repertoire diversity in cancer patients treated with immunorestorative therapies in the context of our ongoing clinical trials. AIDS RELATED 100%