Responsiveness to IL-7 but not to IFN-α is diminished in CD4+ T cells from treated HIV infected patients who experience poor CD4+ T-cell recovery

Abstract

Objective: To assess CD4 T-cell responsiveness to IL-7 and IFN-α in HIV-infected patients who experience poor recovery of CD4 T-cell counts during therapy (immune failure patients).

Design: Responses to IL-7 and IFN-α were compared between HIV-infected immune failure (CD4 cell counts <379 cells/μl) patients and immune success (CD4 cell counts >500 cells/μl) as well as healthy control patients.

Methods: Flow cytometry was used to assess peripheral blood mononuclear cells for IL-7-induced proliferation, CD25 expression, and signaling (signal transducer and activator of transcription 5 phosphorylation and Akt phosphorylation) in CD4 T cells. Freshly isolated cells were characterized by expression of IL-7Rα (CD127) among CD4 T-cell maturation subsets by flow cytometry and sorted CD3 T cells were assessed for expression of IFN-α and interferon stimulated genes (2'-5'-oligoadenylate synthetase-1 and myxovirus resistance A protein) by quantitative real-time PCR. Responses to IFN-α were assessed by induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of IL-7-induced CD4 T-cell proliferation.

Results: IL-7-induced proliferation and CD25 expression were decreased in CD4 T cells from immune failure patients. CD127 expressing CD4 T cells were decreased, whereas expression of 2'-5'-oligoadenylate synthetase-1, myxovirus resistance A protein, and IFN-α mRNA were increased in total CD3 T cells from immune failure patients. CD127 expression correlated with CD25 induction but not proliferation, whereas T-cell IFN-α mRNA was associated with reduced proliferation in CD4 T cells from immune failure patients. IFN-α-mediated induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of proliferation were not diminished in CD4 T cells from immune failure patients.

Conclusion: IL-7 responsiveness is impaired in immune failure patients and may be related to expression of CD127 and IFN-α.

Fig. 1. CD4+ T cell responses to IL-7 are diminished in IF subjects

(A–D) PBMCs were labeled with CFSE and incubated in the presence or absence of rIL-7. After 7 days, cells were assessed by flow cytometry. (A) The gating sequence is provided. Doublets were excluded from all analysis by the FSC-A and FSC-H gate and lymphocytes were identified by forward and side scatter. (B) Representative histograms showing proliferation response by %CSFE low cell population in CD4+ cells. (C) Summary data of proliferation response in CD4+ cells (IF n=11, IS n=10 and HC n= 9) as well as CD45RA+ and CD45RA− CD4 subsets. (D) Summary data showing % divided and proliferation index in the CD45RA− subset in cells from subjects that were analyzed in 1C. (E and F) PBMCs were incubated in the presence or absence of IL-7. After 24 hours, cells were assessed for surface CD25 expression by flow cytometry. (E) Representative histograms showing CD25 expression in IL-7 treated CD4+ T cells in comparison with untreated and isotype control. (F) Summary data of % IL-7 induced CD25+ cells in CD4+ T cells (IF n= 11, IS n= 12, HC n= 9) and maturation subsets (naive defined by CD45RA+CD27+, central memory defined by CD45RA−CD27+ and effector memory defined by CD45RA−CD27−). % IL-7 induced CD25 is the difference in CD25 expression between IL-7 treated and untreated. Abbreviations: immune failure, IF; immune success, IS; healthy control, HC.

Fig. 2. T cells from IF subjects exhibit increased expression of ISGs and IFN-α

(A) Freshly isolated PBMC were examined by flow cytometry for percentages of CD127+ cells in total, naïve, CM and EM CD3+CD4+ T cells. (B–C) T cells were purified from freshly isolated PBMCs and gene expression of ISGs and IFN-α was analyzed by qRT-PCR. (B) Messenger RNA expression of OAS1 and MxA (IF n=10, IS n= 9, HC n= 9) ISGs (C) IFN-α mRNA analyses (IF n=9, IS n= 11, HC n= 7). Abbreviations: 2'-5'-Oligoadenylate Synthetase-1, OAS-1; Myxovirus resistance A protein, MxA; Interferon-alpha, IFN-α; immune failure, IF; immune success, IS; healthy control, HC.

Fig. 3. IL-7 induced CD25 expression is related to expression of CD127

(A) Relationship between CD127 expression and induction of CD25 in response to overnight IL-7 treatment. (B) Relationship between CD127 expression and proliferation (%CFSE) in response to 7 days of IL-7 stimulation. Open symbols represent IS subjects and closed symbols represent IF subjects. Abbreviations: immune failure, IF; immune success, IS; healthy control, HC.

Fig. 4. CD4+ T cell responses to IFN-α were not diminished in IF subjects. PBMCs from subjects were incubated in the presence or absence of IFN-α

All data shown are gated from CD3+CD4+ cells. (A) P-STAT1 induction following 15 minutes of IFN-α treatment (IF n= 12, IS n= 12, HC n= 9). (B-D) PBMCs were CFSE stained and assessed for proliferation and cell death in cells treated with IL-7 in the absence or presence of IFN-α for 7 days. (B) Compares %CFSE low (IF n= 10, IS n= 11, HC n= 9) and (C) are representative dot plots comparing cell death as measured by annexin-V binding CD4+ T cells that were not excluded by viability dye. (D) Summary data of cell death in divided cells (%CFSE low) and undivided cells (CFSE high) (IF n= 10, IS n= 8, HC n= 9). P-values were obtained by Wilcoxon signed rank test. Abbreviations: immune failure, IF; immune success, IS; healthy control, HC.