Activation in vitro with different stimuli of either total PBMCs or purified CD4+ T cells from HIV-1 infected individuals induces viral expression and replication. The number of HIV-1 virions produced by these endogenously infected activated blasts was detected through real-time PCR, while the amount of p24 HIV-1 core antigen released in culture supernatant was determined by ELISA. Using an experimental approach similar to that for activating primary T cells in vitro, we sought to isolate and characterize these productively infected cells starting from highly enriched CD4+ T cells purified from HIV-1 infected viremic patients. Five to seven days of stimulation with phytohemoagglutinin (PHA) and recombinant IL-2 (rIL2) were required to observe a variable but consistent percentage of endogenously HIV-1 infected CD4+ T cell blasts, characterized by the presence of intracellular viral p24 core antigen. We tested the amount of p24 antigen in CD4+ T cell blasts every 3 days for 3 weeks to determine the point of maximal expansion of these infected cells. Within our cohort of HIV-1 infected viremic donors, the highest percentages of p24pos expression in activated CD4+ T cell blasts were detected, on average, at 12 days of activation (median: 9.43%; SD = 6.6) and started to decrease progressively thereafter. ? ? It has also been reported that HIV-1 infection in vitro results in a selective down-modulation of MHC-I molecules in cell lines and in exogenously infected primary CD4+ T cells. To determine whether this phenomenon also occurs in endogenously infected CD4+ T cell blasts expanded from HIV-1 infected individuals, we analyzed the expression of classic and non-classic HLA molecules on p24pos and p24neg blasts. We found that surface levels of HLA-A and -B alleles, calculated as mean fluorescence intensity, were significantly down modulated on p24pos/CD4neg blasts compared to p24neg/CD4pos blasts (p=0.004 for HLA-A alleles; p=0.018 for HLA-BW4 and BW6 alleles). In contrast, the expression of HLA-C and HLA-E molecules did not substantially differ between p24neg and p24neg blasts.? ? Cytolytic activity of NK cells against autologous cells is possible when the usually dominant inhibitory interaction between MHC-I molecules and iNKRs is either absent or weak. The selective down-modulation of HLA alleles on endogenously HIV-1 infected CD4+ T cell-derived blasts would, theoretically, make these cells sensitive to lysis by autologous NK cells. To test this hypothesis, we separated infected from uninfected T cell blasts on the basis of CD4 surface expression, and we assessed the ability of rIL-2 activated autologous NK cells to kill both HIV-1 infected and uninfected T cell blasts. As expected, p24neg/CD4pos blasts with normal levels of MHC-I were highly resistant to NK cell-mediated cytolysis. In contrast, the percentages of infected p24pos/CD4neg blasts lysed by autologous NK cells were significantly higher compared to that of p24neg/CD4pos blasts (p = 0.003). To further determine the importance of the down-modulation of HLA-A and -B alleles in rendering infected blasts sensitive to NK cell lysis, we repeated the same experiment in the presence of specific anti-MHC-I monoclonal antibodies (mAbs). Completely blocking interactions between iNKRs and HLA-I molecules would theoretically render both infected and uninfected CD4+ T cell blasts equally susceptible to NK cell-mediated killing. Indeed, the masking of all MHC-I molecules abolished the inhibitory effect of iNKRs and resulted in a remarkably higher degree of NK cell-mediated lysis of p24neg blasts comparable to that of p24pos blasts. ? ? Despite the significantly higher susceptibility to lysis by autologous NK cells of endogenously HIV-1 infected blasts, compared to uninfected blasts, the degree of NK cell cytolytic activity against p24pos/CD4neg cell blasts was still relatively low and variable from patient to patient (range: 4.2% - 23%; median: 9.6%). Even when interactions between iNKRs and MHC-I were completely blocked, NK cells only partially eliminated both p24pos and p24neg autologous CD4+ T cell blasts (range: 13% 48%; median: 38.4%). If the dominant and negative effect of interactions between iNKRs and MHC-I molecules is partially overcome by the HIV-1 induced selective down-modulation of HLA-A and B alleles, NK cell killing of autologous and endogenously infected CD4+ T cell blasts must occur through activating NK cell receptors. In order to identify those NK receptors that trigger this NK cell-mediated lysis, we analyzed the phenotype and functions of NCRs and NKG2D that, under physiological conditions, represent the major activating receptors that regulate NK cell cytotoxicity. We performed masking experiments to address the role of NCRs in the NK cell lysis of autologous and endogenously HIV-1 infected CD4+ T cell blasts. The blocking of NKp46, NKp30 and NKp44 did not result in a significant decrease in NK cell-mediated lysis of p24pos/CD4neg cell blasts, indicating that the NCRs were not playing a substantial role in this killing. The lack of significant NK cell killing of endogenously HIV-1 infected CD4+ T cell blasts through the NCR activation pathway was also consistent with previously reported data showing that the percentage of NK cells expressing NCRs was significantly decreased on fresh and rIL-2 activated NK cells from HIV-1 viremic patients compared to those from healthy donors. We also analyzed the expression of cellular ligands for NCRs using soluble human NCR-Ig fusion proteins. We found that the surface levels of NCR ligands on p24pos/CD4neg cell blasts were very low, while small fractions of p24neg/CD4pos cell blasts were positive for such ligands. The fact that NCR ligands were expressed at very low levels on naturally HIV-1 infected blasts is consistent with the low degree of NK cell-mediated lysis of p24pos/CD4neg cell blasts through NKp46, NKp30 or NKp44 pathways. ? ? We previously reported that among HIV-1 infected viremic individuals there is a high frequency of a markedly dysfunctional CD56neg subset of NK cells in which surface expression of NKp46 and NKp30 and cytolytic activitiy against tumor cell line targets was found to be very low. In this regard, we found that the degree of NK cell baseline cytolysis of autologous p24pos blasts is inversely correlated with the frequencies of anergic CD56neg NK cells. As previously demonstrated for NCRs, we then performed masking experiments to understand the extent of the contribution of NKG2D to the NK cell-mediated killing of infected p24pos/CD4neg cell blasts. We found no differences in NK cell surface levels of NKG2D in cells from infected and uninfected individuals. We also found that NKG2D ligands, detected through a soluble human NKG2D-Ig fusion protein, were expressed at high levels on endogenously HIV-1-infected CD4+ T cell-derived blasts. In particular, among the previously described NKG2D ligands, UL16-binding proteins (ULBP), especially ULBP-2, were clearly expressed on p24pos blasts, while the levels of MHC class I-related chain A (MIC-A) and MIC-B were almost undetectable. In line with this phenotype, the masking of NKG2D induced a substantial reduction of NK cell cytolysis of p24pos cell blasts from all patients analyzed (p < 0.0001). Moreover, we found a direct and significant correlation between the expression of ULBP-2 on p24pos/CD4neg cell blasts and the NK cell-mediated lysis of autologous HIV-1 endogenously infected T cell blasts. Consistent with results previously published using an in vitro experimental system, our data indicate that the pathway activated by interactions of NKG2D with its ULBP ligands plays an important role in the NK cell killing of endogenously HIV-1 infected CD4+ T cell blasts.
