HIV disease is associated with chronic immune activation and multiple tiers of T cell dysfunction (16
). In this study, we have adapted a multiparameter flow cytometric technique (PhosFlow) (39
) to interrogate T cell signaling pathways in discrete subpopulations of CD4+
T cells obtained from subjects in varying stages of HIV disease and treatment. We show that PhosFlow enables simultaneous analysis of specific signaling pathways within discrete and multiple subpopulations of CD4+
T cells. Using this approach, we wished to know: (a) whether and which defects in T cell signaling might be associated with advanced disease; (b) whether such defects were generalized across the CD4+
T cell lineages; and (c) whether any observed defects were reversed by effective antiviral treatment. Our results indicate that, in subjects with advanced disease, T cell signaling responses to TCR cross-linking, IL-2, and PMA/ionomycin were blunted within many if not all CD4+
T cell subpopulations. Interestingly, changes in phosphorylation of key signaling intermediates were not so much associated with defects in induced phosphorylation per se but with an increased level of basal phosphorylation. When effective antiviral treatment was initiated, basal phosphorylation levels returned to normal, as did signaling responses to multiple T cell stimuli. These observations highlight the indirect effects that HIV infection has on the T cell compartment and may in part provide a mechanistic basis to observed features of T cell dysfunction found in late-stage disease.
PhosFlow analysis showed that, in response to TCR cross-linking, phosphorylation of Lck and Zap70 was most significantly reduced in PROGs compared with LTNP and RESPs, especially in naive T cells for p-Lck and in more differentiated T cell subpopulations for p-Zap70. After stimulation with IL-2, the extent of phosphorylation of Stat5 in PROGs was also diminished in most T cell subpopulations, and especially in CD8+
T cells. Finally, blunted phosphorylation of ERK1/2 was observed in most T cell subpopulations after stimulation of cells from PROGs with PMA/ionomycin. These results are consistent with previous reports in the literature. For example, Cayota et al. (1994) (32
) and Stefanova et al. (1996) (33
) showed that HIV disease progression is associated with defective tyrosine phosphorylation and altered levels of or post-translational modifications of T cell signaling molecules. Likewise, differential display of protein tyrosine kinases in CD4+
T cells revealed dysregulation of multiple protein kinases in the setting of pathogenic SIV infection (34
). Down-regulation of CD3ζ and CD28 on CD8+
T cells has been associated with defects in TCR stimulation (26
), whereas defects in IL-2 receptor expression in HIV disease have been linked to impaired activation of Stat5 and upstream kinases (19
). Of note, all of these findings were made in the context of heterogeneous populations of CD4+
T within PBMCs, making it difficult to determine whether they might simply reflect changes in the relative frequencies of individual T cell subpopulations. In this study, using the single cell analytical platform provided by Phosflow, we show that abnormalities in protein phosphorylation and signaling are found in multiple discrete subpopulations of both CD4+
T cells, suggesting a generalized impact of progressive HIV infection on all. Technical limitations, i.e., primary cells available from HIV+
individuals, did not allow us to directly include more detailed analyses of protein expression levels here.
Potential drivers of such generalized dysregulation of T cell signaling might include the chronically activated state that attends progressive HIV disease (50
) and/or circulating virus (or viral proteins). For instance, cross-linking of CD4 by HIV envelope glycoprotein gp120 and/or gp120-specific Abs has been shown to inhibit CD4+
T cell function and activation (51
). Moreover, gp120 has been found to induce TCR desensitization and to alter signal transduction through Lck, possibly by affecting its association with CD4 (53
). Interestingly, in the present study, only changes in p-Lck, but not p-Zap70, after TCR-stimulation were significantly affected by VL. This suggests that T cells in the presence of high VL and higher levels of circulating gp120 are more prone to lose activation of the immediate-early Lck-mediated TCR signaling and that this altered activation of the CD4-associated Lck kinase does not fully translate to the downstream Zap70 kinase. However, signaling through Lck is also altered in CD8+
T cells, suggesting that gp120 alone does not account for blunted signaling. The more pronounced impairment of p-ERK1/2 after stimulation with PMA/Iono, when compared with p-p38, possibly reflects the fact that blunted changes in p-ERK1/2 are associated with higher VLs, whereas p-p38 is less affected. High VLs do not seem to be the causative agent of blunted IL-2 signaling, as analyzed by change in p-Stat5 after stimulation. To more directly analyze the effect of cellular activation on signaling in HIV infection, we correlated basal phosphorylation and signaling with cellular activation, i.e., as measured by expression of the activation markers CD38 and/or HLA-DR, for a subset of patients (9 LTNP, 3 PROG, and 10 RESP). Data from these patients had all been gathered within 12 mo of the PhosFlow analysis, and the patients had shown no change in clinical status during the intervening time frame. There was a clear trend of higher activation correlated with higher basal phosphorylation and blunted signaling (data not shown). However, with the limited data set available, the correlations were not statistically significant and we are, at this point, not able to directly show a correlation between cellular activation and basal levels of phosphorylation or signaling. However, studies addressing this important question are underway.
The immunoreceptor PD-1 suppresses TCR signaling, likely via a recruitment of SHP phosphatase activity, resulting in decreased phosphorylation of the CD3ζ activation motifs, attenuated Zap70 activation, and inhibition of downstream signal transduction (55
). Recently, up-regulation and expression of PD-1 has been associated with T cell dysfunction, and cellular exhaustion in chronic lymphocytic choriomeningitis virus and HIV infections (42
). Therefore, expression of PD-1 might well account for some of the decreased signaling seen here. PD-1 expression was highest on cells from patients with progressive disease and elevated levels were associated with blunted changes in p-Lck and p-Zap70 in most T cell subpopulations after TCR stimulation. Interestingly, high levels of PD-1 expression were also associated with blunted IL-2 signaling in CD8+
, but not CD4+
, T cells, possibly reflecting different IL-2 signaling networks or requirements in these cell types.
Progressive HIV disease is associated with a chronic inflammatory state that induces T cell activation (48
) and the secretion of multiple proinflammatory cytokines and chemokines. These mediators, in turn, can have profound effects on the expression of cellular proteins involved in cell-cell interactions. For instance, HIV-induced stimulation of IFNα
from plasmacytoid dendritic cells results in up-regulation of MHC class I proteins in vivo, which in turn can interact with TCR of circulating cells (56
). These more global and indirect factors may affect the basal phosphorylation state of key signaling intermediates within CD4+
T cells. Thus, a constitutive activation of Stat1 and Stat5 in PBMCs has previously been reported in the setting of HIV infection (58
). Our results show that levels of Stat5 phosphorylation are highest in CD4+
T cells from patients with progressive disease and that these cells are precisely those with impaired signaling. Similar results were found for Lck and ERK1/2 (but not Zap70 or p38) and, again, high levels of basal phosphorylation of these kinases were associated with lower changes in induced phosphorylation after TCR, IL-2, and PMA/ionomycin stimulation.
To determine whether blunted signaling is the consequence of elevated basal phosphorylation and/or down-regulated signaling pathways, we also compared the MFI of the p-signals after stimulation (data not shown). The results indicate that the “end point levels” of phosphorylation are similar in the three groups. Although in some instances (most evident for p-Stat5 after stimulation with IL-2) PROG had slightly higher end levels, these differences were not as striking and significant as those found in the case of basal phosphorylation (shown in ). These results suggest that blunted responses can, at least in part, be explained by high levels of basal phosphorylation in T cells from HIV PROGs and are consistent with a model in which higher basal phosphorylation levels only allow for blunted changes in phosphorylation of “pre-activated” signaling proteins. Such higher basal phosphorylation might reflect activation and/or perturbed regulation of cellular signaling in the setting of HIV infection. However, other mechanisms, such as differential expression of signaling receptors or proteins, e.g., down-regulated IL-2R, might also be contributory.
Elevated levels of basal phosphorylation and dysregulated signaling might very well be related to or caused by cellular alterations in the cellular redox balance. Thus, HIV disease progression has been shown to be associated with decreasing levels of glutathione (GSH), the major redox buffer in almost all cells (59
). Alterations in GSH levels affect the activity of redox-sensitive enzymes, including protein kinases and phosphatases. Such changes, in turn, appear to impact upon cellular signal transduction pathways (63
). In the specific case of HIV disease, depletion of GSH was shown to result in elevated levels of basal phosphorylation and in cellular dysfunction, e.g., reduced calcium flux and proliferation in response to TCR stimulation (61
). Importantly, GSH deficiency has also been associated with numerous other disease states (64
). It is accordingly conceivable that, in the context of the chronic immune activation found in late-stage HIV disease, a dysregulated redox balance will result in increased levels of basal phosphorylation. If so, specific stimuli may not be able to generate sufficient levels of additional phosphorylation to transduce appropriate intracellular signals. Interestingly, very little is known about how basal phosphorylation levels affect signaling networks, their regulation, and cellular function. In a subset of patients with acute myeloid lymphoma, members of the Stat proteins have been reported to be constitutively activated, and up-regulated basal state of phosphorylation has been connected to the disability to activate further signaling past basal phosphorylation levels (65
). These and our results underscore the need to better understand the role of basal levels of phosphorylation in regulating or perturbing cell signaling in health and in disease.
Finally, we found that the blunted signaling responses in progressive disease resolve upon ART. Although we cannot discriminate between normalization due to cell replacement (e.g., cells produced de novo from progenitor pools) or to reversion (e.g., of previously dysfunctional cells), this observation indicates that the lesion in signaling is reversible, as long as HIV VLs are brought under control.
In summary, we have demonstrated that, in the setting of HIV infection, CD4+ and CD8+ T cell signaling is blunted in cells from untreated subjects with progressive disease compared with LTNP and RESPs. The observed signaling alterations are not restricted to or manifest within a specific T cell subpopulation, suggesting a generalized state of unresponsiveness. Some alterations in cellular signaling correlated with levels of basal phosphorylation, VL, and/or expression of signaling-regulatory protein PD-1. Of these influences, it was the level of basal phosphorylation that appeared to be the most dominant (). Altered signaling found in PROGs was reversible with antiretroviral treatment, indicating that signaling dysfunctions can be restored. More detailed analyses of specific signaling pathways and of levels of basal phosphorylation might suggest ways to correct these T cell signaling dysfunctions and to help restore function of CD4+ and CD8+ T cells.
FIGURE 8 Overview of significances of correlations analyzed using a heat map representation. Values of p from previous correlation analyses are here color-coded (*, p < 0.05 in dark gray; **, p < 0.01 in gray; ***, p < 0.001 in light gray; (more ...)