FACTORS LIMITING ANTI HIV RESPONSES OF CYTOTOXIC T CELLS

越来越明显的是，沿着靶细胞上同源肽-MHC（pMHC）复合物的水平，它们在细胞表面的分布可显著影响T细胞应答的幅度。我们已经证明，MHC I类分子与粘附分子（ICAM-1）共聚集在膜筏中，并且靶细胞上筏完整性的破坏降低了病毒肽呈递的效力。基于这些数据，我们假设沿着靶细胞上同源pMHC复合物的水平，它们在细胞表面的分布也可能影响抗原呈递的功效。为了验证这一假设，我们建议改变分布的HLA-A2分子含有免疫显性肽表位SLYNTVATL（SL 9）从HIV p17 gag蛋白在靶细胞表面上使用不同的途径SL 9交付，并确定是否SL 9-HLA-A2复合物的分布的变化与gag特异性CTL反应的强度相关。这将在4个具体目标中实现：（i）产生可溶性寡聚TCR（ii）模拟SL 9-HLA-A2呈递的生理途径，使用膜易位载体的胶体金颗粒将SL 9递送到靶细胞中;（iii）比较通过“生理途径”递送至细胞表面或用添加至细胞外培养基的合成SL 9肽产生的SL 9-HLA-A2复合物的分布，并确定pHMC分布的变化如何影响gag-HLA-A2的敏感性;特异性CTL应答;（iv）使用寡聚体D3 TCR来测量呈递源自疾病早期和晚期的HLA-A2+ HIV感染患者的SL 9的外周血单核细胞（PBMC）的分数，并评估治疗干预对SL 9在分离自接受治疗的患者的PBMC上的呈递的影响。所产生的数据有望提高我们对病毒肽呈递机制的理解，并有助于开发新方法来监测感染，治疗和疫苗接种期间感染细胞上病毒表位的表达。

It is becoming increasingly evident that along with the level of cognate peptide-MHC (pMHC) complexes on target cells their distribution at the cell surface could substantially influence the magnitude of a T cell response. We have demonstrated that MHC class I molecules are co- clustered with adhesion molecules (ICAM-1) in membrane rafts and that disruption of the raft integrity on target cells decreases the potency of presentation of viral peptides. Based on these data we hypothesize that along with the level of cognate pMHC complexes on target cells their distribution at the cell surface might also influence the efficacy of the antigen presentation. To test this hypothesis we propose to vary the distribution of HLA-A2 molecules containing an immunodominant peptide epitope SLYNTVATL (SL9) from HIV p17 gag protein on the surface on target cells using different pathways on SL9 delivery and to determine whether variations in the distribution of SL9-HLA-A2 complexes correlates with the intensity of gag-specific CTL responses. This will be accomplished in 4 specific aims: (i) to produce soluble oligomeric TCR (from the CTL clone D3) specific to SL9-HLA-A2 complexes on target cells; (ii) to mimic the physiological pathway of SL9-HLA-A2 presentation delivering SL9 into the target cell using colloidal gold particles of membrane-translocating carriers; (iii) to compare the distribution of SL9-HLA-A2 complexes delivered to the cell surface through "physiological pathway" or generated with synthetic SL9 peptide added to the extracellular medium and to determine how variations in the distribution of the pHMCs affect the sensitivity of gag- specific CTL responses; (iv) using oligomeric D3 TCR to measure the fraction of peripheral blood mononuclear cells (PBMC) presenting SL9 derived from HLA-A2+ HIV-infected patients in earlier and late stages of the disease and to evaluate the effect of therapeutic intervention on the presentation of SL9 on PBMC isolated from patients undergoing treatment. The data produced are expected to improve our understanding of the mechanisms of presentation of viral peptides and to aid the development of new approaches to monitor the expression of viral epitopes on the infected cells during infection, treatment, and vaccination.