T CELL ACTIVATION FOR CANCER IMMUNOTHERAPY

我们的实验室一直专注于由进展性肿瘤或肿瘤疫苗引发的来自淋巴结的肿瘤反应性T细胞的产生。基于广泛的动物研究，我们已经证明这些肿瘤引发的LNs可以在体外被抗cd3单抗二次激活，作为替代抗原，并在IL-2中扩增，用于随后的过继免疫治疗。这些抗cd3活化细胞介导免疫特异性肿瘤消退。该培养方法优先激活体内介导抗肿瘤反应的CD8+细胞；不需要CD4+细胞这些效应细胞似乎通过释放1型细胞因子来表现其抗肿瘤作用。ifnγ和GM-CSF)对肿瘤抗原的反应。相比之下，释放2型细胞因子的效应细胞(即。IL-10)在体内不介导肿瘤消退。最近，我们发现我们可以通过添加抗cd28单抗作为共刺激信号进一步刺激肿瘤引发的LN细胞。这导致对肿瘤抗原反应的细胞因子释放增强。此外，我们已经观察到，当纯化的CD3+细胞被激活时，使用这种培养程序的CD4+肿瘤反应性T细胞的优先激活，而不是未分离的LN群体。这些发现表明，细胞群的组成和体外激活条件对随后产生的细胞群有深远的影响。我们的初步结果为我们提供了检测CD4与CD8+ T细胞在过继免疫治疗中的作用的方法；并确定是否可以选择性地培养具有肿瘤反应性的富集T细胞亚群。我们提出以下具体目的：1)研究抗cd3单抗体外激活和抗cd28单抗共刺激后肿瘤引发的LN细胞的抗肿瘤反应性；2)检测抗vbeta单抗体外激活和抗cd28单抗共刺激后肿瘤引发的LN细胞的抗肿瘤反应性；3)探讨肿瘤诱导LN细胞体外活化过程中不同细胞因子对前效应细胞成熟的影响；4)检测肿瘤引发的LN细胞体外激活的其他共刺激信号（即抗cd40或抗41bb）。这些目标的目的是为我们提供一个更好的理解有关肿瘤免疫破坏的细胞效应机制，并选择性地激活和扩大存在于低频率的预先存在的肿瘤反应性T细胞。

Our laboratory has focused on the generation of tumor reactive T cells derived from lymph nodes (LNs) primed by progressive tumors or by tumor vaccinations. Based upon extensive animal studies, we have shown that these tumor-primed LNs can be secondarily activated in vitro by anti-CD3 mAb, as a surrogate antigen, and expanded in IL-2 for subsequent adoptive immunotherapy. These anti-CD3 activated cells mediate immunologically specific tumor regression. This culture method preferentially activates CD8+ cells which have been found to mediate the antitumor responses in vivo; without the requirement of CD4+ cells. These effector cells appear to manifest their antitumor effects by the release of type 1 cytokines (ie. IFNgamma and GM-CSF) in response to tumor antigen. By contrast, effector cells which release type 2 cytokines (ie., IL-10) do not mediate tumor regression in vivo. More recently, we have found that we can further stimulate tumor- primed LN cells with the addition of anti-CD28 mAb as a co- stimulatory signal. This has resulted in enhanced cytokine release in response to tumor antigen. In addition, we have observed the preferential activation of CD4+ tumor reactive T cells utilizing this culture procedure when purified CD3+ cells are activated as opposed to the unfractionated LN population. These findings indicate at the components of the cell population and the in vitro activating conditions can have a profound effect on the subsequent cell population which is generated. Our preliminary results provide us with methods to examine the role of CD4 versus CD8+ T cells in adoptive immunotherapy; and to determine if enriched subpopulations of T cells with tumor reactivity can be selectively cultured. We propose the following specific aims: l) To examine the antitumor reactivity of tumor-primed LN cells after in vitro activation with anti-CD3 mAb and co-stimulation with anti-CD28 mAb; 2)To examine the antitumor reactivity of tumor-primed LN cells after in vitro activation with anti-Vbeta mAbs and co-stimulation with anti-CD28 mAb; 3) To examine the effect of different cytokines during the in vitro activation of tumor-primed LN cells on the maturation of pre- effector cells and; 4) To examine other co-stimulatory signals on the in vitro activation of tumor-primed LN cells (i.e. anti-CD40 or anti-41BB). The objectives of these aims are to provide us with a better understanding regarding the cellular effector mechanisms involved in the immune destruction of tumor, and to selectively activate and expand pre-existing tumor-reactive T cells present at low frequencies.