T CELL TOLERANCE TO ALLOGENIC HEMATOPOIETIC STEM CELLS

本提案的长期目标是确定 用于诱导外周T细胞对同种异体造血干细胞的耐受 干细胞 纯化造血干细胞免疫原性的定义 干细胞可能为实现特异性免疫学提供了新的途径 宽容 这些研究的结果可以指导如何最好地使用 造血干细胞移植作为诱导特异性 对实体器官移植的耐受性 具体目标1：确定抗原 人骨髓CD 34+造血祖细胞的功能。 来自正常人骨髓的CD34+细胞几乎完全是HLA-DR+，B7- 1/B7 - 2阴性。 CD34 +/CD18lo细胞小且无颗粒，具有LTC-IC 活性并且不刺激同种异体T细胞的增殖反应 细胞 相比之下，CD34 +/CD18+细胞较大， 颗粒状，具有最小的LTC-IC活性并刺激同种异体T细胞。 实验将确定是否表达CD18，β 2 白细胞整合素和相关α链之一（CD 11 a、B或c） 是抗原呈递功能的先决条件。 目标二。 定义 外周血CD34+造血抗原提呈功能 通过造血生长刺激动员的祖细胞 因素 来自G-CSF处理的供体的PBMC在以下方面的效率较低： 刺激同种异体T细胞比来自未刺激供体的PBMC。 从G-CSF处理的供体血液中纯化的CD34+细胞可以刺激 同种异体T细胞 有待检验的假设是，G-CSF治疗 正常供者的血供可能会降低经典APC的活性或诱导 功能性抑制剂。 B细胞、单核细胞和树突状细胞将被 在散装和LDA中单独测试，并且将抑制活性 通过混合实验排除。 目标3. 定义共刺激 T细胞应答同种异体MHC分子所需的分子 由红白血病细胞系HEL呈现。 缺乏足够的 适当的粘附或共刺激分子的表达可以 HLA-DR +/CD11alo/LFA-3 +/ICAM-1+红白血病细胞 系（HEL-DR+不刺激同种异体T淋巴细胞增殖。 从成人血液中纯化的静息CD4+被HEL-DR +/B7+活化。 表达CD69和IL-2受体α。 天然CD4 + T细胞 然而，从脐带血中纯化的，除非 自体APC被添加到培养物中，表明其他粘附 受体或共刺激分子从一个完全胜任的APC， 需要激活。 我们将测试抗原呈递 HEL-DR+细胞的功能将通过转染cDNA来重建 编码推定的共刺激分子，包括CD 11 a、B或c。

The long term objective of this proposal is to determine the requirements for induction of peripheral T cell tolerance to allogeneic hematopoietic stem cells. Definition of the immunogenicity of purified hematopoietic stem cells may suggest new approaches to achieve specific immunological tolerance. Results of these studies may guide how to best use hematopoietic stem cell transplants as a means for inducing specific tolerance to solid organ grafts. Specific aim 1: Define the antigen presenting function of human marrow CD34+ hematopoietic progenitor cells. CD34+ cells from normal human marrow are almost entirely HLA-DR+, B7- 1/B7-2 negative. CD34+/CD18lo cells are small and agranular, have LTC-IC activity and do not stimulate proliferative response of allogeneic T cells. In contrast, CD34+/CD18+ cells are large and somewhat more granular, have minimal LTC-IC activity and stimulate allogeneic T cells. Experiments will determine whether expression of CD18, the beta2 leukointegrin, and one of the associated alpha chains (CD11a, b, or c) is a prerequisite for antigen presenting function. Aim 2. Define the antigen presenting function of peripheral blood CD34+ hematopoietic progenitor cells mobilized by stimulation with hematopoietic growth factors. PBMC from G-CSF-treated donors are less efficient in stimulating allogeneic T cells than PBMC from unstimulated donors. Purified CD34+ cells from blood of G-CSF-treated donors can stimulate allogeneic T cells. The hypothesis to be tested is that G-CSF treatment of normal donors may decrease activity of classical APCs or induce a functional inhibitor(s). B cell, monocytes and dendritic cells will be tested individually in bulk and LDA, and inhibitory activity will be ruled out by mixing experiments. Aim 3. Define the costimulatory molecules required for T cell response to allogeneic MHC molecules presented by an erythroleukemia cell line HEL. Lack of adequate expression of the appropriate adhesion or costimulatory molecules could be the reason why an HLA-DR+/CD11alo/LFA-3+/ICAM-1+ erythroleukemia cell line (HEL-DR+ does not stimulate allogeneic T lymphocytes to proliferate. Resting CD4+ purified from adult blood were activated by the HEL-DR+/B7+ line to express CD69 and IL-2 receptor alpha. Native CD4+ T cells purified from umbilical cord blood, however, did not proliferate unless autologous APC was added to the culture, indicating that other adhesion receptors or costimulatory molecules from a fully competent APC were required for activation. We will test whether antigen presenting function of HEL-DR+ cells will be reconstituted by transfection of cDNA encoding putative costimulatory molecules including CD11a, b, or c.