Biology and Therapy of T Cell Depletion

我们实验室的一个主要重点是研究T细胞再生的生物学，并确定新的方法来增强T细胞再生，并在免疫重建期间指导T细胞对特定抗原的反应。我们主要使用小鼠模型和从T细胞耗竭患者获得的临床样本。在免疫重建的基本生物学方面，在前几年，我们通过小鼠模型发表了il - 7调节胸腺依赖性和胸腺非依赖性T细胞免疫重建，并可以通过T细胞增殖、增强抗原呈递和减少程序性细胞死亡的综合作用恢复胸腺T细胞枯竭宿主的免疫。我们还发表了IL7诱导正常食蟹猴外周T细胞稳态的深刻改变，导致体内淋巴细胞总数的显著可逆增加。基于这些和其他数据显示，IL7可以有效地调节非人灵长类动物的外周T细胞稳态，我们在2003年10月开展了IL7的I期临床试验。在过去的一年里，我们专注于进一步研究IL7在小鼠模型中调节抗原特异性反应的作用。我们利用小鼠树突状细胞疫苗模型对次要组织相容性抗原HY进行免疫。该抗原的免疫显性和亚显性表位都已被表征。我们评估了rhIL2， rhIL7或rhIL15在调节内源性表达HY抗原的树突状细胞应答中的功效。我们观察到，在效应细胞CD8群体中，rhIL7和rhIL15的反应都有显著增加，而rhIL2的反应则没有。虽然免疫显性和亚显性种群都有所增加，但对亚显性反应的影响最为显著。IL7也增加了CD4效应物，但IL15和IL2没有。关于记忆细胞群，IL7和IL15都增加了产生的记忆细胞池的大小，尽管与对效应细胞池的影响相比，其影响程度有所减弱。这些结果正准备发表，并首次证明il - 7和il - 15可以扩大疫苗接种的免疫反应，这对这些药物的临床应用具有直接意义。

A primary focus of our laboratory is investigation of the biology of T cell regeneration and the identification of new approaches to enhance T cell regeneration and to direct T cell responses toward specific antigens during the period of immune reconstitution. We primarily use murine modeling and clinical samples acquired from patients with T cell depletion. With regard to the basic biology of immune reconstitution, In previous years, using murine models we published that IL7 modulates thymic-dependent and thymic-independent T cell immune reconstitution and can restore immunity in athymic T cell depleted hosts through a combination of effects of T cell proliferation, enhanced antigen presentation and diminished programmed cell death. We also published that IL7 induced profound alterations in peripheral T cell homeostasis in normal cynomolgus monkeys which resulted in a substantial, reversible increase in total body lymphocyte numbers. Based upon these and other data showing that IL7 potently modulates peripheral T cell homeostasis in nonhuman primates we developed a Phase I clinical trial of IL7 which opened in October 2003. During the past year, we focused on further studies of IL7's effect of modulating antigen specific responses in murine models. We utilized a mouse dendritic cell vaccine model to immunize toward the minor histocompatibility antigen HY. Both immunodominant and subdominant epitopes have been characterized for this antigen. We evaluated the efficacy of rhIL2, rhIL7 or rhIL15 in modulating responses to a dendritic cell which endogenous expresses the HY antigen. We observed potently increased responses in effector cell CD8 populations with both rhIL7 and rhIL15 but not with rhIL2. While both immunodominant and subdominant populations increased, the effects on subdominant responses were most striking. IL7 also increased CD4 effectors but IL15 and IL2 did not. With regard to memory cell populations, both IL7 and IL15 increased the size of the memory cell pool generated although the magnitude of the effect was diminished compared to the effect on effector cells pools. These results are being prepared for publication and represent the first evidence the IL7 and IL15 can serve to broaden immune responses to vaccination which has direct implications for clinical application of these agents. In addition, we have identified flt3 ligand as an agent with potential for clinical application as an immunorestorative. Using murine models of T cell depletion, we have discovered that antigen driven expansion during immune reconstitution requires the presentation of antigen by professional antigen presenting cells. Based upon this, we tested whether one factor limiting immune reconstitution via peripheral expansion might be a limited number of professional antigen presenting cells. Indeed, treatment of mice with flt3 ligand to increase peripheral dendritic cells induced substantial increases immune reconstitution via peripheral expansion. Furthermore, when we evaluated the effects of flt3 ligand on immune reconstitution in thymus-bearing hosts, we also observed substantially increased T cell numbers which were derived via thymic-dependent pathways. This was associated with a significant increase in murine TREC levels. Together, these results identify flt3 ligand as a new immunorestorative agent, capable of enhancing both thymic-dependent and thymic-independent pathways of T cell regeneration. These results have been submitted for publication. Although the current understanding of the biology of T cell depletion emphasizes effects which enhance immune responses to weak antigens, we have observed an increased susceptibility to tolerance following T cell depletion. We hypothesize that the mechanisms at work in this model are critical for maintaining self tolerance in T cell depleted hosts. Thymectomized, T cell depleted (TXY/TCD) C57BL/6 female mice were reconstituted with nave T cells (20X106) and either male dendritic cells or male splenocytes IP. Rapid rejection of male skin grafts occurred in recipients of male dendritic cells but not recipients of male splenocytes. These animals are functionally tolerant to HY since subsequent sensitizations with male dendritic cells were unsuccessful. Tolerance in this model was associated with clonal deletion and an absence of cytokine production to the HY antigen. Splenic B cells were necessary and sufficient to induce tolerance in T cell depleted hosts and the tolerance suppressive since it was not reversed by transfer of nave T cells. Purified CD8+ T cells, but not CD4+ T cells, mediated suppression and neutralization of TGF? had no effect on suppression but T cells from IL-10-/- mice could not mediate this effect. Therefore, when B cells presented antigen to CD8+ T cells present in a host with T cell depletion, a suppressive immune response mediated by IL10 was induced. Since T cell depleted hosts are known to have increased availability of IL-7, we hypothesized that this cytokine might play a role in T cell depletion-induced tolerance. Indeed, neutralization of IL-7 and IL-7R? prevented tolerance induction in the majority of animals. These results provide insight into the mechanisms by which self tolerance is maintained in settings of T cell depletion by identifying that T cell depleted hosts are uniquely susceptible to tolerance induction during T cell regeneration due to stringent APC requirements. While this propensity may prevent autoimmunity during T cell regeneration, it may also limit the success of immune based therapies directed toward infectious and neoplastic antigens in clinical situations involving T cell depletion. We also recently completed immune characterization studies of two murine tumor models to address questions related the biology of the host tumor interface in hosts with normal and depleted T cell numbers. In the first, we used MB49, a bladder tumor which naturally expresses HY, the male associated minor histocompatibility antigen which is the antigen we have studied extensively in skin graft rejection models (above). Using very sensitive and specific measures of immunity in animals inoculated with MB49, we observed that despite progressive tumor growth, there is evidence for immune priming to immunodominant and subdominant class I restricted epitopes as well as immunodominant Class II restricted T cell epitopes. Furthermore, adoptive transfer of T cells from mice with progressive tumors leads to accelerated rejection of HY disparate skin grafts. Therefore, progressive tumor growth is associated with weak immune priming. Using T cell active cytokines, we observed significant increases in the number of tumor reactive populations but this was not sufficient to enhance tumor rejection. These results have been submitted for publication. In the second, we used a mouse model of spontaneously occurring osteosarcoma (K7M2) which has a propensity for distant metastases. This model is very similar to the clinical scenario of osteosarcoma which we observe in our patients: the tumor is implanted into the extremity, the extremity is then amputated following tumor growth and large numbers of animals then develop distant metastases. When we growth of the primary tumor in normal vs. T cell depleted mice, to we observed no differences, however there were substantial differences in the development of metastatic disease. Whereas metastases did not develop in the majority of animals with normal T cell numbers, those animals who were T cell depleted had a very high rate of metastasis. Furthermore, immune reconstitution of these mice by the transfer of normal nave T cells prevented metastases.