EXPANSION OF ANTI-TUMOR T CELLS FROM TUMOR-BEARING HOSTS

对于癌症的有效过继免疫治疗，肿瘤特异性T细胞 淋巴细胞必须从携带肿瘤的患者获得，在体内活化， 体外培养并大量扩增，但肿瘤诱导的抑制细胞 并且肿瘤抗原的不可利用性可能严重限制了扩增 肿瘤反应性T细胞。 在小鼠肿瘤模型中，我们发现T 来自肿瘤引流淋巴结（DLN）的细胞比来自肿瘤引流淋巴结（DLN）的细胞更容易扩增。 来自相同宿主的脾细胞，并且这些细胞具有治疗性 体内活性。 体内低剂量环磷酰胺及其类似物 （马磷酰胺）在体外使用可以抑制抑制细胞的能力， 限制细胞毒性T淋巴细胞（CTL）生长，而不会对CTL产生不利影响 activation. 来自同基因小鼠P815荷瘤宿主（TBH）的CTL 否则不能生长的脾，在体外培养后可以扩增， 使用Mafosfamide治疗。 使用药理学操作的 T细胞信号转导途径，通过激活蛋白激酶 C和增加细胞内Ca++，代替抗原，我们可以扩大 T细胞在体外具有细胞毒性，在体内具有治疗性。 我们有 还从TBH小鼠中获得了肿瘤特异性CTL克隆， 在抗原和外源性白细胞介素2（IL-2）的生长，而且 可以获得类似于来自TBHS的存储器CTL的“静止”状态。 从这个 静息状态下，我们可以清楚地分离抗原依赖性和IL-2- T细胞活化的依赖阶段。 因此，我们有一个独特的机会 来详细研究肿瘤中T细胞活化的调节 特异性、非转化和可调节CTL克隆。 这些克隆的CTL还 在体内具有治疗活性，并将作为模型， 从TBH的淋巴细胞的基本合理的操作，这将最大限度地提高T 细胞生长并保持抗肿瘤活性。 此外，上述方法 抑制细胞抑制和药理学T细胞活化的作用， 适用于来自患有乳腺癌的人类患者的DLN，但是 来自各个节点的细胞的响应性是高度可变的。 本项目旨在：（1）确定不同的T细胞方法 激活可用于扩增CTL克隆并描绘详细的 调节细胞因子（IL-2， IL-4和TGF-β）影响克隆CTL的生长;（2）确定是否 类似的方法，结合Ts的抑制，通过马磷酰胺起作用， 可以应用于扩增获得的T细胞的大量培养物， 直接来自TBH小鼠，从而使细胞产量最大化， 肿瘤活性得以维持;（3）检测通过不同方法获得的T细胞， 体内治疗功效的扩展策略;以及（4） 确定如何识别、分离和扩增具有潜在抗- 肿瘤活性从人乳腺癌患者的DLN，使用类似的 策略，包括马磷酰胺治疗和药理学激活 的T细胞。 拟议的研究应导致一个更明确的。理解 肿瘤特异性CTL激活，调节和生长，以及更多 合理有效的临床过继免疫治疗策略 癌症。

For effective adoptive immunotherapy of cancer, tumor-specific T lymphocytes must be obtained from tumor-bearing patients, activated in vitro and expanded to large numbers, but tumor-induced suppressor cells and the unavailability of tumor antigen may critically limit the expansion of tumor-reactive T cells. In murine tumor models, we have found that T cells from tumor-draining lymph nodes (DLN) are more readily expanded than spleen cells from the same hosts, and that these cells have therapeutic activity in vivo. Low dose cyclophosphamide in vivo and an analogue (mafosfamide) used in vitro can inhibit the ability of suppressor cells to limit cytotoxic T lymphocyte (CTL) growth, without adversely affecting CTL activation. CTL from syngeneic murine P815 tumor-bearing host (TBH) spleens, which would otherwise not grow, could be expanded after in vitro treatment with mafosfamide. Using pharmacologic manipulation of the pathways of T cell signal transduction, via activation of protein kinase C and increasing intracellular Ca++, in place of antigen we could expand T cells which were cytotoxic in vitro and therapeutic in vivo. We have also derived tumor-specific CTL clones from TBH mice which not only depend upon both antigen and exogenous interleukin 2 (IL-2) for growth, but also can attain a "resting" state similar to memory CTL from TBHS. From this resting state, we can distinctly separate the antigen-dependent and IL-2- dependent stages of T cell activation. Thus, we have a unique opportunity to examine in detail the regulation of T cell activation in a tumor- specific, non-transformed and regulable CTL clone. These cloned CTL also have therapeutic activity in vivo and will serve as a model on which to base rational manipulations of lymphocytes from TBH which will maximize T cell growth and preserve anti-tumor activity. Moreover, the above methods of suppressor cell inhibition and pharmacologic T cell activation are applicable to DLN from human patients with breast cancer, but the responsiveness of cells from individual nodes is highly variable. This project aims to: (1) Determine how different methods of T cell activation can be used to expand CTL clones and to delineate the detailed cellular and molecular mechanisms by which regulatory cytokines (IL-2, IL-4 and TGF-Beta) affect the growth of cloned CTL; (2) Determine whether similar methods, combined with inhibition of Ts, function by mafosfamide, can be applied to the expansion of bulk cultures of T cells obtained directly from TBH mice, so that the yield of cells is maximized and anti- tumor activity is maintained; (3) Test the T cells obtained by different, strategies of expansion for therapeutic efficacy in vivo; and (4) Determine how to identify, isolate and expand T cells with potential anti- tumor activity from the DLN of human breast cancer patients, using similar strategies, including mafosfamide treatment and pharmacologic activation of T cells. The proposed studies should lead to a clearer. understanding of tumor-specific CTL activation, regulation and growth and to more rational and more effective strategies for clinical adoptive immunotherapy of cancer.