FACTORS LIMITING ANTI HIV RESPONSES OF CYTOTOXIC T CELLS

越来越明显的是，随着靶细胞上同源肽- mhc （pMHC）复合物的水平，它们在细胞表面的分布可能会显著影响T细胞反应的强度。我们已经证明MHC I类分子与粘附分子（ICAM-1）在膜筏中共聚集，并且靶细胞上筏完整性的破坏降低了病毒肽呈现的效力。基于这些数据，我们假设，随着同源pMHC复合物在靶细胞上的水平，它们在细胞表面的分布也可能影响抗原呈递的效果。为了验证这一假设，我们提出通过不同的SL9递送途径改变含有免疫优势肽表位SLYNTVATL （SL9）的HLA-A2分子在靶细胞表面的分布，并确定SL9-HLA-A2复合物分布的变化是否与gag特异性CTL反应的强度相关。这将在4个特定目标中实现：(i)在靶细胞上产生针对SL9-HLA-A2复合物的可溶性低聚TCR（来自CTL克隆D3）；（ii）模拟SL9- hla - a2呈递的生理途径，利用膜转运载体的胶体金颗粒将SL9递送至靶细胞；（iii）比较通过“生理途径”传递到细胞表面的SL9- hla - a2复合物的分布，或将合成的SL9肽添加到细胞外培养基中产生的SL9- hla - a2复合物的分布，并确定phmc分布的变化如何影响gag特异性CTL反应的敏感性；（iv）使用寡聚D3 TCR测量疾病早期和晚期HLA-A2+ hiv感染患者外周血单个核细胞（PBMC）呈现SL9的比例，并评估治疗干预对接受治疗的患者分离的PBMC呈现SL9的影响。所产生的数据有望提高我们对病毒肽呈现机制的理解，并有助于开发新的方法来监测感染细胞在感染、治疗和疫苗接种期间病毒表位的表达。

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.