Introduction
Despite effective combination antiretroviral therapy (cART), HIV-infected individuals (HIV+) are at higher risk than HIV-uninfected (HIV-) people for age-related diseases and geriatric syndromes [
1,
2]. Among the latter, frailty is of particular interest considering its high prevalence in older adults [
3], association with dysfunction of multiple physiological systems [
4‐
6], and portent of poor health outcomes including death [
6].
The pathophysiology of frailty is not well understood [
5,
6], but chronic inflammation, as reflected in elevated circulating levels of proinflammatory markers such as interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α) [
7‐
9], appears to be a contributing factor in both HIV- and HIV+ people. Increasing age is characterized by elevated levels of these markers, as is treated HIV infection even after effective viral suppression [
7,
8]. However, the cause of chronic inflammation in these circumstances remains unknown.
One possible driver of chronic inflammation is persistent cytomegalovirus (CMV) infection. HIV- people who are CMV-seropositive have a higher prevalence of frailty than those who are -seronegative [
10], and in one study older women who had detectable CMV DNA in peripheral blood monocytes had higher serum concentrations of IL-6 than those who did not [
11]. Among HIV+ people, higher titers of anti-CMV IgG antibodies have been associated with subclinical cardiovascular disease [
12], another condition linked to chronic inflammation [
8]. In both HIV- and HIV+ people, chronic CMV infection triggers an unusually robust T cell response, with an expansion of CMV-specific T cells that often comprises 10-20%, or even more, of the circulating T cell pool [
13‐
15]; and we found that the magnitude of this response was strongly correlated with serum concentrations of some inflammatory markers in men in the Multicenter AIDS Cohort Study (MACS) [
16]. Indeed, in that study the total percentage of CD4 T cells producing IL-2 in response to a broad panel of CMV antigens predicted onset of frailty in HIV- but not HIV+ nonfrail men [
16]. These observations suggest that CMV-specific T cells play an important role in the pathogenesis of chronic inflammation and frailty.
CMV-specific CD4 and CD8 T cells can produce many different combinations of cytokines [
17‐
26], and production of different cytokines determines functionality of T cells [
27] and relates to control of viral infections [
28]. No studies have evaluated the relationship between polyfunctional CMV-responsive T cells and onset or maintenance of frailty. Therefore, we studied the relationship between subsets of CMV-responsive CD4 and CD8 T producing IFN-γ, TNF-α, and/or IL-2, alone or in combination, and onset of frailty in HIV- and virally suppressed HIV+ men in the MACS. Since frailty can wax and wane, especially in HIV+ men [
29], we also studied the relation between CMV-responsive T cells and maintenance of frailty.
Discussion
This study investigated the relationship between polyfunctional CMV-specific T cell responses and onset and maintenance of frailty in HIV- and virologically suppressed HIV+ men. This study extends our previous analysis of the same population [
16] by evaluating: 1) the polyfunctional T cell cytokine response to CMV antigens, and 2) its relationship with maintenance as well as onset of frailty.
We found that six out of the 14 possible functional subsets of CMV-responsive CD4 and CD8 T cells accounted for almost all CMV-responsive cells. This predominance was not substantially affected by HIV or frailty status. Of these six subsets, percentages of two, CD4 T cells producing only IFN-γ (IFN-γ-SP CD4 T cells) and CD8 T cells producing IFN-γ, TNF-α, and IL-2 (TP CD8 T cells), significantly predicted onset and maintenance of frailty in both the HIV- and the HIV+ men.
In HIV- men who were nonfrail at baseline, higher percentages of IFN-γ-SP CD4 T cells and lower percentages of TP CD8 T cells predicted faster onset of frailty. Further, a) men who had both high percentages of IFN-γ-SP CD4 T cells and low percentages of TP CD8 T cells progressed to frailty faster than other men; b) such men comprised most of those who progressed to frailty; and c) serum levels of CRP, which have been associated with CMV infection and frailty [
8,
34], were correlated positively with percentages of IFN-γ-SP CD4 T cells and negatively with percentages of TP CD8 T cells. Since polyfunctional T cells have been shown to exert stronger anti-CMV effects than single cytokine-producing T cells [
18,
24,
35], these data suggest that insufficient control of CMV infection may contribute to the development of frailty, and that the T cell response to CMV may contribute to the well-known association of inflammation with frailty.
This interpretation is strengthened by the findings that a large percentage of IFN-γ-SP CD4 T cells were elicited by peptides encoded by CMV UL55 (gB), and higher percentages of UL55-specific IFN-γ-SP CD4 T cells predicted faster onset of frailty. UL55-specific CD4 T cells have been reported to be more cytolytic than CD4 T cells specific for other CMV proteins (i.e., UL83 (pp65), UL86, UL115 (gL), and UL75 (gH)) [
20,
21,
26], and to express high levels of the vascular endothelium homing receptor CX3CR1 [
26,
36]. Considering the importance of TP CD8 T cells in controlling CMV reactivation [
18], low levels of these cells may lead to more CMV reactivation, which could in turn trigger higher levels of UL55-specific IFN-γ-SP CD4 T cells. Thus, one could postulate that suboptimal control of CMV reinfection or reactivation could lead to increased inflammation and vascular damage [
26,
36,
37], both of which can predispose to frailty [
8,
10]. The strong response of IFN-γ-SP CD4 T cells to UL55 may explain our finding that in HIV- men percentages of IFN-γ-SP CD4 T cells were correlated positively with serum levels of CRP and negatively with serum levels of IL-10. Since IL-2 can increase proliferation and survival of UL55-specific cytolytic CD4 T cells [
38], this could explain why high levels of IL-2-producing CD4 T cells and IFN-γ-SP CD4 T cells together predicted progression to frailty more strongly than either subset individually.
Unexpectedly, in HIV+ men it was lower, rather higher, percentages of IFN-γ-SP CD4 T cells that predicted faster onset of frailty. The reason for this difference is unclear. However, the differences between HIV- and HIV+ nonfrail men in antigenic specificity of these cells and their correlations with serum inflammatory markers may offer clues. In contrast to HIV- men, in HIV+ men most IFN-γ-SP CD4 T cells were not specific to UL55, and percentages of these cells specific to UL55 did not predict onset of frailty. Also, the percentages of these cells were not correlated with CRP. However, these percentages were positively correlated with the serum levels of the anti-inflammatory cytokine IL-10 and of IFN-γ. IL-10 was protective against frailty in a mouse model with IL-10 knockout [
39], and circulating IFN-γ inhibits CMV replication [
40]. Thus, in HIV+ men lower levels of these cells could predispose to induction of frailty-associated inflammation by CMV. Other differences between HIV+ and HIV- men, including numerical and functional differences in T cell subsets, and/or potential sites and nature of CMV antigen production, could also have contributed to the difference in CMV responses.
Lower percentages of TP CD8 T cells were associated with faster onset of frailty in both HIV- and HIV+ men, although this was statistically significant only for the former. This weaker effect in HIV+ men may indicate that the protective effect of TP CD8 cells is disrupted by chronic HIV infection, or it could reflect the small sample size of the study. Nevertheless, the majority (5 out of 6) of HIV+ men with progression to frailty had low percentages of both IFN-γ-SP CD4 T cells and TP CD8 T cells, suggesting a combined effect of these two cell subsets on onset of frailty in HIV+ men as in HIV- men. In HIV+ men, however, both of these cell subsets may protect against CMV reactivation and prevent CMV-induced inflammation. CMV-specific CD4 T cells have been shown to be required for dendritic cell-mediated activation of cognate CD8 T cell responses, partly through production of IFN-γ by CD4 T cells [
41]. Therefore, the combined effect of both T cell subsets may suggest the importance of CD4 help in promoting and sustaining effective anti-viral CD8 T cell responses. This interpretation may also explain the weaker effect of TP CD8 T cells we observed in HIV+ men, as it is possible that HIV provirus in CMV-specific CD4 T cells could become lytic after these cells are activated by CMV antigens [
42].
For both HIV- and HIV+ men who were frail at baseline, lower levels of IFN-γ-SP CD4 T cells predicted greater maintenance of frailty, while lower levels of TP CD8 T cells were predictive only in HIV- men. These results provide additional evidence suggesting the importance of these two T cell subsets in the pathogenesis of frailty. They also suggest that chronic HIV infection may disrupt the protective effect of TP CD8 T cells, and that controlling CMV reactivation may be key for reversal of frailty, regardless of HIV infection.
We found that the serum inflammatory markers CRP, IL-10, and IFN-γ, which were correlated with the percentages of the two predictive CMV-specific T cell subsets, also predicted onset and/or maintenance of frailty. The predictions were restricted to either HIV- or HIV+ men, and directions of the predictions were consistent with the CMV-specific T cell subsets with which they were correlated. The directions of the predictions by CRP and IL-10 were also consistent with previous reports [
43,
44].
Overall, the results of this study support the hypothesis that CMV-induced inflammation is an important contributory mechanism for the observed association between CMV-specific T cells and frailty. However, no causation can be inferred from the cross-sectional correlations between inflammatory markers and CMV-specific T cells. Moreover, an additional hypothesis that is consistent with the data is that inflammation activates CMV replication which in turn elicits the T cell responses observed in this study.
This study had several limitations. The sample size was small, and the findings, though statistically significant, should be confirmed in larger studies. We did not adjust our statistical analyses for multiple comparisons, because this increases the likelihood of false-negative results with a small sample size [
45]. Although not adjusting can lead to false-positive results, the likelihood of this is reduced by the consistency of the directions of associations between functional T cell subsets and onset or maintenance of frailty across different tertile comparisons, and of differences in the proportions of follow-up visits at which the participants were frail. The study is also limited by unavailability of data on clinical conditions that could have affected the development of frailty, and by the lack of women in the study population. Another limitation is that while half of the study population was known be CMV-seropositive, the serostatus of the other half was not known. However, since all had positive CMV-specific T cell responses and nearly all HIV-infected people are CMV-seropositive [
46], it is highly likely that all the men studied were CMV-seropositive. This study also had some notable strengths, including: 1) the prospective study design and long follow-up period, which reduce bias and permit temporal inference; 2) exploration of both onset and maintenance of frailty since few studies have investigated the maintenance of frailty; 3) a large panel of peptide pools spanning 19 CMV ORFs was used to assess CMV-specific T cells, permitting a much more comprehensive detection of these cells than in most studies, which have assessed only responses to UL83 and/or UL123, which usually represent a minority of anti-CMV T cell responses [
14,
15,
47].
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