In this study, we report a novel subset of dysfunctional TA-specific CD8+ T cells in patients with advanced melanoma. We show that NY-ESO-1-specific PD-1+BTLA+Tim-3− CD8+ T cells exhibit partial dysfunction, producing more IFN-γ, TNF and IL-2 than BTLA+PD-1+Tim-3+ cells but less IFN-γ than BTLA−PD-1+Tim-3− and BTLA−PD-1−Tim-3− NY-ESO-1-specific CD8+ T cells. PD-1+BTLA+Tim-3− NY-ESO-1-specific CD8+ T cells represent the largest molecularly-defined dysfunctional T cell subset among circulating NY-ESO-1-specific CD8+ T cells.
In patients with advanced melanoma, we have shown that the large majority of circulating NY-ESO-1-specific CD8+ T cells upregulate PD-1 expression and that PD-1 regulates the expansion of NY-ESO-1-specific CD8+ T cells (3
). Along with previous studies of HIV-specific CD8+ T cells which have shown that PD-1 blockade fails to completely restore virus-specific CD8+ T cell dysfunction (17
), we observed that PD-1 blockade did not increase TA-specific T cell function on a cell-per-cell basis. Several hypotheses have been proposed to explain the failure of PD-1 blockade to completely reverse T cell dysfunction of PD-1+
exhausted T cells. First, virus-specific exhausted T cells express variable levels of PD-1, and PD-1high
T cell subsets appeared to be less responsive to PD-1 blockade than PD-1low/int
CD8+ T cells (15
). In line with this observation, we have found that PD-1 expression was higher on partially dysfunctional PD-1+
TA-specific CD8+ T cells than on BTLA−
cells but lower than on highly dysfunctional BTLA+
cells. Second, exhausted T cells in chronic viral infections have been shown to upregulate multiple inhibitory receptors, including PD-1, 2B4, CD160 and LAG-3 (2
). The co-expression of these receptors was associated with lower T cell function and more severe infection, and the blockade of multiple inhibitory pathways appeared to better reverse T cell dysfunction. Consistent with these observations, we have shown that a subset of highly dysfunctional NY-ESO-1-specific CD8+ T cells upregulate both PD-1 and Tim-3 in patients with advanced melanoma (5
). In the present study, we further demonstrate that PD-1+
TA-specific CD8+ T cells represent two distinct subsets of TA-specific CD8+ T cells with increased T cell dysfunction, illustrating the hierarchical loss of T cell function in the context of chronic antigen simulation in patients with advanced melanoma.
Such information may be useful to further the studies of the gene programs driving T cell exhaustion in cancer patients and provide potential novel targets to reverse T cell exhaustion (18
As previously reported and in sharp contrast to PD-1 and Tim-3, we observed a positive correlation between the upregulation of BTLA and the upregulation of CCR7, CD45RA and CD27 by total CD8+ T cells, supporting that BTLA upregulation is inversely correlated with CD8+ T cell maturation (14
). Several lines of evidence suggest that the factors controlling BTLA upregulation by TA-specific dysfunctional T cells are distinct from those controlling PD-1 and Tim-3 expression. First, as opposed to PD-1+
exhausted LCMV-specific CD8+ T cells (2
), the large majority of the PD-1+
NY-ESO-1-specific CD8+ T cells in patients with advanced melanoma upregulate BTLA. Second, and in striking contrast with PD-1 expression, we observed no significant upregulation of BTLA expression between PD-1+
TA-specific CD8+ T cells, ruling against a correlation between BTLA expression and the severity of T cell dysfunction in patients with advanced melanoma. Third, NY-ESO-1-specific CD8+ T cells upregulated PD-1 and Tim-3 but not BTLA expression upon prolonged stimulation with cognate antigen. Collectively, these findings are consistent with the idea that the upregulation of BTLA by dysfunctional TA-specific CD8+ T occurs independently of functional exhaustion upon high antigen load. The molecular mechanisms driving BTLA upregulation by antigen-specific CD8+ T cells in patients with advanced melanoma but not in chronic viral infections remain to be identified. Adaptive T cell immune responses to chronic viral infections and cancers are both shaped by high antigen load and the immunosuppressive environment. However, one major difference is the occurrence of T cell anergy, which appears to be an early event in the course of tumor progression possibly due to poor antigen presentation at tumor sites (21
). In this perspective, it is conceivable that upon chronic antigen expression at tumor sites, subsets of TA-specific T cells become dysfunctional through both exhaustion upon high antigen load and anergy upon suboptimal priming. In contrast, T cells directed against exogenous antigens in chronic viral infections may become dysfunctional mainly by functional exhaustion. In support of this hypothesis, exhausted T cells in chronic LCMV infections do not upregulate anergy-associated genes used to define molecular signatures of anergy in vitro and in vivo such as grail, Egr-2 and Egr-3 (23
). In addition, high levels of BTLA expression have been reported on anergic T cells directed against an antigen expressed by somatic tissues (13
). Therefore and together with these studies, our findings of BTLA upregulation by PD-1+
TA-specific CD8+ T cells indicate major differences in the profile of inhibitory receptors upregulated by dysfunctional antigen-specific T cells in cancer versus chronic viral infections.
One critical finding is that BTLA blockade adds to PD-1 blockade to enhance NY-ESO-1-specific CD8+ T cell expansion and function. BTLA blockade, in combination with PD-1 and Tim-3 blockades further augment the frequencies of IL-2-producing, proliferating and total NY-ESO-1-specific CD8+ T cells among total CD8+ T cells upon prolonged antigen stimulation.
In summary, we report a novel major subset of dysfunctional TA-specific CD8+ T cells, which upregulate BTLA and PD-1 in patients with advanced melanoma. The precise mechanisms driving BTLA upregulation by TA-specific CD8+ T cells appear to be independent of functional exhaustion and need to be further investigated. Our findings support the targeting of BTLA, PD-1 and Tim-3 pathways to reverse tumor-induced T cell dysfunction in patients with advanced melanoma.