Platelet-medicated deregulation of dendritic cell function- implications for anti-HIV immune responses and immunotherapy

摘要 我们将检验血小板-单核细胞和血小板-DC复合物的破坏将导致 改善DC的功能，从而增加a）HIV的自发控制和B） 基于DC的免疫疗法的治疗效果。我们已经报道，艾滋病毒感染导致高达2倍的 循环血小板-单核细胞复合物（PMC）的增加导致单核细胞的不期望的活化。 我们最新的体外初步数据显示，来源于PMC的DC（MoDC）表达水平降低的几种细胞因子。 对DC功能至关重要的分子（例如CD 206、DC-SIGN、CD 80、CD 86、CCR 7），并显示减少的抗原摄取 能力，诱导幼稚CD 4和CD 8 T细胞增殖的能力，以及在免疫后降低IL-12分泌的能力。 用CD 40 L刺激。共培养上清液中TGF β 1水平的增加表明这种分子 可能介导血小板对MoDC的抑制作用。我们还检测到了 HIV+和HIV-个体全血中的血小板和循环DC（cDC），与 CD 40和CD 80表达减少，表明与其他白细胞相似，血小板可以影响 在这些观察的指导下，在目标1中，我们将确定血小板对内源性DC功能的影响。 内源性血液分离的cDC 1、cDC 2、pDC和MoDC的免疫原性功能。为此，艾滋病毒感染者， 接受或未接受抗血小板治疗的cART治疗和未感染个体将使用我们的活性 协议.将分析循环DC与血小板的相互作用以及共刺激因子的表达。 分子和趋化因子受体。这些发现将与血小板活化的测量相关， 通过ELISPOT的HIV特异性免疫应答。此外，cDC（直接从血液中分离）或MoDC将在体外培养。 与血小板复合以评价其免疫刺激和抑制因子的产生，并用于 评估抗原提呈至初始CD 4和CD 8 T细胞，向淋巴结定向迁移 趋化因子（CCL 21，CCL 19），体外诱导针对HIV和对照抗原的Th 1和CTL应答， 细胞毒功能目的2，将确定TGF β 1和PGE 2信号转导在血小板介导的调节中的作用， MoDC和cDC功能。将使用TGF β 1和PGE 2的阻断抗体/抑制剂。识别 与血小板DC调节作用相关的关键信号事件将使我们能够开发新的策略 改善感染个体固有的抗HIV免疫反应，以及潜在的其他慢性疾病 包括癌症

ABSTRACT We will test the hypothesis that the disruption of platelet-monocyte and platelet-DC complexes will result in improved function of DCs, thereby increasing the efficacy of a) spontaneous control of HIV and b) the therapeutic effects of DC based immunotherapy. We have reported that HIV infection results in up to 2-fold increase in circulating platelet-monocyte complexes (PMCs) resulting in undesirable activation of monocytes. Our latest in vitro preliminary data show that DCs derived from PMCs (MoDCs) express reduced levels of several molecules critical to DC function (e.g. CD206, DC-SIGN, CD80, CD86, CCR7) and show reduced antigen uptake capacity, ability to induce the proliferation of naïve CD4 and CD8 T cells and decreased IL-12 secretion upon stimulation with CD40L. Increased levels of TGF in co-culture supernatants are suggestive that this molecule may mediate the suppressive effects of platelets on MoDCs. We also detected direct complexes between platelets and circulating DCs (cDCs) in whole blood of HIV+ and HIV- individuals, that were associated with decreased expression of CD40 and CD80 suggesting that, similar to other leukocytes, platelets can influence endogenous DC function.Guided by these observations, in Aim 1 we will determine the effect of platelets on the immunogenic functions of endogenous blood-isolated cDC1, cDC2, pDC and MoDC. To this end, HIV infected, cART treated and uninfected individuals on or not on anti-platelet therapy will be enrolled using our active protocols. Circulating DCs will be analyzed for their interaction with platelets and expression of costimulatory molecules and chemokine receptors. These findings will be correlated with measures of platelet activation and HIV specific immune responses by ELISPOT. Further, cDCs (directly isolated from blood) or MoDCs will be complexed with platelets to evaluate their production of immunostimultory and suppressive factors, and used to evaluate antigen presentation to naïve CD4 and CD8 T cells, migration towards lymph-node-directing chemokines (CCL21, CCL19), in vitro induction of Th1 and CTL responses against HIV and control antigens and cytotoxic function. Aim 2, will determine the role of TGF and PGE2 signaling in platelet-mediated modulation of MoDC and cDC function. Blocking antibodies/inhibitors of TGF and PGE2 will be used. Identification of the key signaliing events associated with DC-modulating effects of platelets will allow us to develop new strategies to improve inherent anti-HIV immune responses in infected individuals, and, potentially other chronic disorders including cancer.