Directing T Cell Responses during Immune Reconstitution

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。我们的方法包括小鼠模型、非人类灵长类动物研究和来自T细胞耗竭患者的临床样本研究。关于免疫重建的基本生物学，我们在2001年发表了il - 7是胸腺依赖性和胸腺非依赖性T细胞免疫重建的有效调节剂。我们还发表了il - 7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，有效地恢复胸腺T细胞枯竭宿主的免疫力。在同基因小鼠模型中观察到这些结果后，我们研究了IL7在异基因BMT环境下增强T细胞的潜力。我们注意到，在这种情况下，IL7显著恶化移植物抗宿主病，并且与GVH反应相关的毒性作用抵消了IL7对胸腺生成的任何有益作用。Sinha M, Fry TJ, Fowler DH, Miller G, and Mackall CL， IL-7加重移植物抗宿主病。《血》，2002年出版。

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. Our methods involve modeling in mice, studies in non-human primates and studies involving clinical samples derived from patients with T cell depletion. With regard to the basic biology of immune reconstitution, we published in 2001 that IL7 is a potent modulator of both thymic-dependent and thymic-independent T cell immune reconstitution. We also published that IL7 can potently 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. Subsequent to these observations in syngeneic murine models, we studied the potential for IL7 to enhance T cells in the setting of allogeneic BMT. We noted that IL7 significantly worsens graft versus host disease in this setting and that the toxic effects related to the GVH reaction abrogated any beneficial effect of IL7 on thymopoiesis. These results are currently in press: Sinha M, Fry TJ, Fowler DH, Miller G, and Mackall CL, IL-7 Worsens Graft-vs-Host Disease. Blood, 2002, In Press. In addition, because of the potential for clinical application of IL7 as an immunorestorative, we participated in trials of IL-7 administration to non-human primates through collaborations with the Monoclonal Antibody and Recombinant Protein Facility in Frederick, MD and Cytheris Inc. in Paris, France through an MTA/CRADA with the NCI. These studies revealed that a short course of rhIL7 administered to normal cynomolgus monkeys led to dramatic increases in circulating CD4+ and CD8+ T cells and the development of clinically apparent but reversible lymphadenopathy. We undertook further studies to determine whether this potent biologic response reflected thymopoietic effects of IL7 or rather an increase in peripheral expansion. We demonstrated using multicolor flow cytometry, that IL7 increased both "naive" and "memory" T cell subsets but that it also transiently upregulated CD11a on naive CD8+ cells. Furthermore, IL7 induced cycling of both naive and memory subsets as evidenced by increased Ki-67 expression in CD4+ and CD8+ T cells as well as increased BrDu incorporation during ex vivo culture of cells collected from IL7 treated animals. Furthermore, TREC levels in the IL7 treated primates were substantially reduced. Thus, 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. We also evaluated IL7 therapy in T cell depleted SIV infection rhesus monkeys and observed similar effects. Furthermore, antiretroviral therapy was continued and we observed no increase in viral load during these studies. These studies have been submitted for publication. Therefore, these results show that IL7 potently modulates peripheral T cell homeostasis in nonhuman primates and provide promising evidence that IL7 will substantially increase T cell numbers in both T cell replete and T cell depleted humans. We are currently in the process of developing a Phase I clinical trial of IL7 to be tested in patients with refractory cancer wherein we will evaluate the biology of IL7 therapy in humans. In addition, we have identified two new agents with the potential for clinical application as immunorestorative agents. The first, flt3 ligand is known to enhance dendritic cell development. However, through our murine models of T cell depletion, we have made the important observation that antigen driven expansion during immune reconstitution requires the presentation of antigen by professional antigen presenting cells. Based upon this hypothesis, we tested whether one factor limiting immune reconstitution via peripheral expansion might be a limited number of professional antigen presenting cells. We thus treated mice with doses of flt 3 ligand which are known to substantially increased the number of dendritic cells present in lymphoid tissues. Importantly, this dose of flt 3 ligand has no known direct effect on mature T cells. These results revealed substantial increases in cells derived via peripheral expansion which was associated with improved immune competence. 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 flt 3 ligand as a new immunorestorative agent, capable of enhancing both thymic-dependent and thymic-independent pathways of T cell regeneration. These results are currently being prepared for publication. Secondly, we have observed that the HIV protease inhibitor, Indinavir, is active as an immunorestorative agent in mouse models, even in the absence of HIV infection. This effect is similar to that observed when programmed cell death is inhibited by consitutive bcl-2 expression and in vitro studies by other groups have provided evidence that indinavir is capable of inhibiting programmed cell death in mature T cells. We are currently in the process of incorporating indinavir into our clinical trials of immune reconstitution in order to assess whether substantial improvement in T cell regeneration is observed. We are also engaged in two murine tumor models which will serve to answer basic questions regarding the biology of the host tumor interface in hosts with normal T cell numbers and which will serve as a basis for developing immune based therapies for patients in the setting of minimal residual neoplastic disease. In the first, we are using a bladder tumor for which a unique tumor antigen has been defined. This antigen, termed HY, is 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 have observed that despite progressive tumor growth, that there is evidence for immune priming to immunodominant and subdominant class I restricted epitopes as well as immunodominant Class II restricted T cell epitopes. Therefore, progressive tumor growth does not necessarily imply T cell tolerance but rather appears to be associated with weak immune priming. We are currently investigating whether the coadministration of T cell active cytokines to tumor bearing animals can enhance this weak tumor antigen directed immunity and whether such enhanced responses can contribute to tumor control. Secondly, we are using a mouse model of osteosarcoma. In this model, the tumor is implanted into the extremity and the animal subsequently receives an amputation after tumor growth. We then attempt to modulate immune reconstitution and evaluate the effects on tumor recurrence. This model is very similar to the clinical scenario of osteosarcoma which we observe in our patients. We have made the surprising observation that non-specific immune reconstitution, in the absence of any specific tumor vaccine, is capable of preventing tumor recurrence following amputation (Melchionda et al, submitted to ASH 2001). Furthermore, we find no evidence that lymphocytes present during the phase of primary tumor growth are tolerized to the tumor antigens. Together, these studies imply that therapies aimed at hastening T cell immune reconstitution in cancer patients may prevent tumor recurrence and have broad implications for clinical oncology, especially as more agents which can modulate T cell regeneration are identified. AIDS Related 100%