CD8 T cell responses are critical to control HIV replication and progression to AIDS [1,2]. However, despite seemingly vigorous responses in HIV-infected patients, they fail to prevent establishment of chronic infection and exhibit altered differentiation patterns, impaired lytic capacity, and increased susceptibility to apoptosis [3–5]. The proliferation, differentiation, function, and maintenance of these cells is largely controlled by cytokines, particularly those sharing the common gamma chain (γc) receptor subunit, including interleukin (IL)-2, IL-7, IL-15, through cytokine-induced activation of the signal transducer and activator of transcription (STAT) signaling pathway [6,7]. Although the mechanism remains unclear, IL-7 receptor-α (IL-7Rα) is downregulated in CD8 T cells from HIV-positive patients, a phenomenon associated with an increased proportion of effector-like, low-IL-7Rα-expressing (IL-7Rαlow) CD8 T cells that was partially restored under antiretroviral therapy (ART) [3,8–10]. Therefore, as we showed previously for IL-2 , CD8 T-lymphocyte alterations in HIV-positive patients may be related to STAT signaling defects in response to multiple other cytokines. Herein, we further investigated cytokine-dependent STAT signaling ex vivo in HIV-positive patient CD8 T cells and the potential molecular mechanism responsible for the reduced IL-7Rα expression observed.
HIV-negative controls (n = 9) and chronically infected HIV-positive patients were from the Ottawa Hospital and included ART (>1 year; n = 17) and off-therapy (>6 months; n = 17) patients. Viral loads were 10(1–500) × 103 [mean(range)] and less than 50 copies/ml whereas CD4 cell counts were 267(46–733) and 566(288–1002) cells/μl in off-therapy and ART patients, respectively.
Antibodies and cytokines
The antihuman monoclonal antibodies (mAbs) used were phycoerythrin-labeled anti-IL-7Rα [R&D Systems (R&D), Minneapolis, Minnesota, USA], anti-CD3, antiphospho-tyr-STAT (P-STAT)-6 [BD Biosciences (BD), Mississauga, Ontario, Canada]; Alexa488-conjugated anti-P-STAT5, anti-P-STAT3; fluorescein isothiocyanate (FITC)-labeled anti-programmed death (PD)-1 and PECy-7-labeled anti-CD8 (BD). Corresponding antibody isotypes served as controls. Recombinant human IL-2, IL-7, IL-15, IL-10, and IL-4 were purchased (R&D).
Peripheral blood mononuclear cells (PBMCs) were stimulated with the specified cytokines, stained with anti-CD3 or anti-CD8 or both along with anti-IL-7Rα or anti-PD-1 or anti-P-STAT Abs, essentially as described [11–14].
CD8+/IL-7Rα+ and CD8+/IL-7Rα-negative T cells were purified (>85%, data not shown) from PBMCs by negative selection using the CD8+ T Cell Isolation Kit II followed by positive selection using phycoerythrin-labeled anti-IL-7Rα Abs and anti-phycoerythrin-Microbeads (Miltenyi Biotec, Auburn, California, USA).
Total cellular RNA was extracted (RNeasy kit; Qiagen, Mississauga, Ontario, Canada), and cDNA was reverse transcribed [cDNA Archive kit; Applied Biosystems (ABI), Streetsville, Ontario, Canada]. cDNA was amplified using TaqMan gene expression assays [IL-7Rα, growth factor independent (Gfi)-1, GA binding protein (GABP) α, and β-actin] in a 7500 real-time-PCR system (ABI). Relative mRNA expression levels were determined by the comparative Ct method .
Significance for IL-7Rαhigh to IL-7Rαlow cell comparisons was established by calculating 95% confidence intervals. The t-test and Bonferroni test were used for between group and multiple comparisons, respectively. Pearson's r (two-tailed) was used to determine correlations between continuous variables. P < 0.05 or less was considered significant.
IL-7Rαlow CD8 T cells fail to activate STAT5 in response to IL-7 but maintain Jak/STAT signaling following IL-2, IL-15, IL-4, and IL-10 stimulation
We evaluated the capacity of CD8 T lymphocytes (IL-7Rαhigh and IL-7Rαlow) in chronically infected HIV-positive patients to recruit the STAT signaling pathway in response to a panel of cytokines implicated in their growth and differentiation. The focus was on γc-sharing cytokines (IL-2, IL-4, IL-7, IL-15) and IL-10, which exhibits both stimulatory and inhibitory effects on CD8 T cells [6,16]. Interestingly, IL-7 stimulation revealed a substantial percentage of CD8 T cells from HIV-positive patients that failed to activate (tyr-phosphorylate) STAT5 (P-STAT5-negative) by flow cytometry (Fig. 1a). In contrast, P-STAT5 was induced by IL-7 in the majority of CD8 T cells from HIV-negative controls, but P-STAT5-negative cells were detectable, albeit at a significantly lower frequency compared with HIV-positive individuals (Fig. 1a). Moreover, impaired P-STAT5 activation was apparently unique to IL-7, as stimulation with IL-2 and IL-15 did not reveal the same P-STAT5-negative population (Fig. 1a). Furthermore, P-STAT6 and P-STAT3 induction by IL-4 and IL-10, respectively, remained unimpaired (Fig. 1a). Overall, P-STAT5-negative cells in response to IL-7 represented on average 15.6 ± 7.1% in HIV-negative controls, increased in off-therapy patients (56.0 ± 14.2%) and, in patients undergoing ART (34.2 ± 13.5%), values remained higher, but returned toward that of HIV-negative controls (Fig. 1b). Furthermore, the capacity of CD8 T cells to activate STAT5 in response to IL-7 strongly correlated (r = 0.97, P < 0.001) with cell surface IL-7Rα expression (Fig. 1c). There were no significant differences in expression of the other IL-7R complex component, γc (data not shown).
Reduced levels of growth factor independent-1 in IL-7Rαhigh vs. IL-7Rαlow CD8 T cells
To investigate the molecular mechanism by which IL-7Rα expression may be downregulated in CD8 T lymphocytes from HIV-positive patients, mRNA expression of IL-7Rα and a number of reputed transcriptional regulators of the IL-7Rα gene was studied by real-time PCR. In Fig. 2(a), relative mRNA expression of these genes is presented as fold change, comparing purified IL-7Rαhigh to IL-7Rαlow CD8 T cells from HIV-positive patients. Gfi-1 mRNA expression was significantly reduced (95% confidence) in IL-7Rαhigh vs. IL-7Rαlow cells of HIV-positive patients, and this was paralleled by an increase in IL-7Rα mRNA expression. Expression of the transcription factor GABPα was not significantly altered in IL-7Rαhigh vs. IL-7Rαlow cells. Gfi-1B, the other Gfi repressor family member, was not consistently detectable in these two subpopulations (data not shown). Similar results were obtained when IL-7Rαhigh and IL-7Rαlow cells, purified from HIV-negative controls, were compared (Fig. 2b).
Recent studies [17–19] suggesting the role of inhibitory receptor PD-1 in HIV-specific CD8 T cell dysfunction prompted the evaluation of PD-1 expression in IL-7Rαlow and IL-7Rαhigh subpopulations. Although the percentage of PD-1+ cells was increased in HIV-positive patients compared with HIV-negative controls, there was no significant difference in %PD-1+ cells between IL-7Rαlow and IL-7Rαhigh subsets (Fig. 2c).
It is well established that IL-7Rα expression is reduced in CD8 T lymphocytes from HIV-positive vs. HIV-negative controls [3,8–10,20–24], a phenomenon not exclusive to HIV infection [25–27]. As we (Fig. 1) and others have observed, this is particularly evident in untreated viremic patients, with ART exerting at least partial restoration [3,9,23]. In HIV-positive patients, IL-7Rαlow cells have been suggested to represent T effector-memory cells, as defined by cell surface phenotype (CCR7−, CD62L−, CD45RA+, or RA−) and diverse functional features including enhanced susceptibility to apoptosis, inferior ex-vivo proliferative capacity, increased IFN-γ but reduced IL-2 production, compared with their IL-7Rαhigh-expressing counterparts . Furthermore, elevated PD-1 expression in HIV-specific CD8 T cells has been associated with functional impairment, reduced survival and an activated, early/intermediate differentiation phenotype [17–19,28] with lower percentage IL-7Rα-positivity , compared with cytomegalovirus-specific cells in viremic patients. We also noted increased PD-1 expression on bulk CD8 T cells from HIV-positive vs. HIV-negative patients, but in both IL-7Rαhigh and IL-7Rαlow subsets, suggesting that IL-7Rα expression may be related to activation/differentiation stage rather than PD-1 levels.
The responsiveness of IL-7Rαlow-expressing CD8 T cells to cytokines involved in regulating their growth and differentiation has remained largely unexplored until recently. Our results suggest that impaired responsiveness to IL-7, as indicated here by attenuated P-STAT5 induction, was due to low/absent IL-7Rα cell surface expression, observations consistent with other studies showing reduced function and survival of T cells from HIV-positive patients in response to IL-7 [20,23,29,30]. However, it is interesting to note here that these cells retained the capacity to activate the STAT pathway in response to IL-2, IL-15, IL-4, and IL-10. Notably, the level of cytokine-dependent STAT activation in the remaining IL-7Rαhigh subset was not significantly affected between study groups. Preliminary results also suggest that in response to IL-21, another γc-sharing cytokine with T cell stimulatory activity , STAT1, STAT3, and STAT5 activation was unaffected in these patient subsets (data not shown). The intact IL-2-induced STAT5 signaling in CD8 T cells from patients off-therapy for more than 6 months is distinct from our previous findings in CD8 T cells from a subset of patients completely naive to therapy and may reflect a more progressive disease in the patient cohort studied previously . This is consistent with a recent report extending our findings of defective IL-2-induced STAT5 activation to all CD8 T cell subpopulations (naive, memory, effector) from patients with progressive disease compared with long-term nonprogressors and those responsive to ART .
IL-7Rαlow cells in HIV-positive individuals may arise as a result of chronic antigen exposure or signals received from elevated IL-7 serum levels or both, particularly in viremic patients [3,24,33]. We speculate that amidst chronic stimulation and reduced IL-7Rα expression, responsiveness to other critical CD8 T cellcytokines, reportedly dysregulated in HIV-positive patients [10,34–37], may thus predominate and contribute to the altered functionality, expansion, and/or maintenance of IL-7Rαlow cells in the periphery of HIV-positive patients.
The molecular mechanisms regulating IL-7Rα expression in CD8 T cells, particularly from HIV-positive patients, are not well understood. However, the role of transcriptional regulators, including the transcriptional repressor Gfi-1 and the Ets family transcription factor GABPα, has been studied in T cells [38–41]. Gfi-1 and GABPα are negative and positive regulators of IL-7Rα expression in mouse T cells, respectively, and were recently implicated in the formation of IL-7Rαhigh and IL-7Rαlow CD8 T cells responding to lymphocytic choriomeningitis virus (LCMV) infection [39–41]. In humans, effector/effector-memory CD8 T cells comprise the majority of IL-7Rαlow cells, whereas naive and memory subsets are IL-7Rαhigh. In contrast to mice, no significant differences were found in GABPα and Gfi-1 mRNA expression when comparing human naive, IL-7Rαhigh and IL-7Rαlow effector-memory cytotoxic T lymphocyte (CTLs), as defined by their CD45RA+ CCR7− phenotype . This differed from our findings of increased Gfi-1 mRNA expression in IL-7Rαlow vs. IL-7Rαhigh cells, data concordant with IL-7Rα mRNA and protein expression in these subsets. The source for this discrepancy may be that we purified CD8 T cells based solely on their expression of IL-7Rα rather than also substratifying based on effector-memory phenotype. Importantly, we confirmed our TaqMan real-time-PCR results using a SybrGreen-based real-time-PCR assay  and established the Gfi-1 amplicon's identity by sequencing (data not shown). Our mRNA expression data do not exclude Gfi-1B and GABPα, but failed to support a role for these reputed IL-7R transcriptional regulators in distinguishing IL-7Rαlow from IL-7Rαhigh cells [39,42]. GABPα mRNA expression did not vary significantly between IL-7Rαhigh and IL-7Rαlow cells, consistent with the previous report in humans . Expression of Gfi-1B was not detected in human CD8 T cells and may reflect its predominant role in the regulation of human erythroid development [43,44].
In conclusion, we demonstrated that the IL-7Rαlow effector-like CD8 T lymphocytes, found at increased proportions in HIV-positive patients, failed to activate STAT5 in response to IL-7, which strongly correlated with their lack of IL-7Rα but not γc expression. Interestingly, these cells, similar to their IL-7Rαhigh counterparts, were capable of activating the STAT pathway in response to other cytokines involved in CD8 T cell growth and differentiation including IL-2, IL-15, IL-4, and IL-10. Although more direct functional evidence for this is required, our results suggest for the first time that a mechanism involving Gfi-1 may be operative in human CD8 T cells for the transcriptional repression of IL-7Rα, but a mechanism that is not restricted to HIV-positive patients alone.
The authors thank the study participants and Jonathan Angel, Nancy Lamoureux, and Mary-Ellen Arsenault (Immunodeficiency Clinic, Ottawa Hospital – General Campus) for patient recruitment. M.K. acknowledges grant support for this work from the Canadian Institutes of Health Research, the Canadian Foundation for Innovation, the Ministry of Economic Development and Trade (Ontario), the J.P. Bickell Foundation, and the CHEO-Research Institute. A.B. is supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council. A.K. is the recipient of a Career Scientist award from the Ontario HIV Treatment Network. Niranjala Gajanayaka (IDVRC-CHEO-Research Institute) provided technical assistance for certain experiments. Nick Barrowman (Clinical Research Unit, CHEO-Research Institute) is acknowledged for his input on statistical analysis of the data. Author contributions: A.B. played a major role in the performance of the experiments, data analysis, and contributed to the drafting and revision of the article. K.A., A.A., and N.S. contributed to data acquisition and provided intellectual input on initial study design, data interpretation and manuscript preparation. D.S. provided key data pertaining to the interpretation of the Gfi-1 RT-PCR results and had an input on manuscript preparation and revision. F.D.M. had an input on data acquisition and manuscript preparation. A.K. contributed intellectually to the study design, data interpretation, and manuscript preparation and revision. M.K. conceived and designed the study, acquired funding, supervised the work, and played a principal role in the drafting and revision of the paper. All authors approved the final version of the manuscript and declare that they have no conflicts of interest.
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