| Non-specific T cell hyperactivation is associated with HIV-1 disease progression, but its mechanisms are poorly defined. Recent findings have demonstrated that T cell activation is substantially impacted by cell surface-expressing co-inhibitory molecules such as programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA, CD272), as well as other receptors. These co-signaling pathways play distinct and overlapping modulatory roles during sequential (or different) stages of the T-cell response or for different lymphocyte subsets so that immune responses occur in the correct intensity and pattern. Recent studies have highlighted the role of immuno-inhibitory molecules such as PD-1, CTLA-4 and Tim-3, which mediate CD4 and CD8 T cell exhaustion during chronic HIV-1 infection. Although the pioneering findings indicate that co-inhibitory signaling mediates T-cell exhaustion and partially contributes to HIV-1 persistence, it is still unknown whether these inhibitory pathways experience functional deficits that may be responsible for the loss of the delicate balance between immune over-activation and efficient viral containment in chronic HIV-1 infection. BTLA, the most recently recognized member of the CD28 family, which interacts with its ligand, herpesvirus entry mediator (HVEM), predominantly negatively regulates immune responses. Recent investigations further demonstrated that BTLA is down-regulated in allergen-specific immunotherapy-untreated patients with Japanese cedar pollinosis, and BTLA gene polymorphism is associated with a high risk of rheumatoid arthritis development. However, little is known regarding the role of BTLA in viral infection, in particular in chronic HIV-1 infection.Eighty-five HIV-1-infected individuals were enrolled in our study. They were divided into three groups including long-term non-progressors (LTNPs), typical progressors (TPs) and AIDS patients according to CD4 T-cell count and serum HIV-1 RNA load. Twenty-six uninfected subjects were employed as healthy controls (HCs). To define the pattern of BTLA expression in lymphocytes, our experiments showed that BTLA was mainly expressed by peripheral B cells, T cells, NKT cells and monocytes rather than NK cells. In particular, BTLA was found to be constitutively expressed by CD4 T cells and CD8 T cells. We found that BTLA expression levels in both CD4 and CD8 T cells were significantly down-regulated in HIV-1-infected subjects compared with HCs. Importantly, HIV-1-infected individuals displayed large differences in BTLA expression levels in both CD4 and CD8 T cells: LTNPs exhibited higher levels of BTLA expression than did TPs and AIDS patients; while the lowest levels of BTLA expression in both CD4 and CD8 T cells were observed in AIDS patients. The data clearly indicated that chronic HIV-1 infection may lead to overall progressive BTLA down-regulation in CD4 and CD8 T cells. We further analyzed BTLA mRNA expression in HC and HIV-1 infected subjects. It was found that BTLA mRNA expression was also significantly lower in the peripheral blood of TPs and AIDS patients than HCs. Thus, BTLA down-regulation in total T cell subsets is associated with the progression of HIV-1 infection and may serve as a marker of disease progression.Next, we investigated difference in BTLA expression among HIV-1-, CMV- and Flu-specific pentamer T cells from HIV-1-infected and HC subjects. We observed that BTLA expression in both the CMV- and Flu-specific pentamer cells was significantly reduced in LTNP and TP/AIDS subjects compared with HCs. These data suggest that BTLA expression is also down-regulated in HIV-specific and non-HIV-specific CD8 T cells in progressive HIV infection.We also analyzed the relationships of BTLA expression in CD4 T cells with two markers of HIV-1 disease progression: plasma viral load and peripheral CD4 T cell count. Our results showed that both BTLA percentage and MFI (mean fluoresce intensity) in total CD4 T cells positively correlated with peripheral CD4 T cell counts, but inversely correlated with plasma viral loads. We further analyzed the associations of BTLA expression with other markers such as CD38, PD-1, CD127 and Ki67, which have been demonstrated to correlate with HIV-1 disease progression. We found that BTLA expression in total CD4 T cells was negatively correlated with CD38, PD-1 and Ki67 expression levels, but positively correlated with CD127 expression in TPs. These data suggest that BTLA down-regulation in CD4 T cells correlates with disease progression in chronic HIV-1 infection.Through examination of the distribution of BTLA expression in Tna?ve, Tcm and Tem subsets on the basis of CD45RO and CD27 expression, we found that CD4 T cells from HIV-1-infected TPs and AIDS patients were constituted with higher percentages of Tcm and Tem subsets and lower percentage of Tnaive subsets than those from the HC and LTNP subjects. These data probably indicate that chronic HIV-1 infection leads to a progressive decline in naive CD4 T cell subsets but a continuous increase in memory CD4 T cell subsets, and thus altered the distribution of memory CD4 T cell population. More importantly, we found that BTLA expression was more dominant in Tna?ve cells, but gradually decreased in the Tna?ve, Tcm and Tem populations regardless of disease status. BTLA expression was significantly down-regulated in all of three CD4 T cell subsets in HIV-1-infected subjects in comparison to the HCs. These data indicate that persistent HIV-1 infection leads to a progressive loss of BTLA expression as an early change, which precede a reduction of the na?ve CD4 T cell subset and an expansion of the memory CD4 T cell subset.To determine whether BTLA expression in CD4 T cells influences their activation status, we analyzed BTLA+ and BTLA– CD4 T cell phenotypic profiles in HC and HIV-1-infected subjects. It was found that HC subjects displayed a juvenile phenotype in BTLA+CD4 T cells which were constituted with large percentage of the Tnaive and small percentage of the Tcm and Tem, while BTLA– CD4 T cells that displayed a relatively mature phenotype indicated by large percentage of the Tcm and Tem and small percentage of the Tniave subsets. Interestingly, this distribution was disturbed within BTLA+ but not BTLA– CD4 T cells in HIV-1-infected subjects. Interestingly, this distribution was disturbed within BTLA+ but not BTLA– CD4 T cells in HIV-1-infected subjects. We found that BTLA+ CD4 T cells were constituted with a smaller fraction of Tna?ve subset and more of the Tem subset, in particular in HIV-infected TP and AIDS patients. These data suggest that persistent HIV-1 infection might substantially alter the profiles of memory subsets within BTLA+ CD4 T cells. We further analyzed the associations of BTLA expression with CD38, CD95, PD-1, Ki67, and perforin. Data showed that chronic HIV-1 infection markedly increased CD38, CD95, PD-1, Ki67 and perforin expression and decreased CD127 expression in BTLA+ CD4 T cell subsets, in particular TPs and AIDS patients, in contrast with HCs. For the BTLA– CD4 T cell population, HIV-1 infection seldom influences the expression of these markers, except for CD38. This comprehensive analysis suggests that HIV-1 infection significantly skews BTLA+ rather than BTLA– CD4 T cells towards differentiation and maturation. Thus, HIV-1 infection not only reduces BTLA expression, but also significantly impacts the immune status of BTLA+ CD4 T cells. We found that HIV-1 NL4-3 viral could down-regulate BTLA expression in CD4 T cells in vitro. In addition, HIV-1 NL4-3-induced BTLA down-regulation in CD4 T cells was viral load-dependent. BTLA down-regulation in CD4 T cells was more pronounced when the cells were cultured with high doses of the virus supernatant. Notably, we found that addition of the anti-viral agent AZT failed to reverse HIV-1-induced BTLA loss at the indicated time point. These data indicate that HIV-1 virus can directly stimulate CD4 T cells to down-regulate BTLA expression, which may reduce HVEM-mediated inhibition and cause more non-specific activation of T cells via a positive loop.The BTLA-mediated inhibitory pathway plays an important role in maintaining basic immune tolerance by inhibiting T cell activation, cytokine production and proliferation. In order to investigate the functional relevance of diminished BTLA expression in CD4 T cells, we used the agonistic anti-BTLA mAb to compare the BTLA-mediated functional inhibition of CD4 T cells in HIV-1-infected subjects and uninfected persons. We found that BTLA-mediated suppression of CD4 T cell activation (marked by CD25, CD38 and CD69) and cytokine production (IL-2 and INF-γ) was significantly impaired during chronic HIV-1 infeciton.Some studies showed that BTLA cross-linking can inhibit the proliferation of T cells. But in our study we found that inhibition of T cell proliferation exhibited a high heterogeneity. BTLA cross-linking inhibited T cells proliferation in some subjects but promoted proliferation in other subjects. So further studies should be performed to determine the exact function of BTLA in influencing the T cell proliferation.Some investigations have demonstrated that PD-1+ T cells are more prone to apoptosis. So we considered that whether BTLA signaling were associated with CD4 T cell apoptosis. We found that annexin V+ CD4 T cells were mainly the BTLA– cells, and BTLA+ CD4 T cells were seldom to apoptosis. Also BTLA expression on CD4 T cells was negatively with annexin V+ CD4 T cells. These data indicate that BTLA–CD4 T cells are more prone to apoptosis that BTLA+CD4 T cells.Taken together, these data indicate that chronic HIV-1 infection not only progressively down-regulate in BTLA expression in CD4 T cells but also leads to an impairment of BTLA-mediated inhibitory functions on CD4 T cells. This functional loss of BTLA might facilitate immune hyper-activation, which is potentially favorable for HIV-1 proliferation and persistence. Our findings likely indicate that BTLA may present a novel predictor for early progression, which provide further insight into the mechanisms by which non-specific immune activation disrupts T-cell homeostasis and contributes to AIDS disease progression. |
