T CELL ACTIVATION FOR CANCER IMMUNOTHERAPY

我们的实验室一直专注于肿瘤反应性T细胞的产生，这些T细胞来源于由进行性肿瘤或肿瘤疫苗引发的淋巴结（LN）。基于广泛的动物研究，我们已经表明，这些肿瘤致敏的LN可以在体外被抗CD 3 mAb作为替代抗原二次激活，并在IL-2中扩增，用于随后的过继免疫治疗。 这些抗CD 3活化细胞介导免疫特异性肿瘤消退。该培养方法优先激活已发现在体内介导抗肿瘤应答的CD 8+细胞;而不需要CD 4+细胞。这些效应细胞似乎通过释放1型细胞因子（即，IFN γ和GM-CSF）对肿瘤抗原的应答。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，我们已经发现，我们可以通过添加抗CD 28 mAb作为共刺激信号来进一步刺激肿瘤致敏的LN细胞。这导致响应于肿瘤抗原的细胞因子释放增强。 此外，我们已经观察到，当纯化的CD 3+细胞被激活时，与未分级的LN群体相反，利用该培养程序的CD 4+肿瘤反应性T细胞的优先激活。这些发现表明，细胞群的组分和体外活化条件可对随后产生的细胞群产生深远影响。我们的初步结果为我们提供了方法来检查CD 4与CD 8 + T细胞在过继免疫治疗中的作用，并确定是否可以选择性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）检测肿瘤致敏LN细胞经抗CD 3单抗体外活化并与抗CD 28单抗共刺激后的抗肿瘤反应性：2）检测肿瘤致敏LN细胞经抗V β单抗体外活化并与抗CD 28单抗共刺激后的抗肿瘤反应性; 3）检查在肿瘤致敏的LN细胞的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）检查其他共刺激信号对肿瘤致敏的LN细胞（即抗-CD 40或抗-41 BB）的体外活化。这些目标的目的是让我们更好地了解参与肿瘤免疫破坏的细胞效应机制，并选择性地激活和扩增以低频率存在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.