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. 2015 Jun;97(6):1139-46.
doi: 10.1189/jlb.4A0714-345RR. Epub 2015 Mar 17.

Interferon-α inhibits CD4 T cell responses to interleukin-7 and interleukin-2 and selectively interferes with Akt signaling

Thao P Nguyen  1 , Doug A Bazdar  1 , Joseph C Mudd  1 , Michael M Lederman  1 , Clifford V Harding  1 , Gareth A Hardy  1 , Scott F Sieg  2
Affiliations

Interferon-α inhibits CD4 T cell responses to interleukin-7 and interleukin-2 and selectively interferes with Akt signaling

Thao P Nguyen et al. J Leukoc Biol. 2015 Jun.

Abstract

Persistent type I IFN production occurs during chronic viral infections, such as HIV disease. As type I IFNs have antiproliferative activity, it is possible that chronic exposure to these cytokines could adversely affect T cell homeostasis. We investigated the capacity of IFN-α to impair T cell proliferation induced by the homeostatic cytokine, IL-7, or another common γ-chain cytokine, IL-2, in cells from healthy human donors. We found that IL-7- or IL-2-induced proliferation of CD4(+) T cells was partially inhibited in the presence of IFN-α. The CD4(+) T cells that were exposed to IFN-α also displayed attenuated induction of IL-2 and CD40L following TCR stimulation. Analyses of signaling pathways indicated that IL-7 and IL-2 induced a delayed and sustained P-Akt signal that lasted for several days and was partially inhibited by IFN-α. In contrast, IL-7-induced P-STAT5 was not affected by IFN-α. Furthermore, IFN-α had no detectable effect on P-Akt that was induced by the chemokine SDF-1. Both inhibitors of P-Akt and P-STAT5 blocked IL-7-induced T cell proliferation, confirming that both signaling pathways are important for IL-7-induced T cell proliferation. These results demonstrate that IFN-α can selectively inhibit cytokine-induced P-Akt as a potential mechanism to disrupt homeostasis of T lymphocytes.

Keywords: cytokines; homeostatic proliferation.

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Figures

Figure 1.
Figure 1.. IFN inhibits IL-7-induced proliferation.
PBMCs or negatively selected purified CD4+ T cells were CFSE labeled and incubated with IL-7 (5 ng/ml) ± IFN-α (500 U/ml). Flow cytometric analyses were performed after 7 days to assess cell proliferation. Cells were gated based on forward- and side-scatter characteristics to exclude debris and forward-scatter height versus area to exclude doublets. Viability dye stain was used to exclude dead cells. Histograms represent CD4+ lymphocytes. (A) CFSE dye dilution of gated CD4+ cells at 7 days is shown on the x-axis for the various conditions. (B) Summary data of CFSE dye dilution from studies of PBMC gated for CD4+ T cells (left) and of purified CD4+ T cell cultures (right). Each pair of connected symbols represents data from a different donor. (C) Assessment of CD25 expression in CD4+ T cells incubated overnight in medium alone, IL-7 (5 ng/ml) or IL-7 plus IFN-α (500 U/ml). Cells incubated in IFN-α alone showed no change in CD25 expression (not shown). Connected symbols represent data from a single donor. Results are shown for 3 different donors.
Figure 2.
Figure 2.. Preincubation of CD4+ T cells in IFN-α plus IL-7 reduces T cell function compared with preincubation of cells in IL-7 alone.
PBMCs or purified CD4+ T cells were CFSE labeled and incubated with IL-7 (5 ng/ml) ± IFN-α (500 U/ml) for 7 days. Cells were then stimulated with SEB (PBMC) or CytoStim beads (purified CD4+ T cells) to induce intracellular cytokines or CD40L expression. Cells were gated based on forward- and side-scatter characteristics to exclude debris and forward-scatter height versus area to exclude doublets. Viability dye stain was used to exclude dead cells. CFSE dye dilution and intracellular CD40L expression among CD4+ cells were determined by flow cytometry. (A) Representative histograms depict IL-2, CD40L, and IFN-γ expression (y-axis), which were induced by SEB stimulation and CFSE dye dilution (x-axis). The CFSE dye dilution resulted from preincubation in IL-7 or IL-7 + IFN-α. The addition of SEB did not cause further proliferation, as the assay was performed over hours and the Golgi plug reagent had been added during the SEB incubation period (not shown). (B) Summary data of cytokine induction in PBMCs that had been incubated in IL-7 or IL-7 + IFN-α before SEB stimulation. (C) Summary data that use purified CD4+ T cells from 4 different donors, showing induction of CD40L expression in total and in CFSElow cells at day 7 following preincubation of cells in IL-7 or IL-7 + IFN-α (P = 0.068 in each experiment).
Figure 3.
Figure 3.. IFN-α causes cell death, primarily in nondividing T cells, in IL-7-treated cell cultures.
PBMCs were CFSE labeled and incubated with IL-7 (5 ng/ml) ± IFN-α (500 U/ml) for 7 days. (A) Apoptosis was assessed by first gating out debris, then gating on CD4+ cells, and then CD3+ cells. SSC-A, Side-scatter-area; FSC-A, forward-scatter-area. (B) Histograms depict cell proliferation by CFSE dye dilution (x-axis) and apoptosis by Annexin V binding (y-axis) in IL-7 and IL-7 + IFN-α-treated cells. (C) Data summarize the percentages of apoptotic CFSElow cells among the CD4+CD3+ cells (left) and the percentage of CFSElow cells among only the apoptotic CD4+CD3+ cells (right).
Figure 4.
Figure 4.. IFN-α impairs P-Akt but not P-STAT5 signaling in IL-7-treated cells.
(A) PBMCs were incubated in medium alone, medium + IL-7 (5 ng/ml), or medium + SDF-1 (10 ng/ml) for various periods of time as indicated, and then cells were assessed for expression of P-Akt by intracellular flow cytometry. (B) Flow cytometry histograms (left) in CD3+CD4+ cells, showing induction of P-STAT5 (y-axis) and P-Akt (x-axis) at 3 days of incubation in PBMC treated with medium alone, medium + IL-7 (5 ng/ml), or medium + IL-7 and IFN-α (500 U/ml). No induction of P-Akt was observed in cells incubated with IFN-α alone (not shown). Summary data from 6 donors are provided (right). (C) PBMCs were incubated with IL-7 for 2 days, and then some cells were treated with an inhibitor of PI3K signaling (wortmannin). After an additional overnight incubation, cells were assessed for P-Akt expression. (D) PBMCs from 5 different donors were treated with IL-7 for 3 days and assessed for P-Akt expression by intracellular flow cytometry (labeled “IL-7”). In comparison, some cells were incubated with IL-7 for 3 h (IL-7, 3 h) or 1 day (IL-7, 1 day), washed, and then returned to culture in the absence of IL-7 until analysis for P-Akt at Day 3.
Figure 5.
Figure 5.. Impaired responses to IL-2 but not SDF-1 in CD4 cells exposed to IFN-α.
(A) PBMCs were CFSE labeled and incubated in medium alone, medium + IL-2 (50 ng/ml), or medium + IL-2 + IFN-α (500 U/ml). Histograms show expression of P-STAT5 (y-axis) and P-Akt (x-axis) after 3 days of incubation among CD3+CD4+ cells, and cells incubated in IFN-α looked similar to cells incubated in medium alone (not shown). (B) Results are shown for cells from 5 different donors comparing P-Akt and P-STAT5 induction in CD3+CD4+ cells incubated for 3 days in the presence of IL-2 or IL-2 + IFN-α. (C) PBMCs were preincubated in medium alone or in medium + IFN-α (1000 U/ml) for 2 days before stimulating cells with SDF-1 (10 ng/ml) for 1 min. The percent of P-Akt+ cells is indicated for cells stimulated with SDF-1. (D) PBMCs were incubated for 7 days with IL-2 or IL-2 + IFN-α. The percentage of CFSElow cells was determined by flow cytometry (debris, doublets, and dead cells were removed from the analysis). Each pair of connected symbols represents cells from a different donor (n = 6).
Figure 6.
Figure 6.. Inhibition of PI3K or STAT5 reduced cell proliferation, decreased functionality, and enhanced cell death in cells treated with IL-7.
(A) CFSE-labeled PBMCs were incubated in medium alone, medium + IL-7, medium + IL-7 + PI3K inhibitor, and IL-7 + STAT5 inhibitor and then assessed 7 days later for proliferation (CFSE dye dilution, x-axis) and response to SEB stimulation (CD40L induction, y-axis). Data are representative of results from 3 different donors. (B) Viability was assessed in the above cultures by gating on forward- and side-scatter to eliminate debris and monocytes, followed by forward-scatter-height versus forward-scatter-area to exclude doublets (not shown). Then, cells were gated on CD4 cells to assess viability dye stain. The percentage of dead CD4+ cells is shown on the y-axis under the various conditions (x-axis). Connected symbols represent data from a single donor. Data from 3 different donors are shown as different symbols.
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