Chronic exposure to type-I IFN under lymphopenic conditions alters CD4 T cell homeostasis

Abstract

HIV infection and the associated chronic immune activation alter T cell homeostasis leading to CD4 T cell depletion and CD8 T cell expansion. The mechanisms behind these outcomes are not totally defined and only partially explained by the direct cytopathic effect of the virus. In this manuscript, we addressed the impact of lymphopenia and chronic exposure to IFN-α on T cell homeostasis. In a lymphopenic murine model, this interaction led to decreased CD4 counts and CD8 T cell expansion in association with an increase in the Signal Transducer and Activator of Transcription 1 (STAT1) levels resulting in enhanced CD4 T cell responsiveness to IFN-α. Thus, in the setting of HIV infection, chronic stimulation of this pathway could be detrimental for CD4 T cell homeostasis.

Conflict of interest statement

AWR is affiliated with AIDS Monitoring Labs, Leidos Biomedical Research, Inc. This does not alter our adherence to all PLOS Pathogens policies on sharing data and materials.

Figures

Figure 1. t-STAT1 in CD4 and CD8 T cell subsets from HIV-infected patients.

PBMCs from a longitudinal cohort of HIV-infected individuals (n = 10) were analyzed by flow cytometry before and after suppressing viremia to a) Gating strategy to assess t-STAT1 expression in T cell subsets, using CD27 and CD45RA as markers of naïve (CD45RA+ CD27+) and memory (CD45RA− CD27+) CD4 (CD3+ CD4+) or CD8 (CD3+ CD4−) T cells. (b) The Median Fluorescence Intensity (MFI) of t-STAT1 in the different T cell subsets was compared before and after treatment and viremia suppressed to <50 copies/ml using a Wilcoxon signed-rank test.

Figure 2. In vitro culture with IL-7 increases IFN-α-induced STAT1 activation (p-STAT1).

PBMCs from healthy donors (n = 12) were cultured 3 days in media alone (black symbols), rhIL-7 (10 ng/ml; blue symbols) or rhIFN-α (100 U/ml; red symbols). After 3 days of culture, the cells were harvested, washed and rested overnight. Rested cells were stimulated in vitro with rhIFN-α (100 U/ml) for 30 minutes and analyzed for intracellular expression of t-STAT1 (a) and phosphorylated STAT1 (b) in naïve and memory T cell subsets. A Wilcoxon signed-rank test was performed for comparisons of the MFI of t-STAT1 and p-STAT1 between groups.

Figure 3. In vitro culture with IL-7 increases IFN-α-induced activation of STAT1, STAT2 and STAT3 in CD4 but not CD8 T cells.

Isolated CD4 (a) and CD8 (b) T cells from a healthy donor were cultured and treated as described in Figure 2. Cell lysates were analyzed by Western blotting with antibodies specific to p-STAT1, t-STAT1, p-STAT2, t-STAT2, p-STAT3, t-STAT3, p-STAT5 and t-STAT5. An antibody to actin was used to confirm even protein loading. Numbers represent the ratio of the densitometry values of band densities on western blots calculated using the values of the unstimulated cells cultured for 3 days with media only as the baselines. Results are representative of at least 3 different donors.

Figure 4. t-STAT1 expression is inversely associated with CD4 T cell counts and IL-7 serum levels in HIV-infected patients undergoing cART.

PBMCs from healthy controls (HC, n = 22) and HIV-infected patients with viremia suppressed to a) The MFI of t-STAT1 in CD4 and CD8 T cells was compared between HIV+ and HC divided according to their CD4 T cell counts using a nonparametric Mann-Whitney test. (b) Relationship between t-STAT1 levels and CD4 T cell counts. (c) Relationship between IL-7 serum levels and CD4 T cell counts. (d) Relationship between t-STAT1 expression and IL-7 serum levels. (e) Relationship between STAT1 phosphorylation after in vitro stimulation with IFN-α and t-STAT1 levels. The correlations between the levels of t-STAT1, p-STAT1, IL-7 and CD4 T cell counts were analyzed with the non-parametric Spearman test.

Figure 5. In vivo exposure to lymphopenic conditions enhances t-STAT1 expression and IFN-α responsiveness.

Lymphoreplete B6 CD45.1 (n = 6) and lymphopenic RAG−/− CD45.1 (n = 6) mice were injected intravenously (i.v.) with 9×106 of CellTrace Violet-labeled lymph node (LN) cells isolated from congenic B6 CD45.2 mice. Analysis of transferred cells in LNs and spleen was performed on day 7 after transfer. The levels of t-STAT1 and p-STAT1 of donor T cells were evaluated by flow cytometry in LNs and spleen as function of CellTrace Violet Fluorescence after in vitro stimulation with rmIFN-α (500 U/ml). (a) The percentages of donor T cells CellTrace Violet+ t-STAT1high, CellTrace Violet− t-STAT1high, CellTrace Violet− t-STAT1low and CellTrace Violet+ p-STAT1high, CellTrace Violet− p-STAT1high and CellTrace Violet− p-STAT1low are indicated. The MFIs of t-STAT1 (b) and p-STAT1 (c) in CD4 and CD8 donor T cells seven days after adoptive transfer into replete B6 (gray symbols) and lymphopenic RAG−/− (black symbols) hosts were compared using a nonparametric Mann-Whitney test. Data are from one representative experiment out of three, including an average of 5 mice per group.

Figure 6. Lymphopenia-induced t-STAT1 upregulation in T cells is partially dependent on IL-7.

(a) Lymphoreplete B6 CD45.1 (n = 5; gray symbols), lymphopenic RAG−/− (n = 5; black symbols) and IL-7−/− x RAG−/− (n = 4; blue symbol) mice were injected i.v. with 9×106 of CellTrace Violet-labeled LN cells isolated from congenic B6 CD45.2 mice. Analysis of transferred cells in LNs and spleen was performed on day 5 after transfer and levels of t-STAT1 of donor T cells were evaluated by flow cytometry as function of CellTrace Violet Fluorescence. The percentages of donor T cells CellTrace Violet+ t-STAT1high, CellTrace Violet− t-STAT1high and CellTrace Violet− t-STAT1low are indicated. (b) The MFIs of t-STAT1 in CD4 and CD8 donor T cells in the different hosts were compared using a nonparametric Mann-Whitney test. Data are from one representative experiment out of three, including an average of 4 mice per group. (c) Non irradiated (n = 3, gray symbols) and irradiated B6 CD45.1 mice were adoptively transferred as described above. Irradiated B6 hosts were treated with either anti-IL-7 and anti-IL-7R (α-IL-7+α-IL-7R; n = 3, open red symbols) or the respective isotype matched control (n = 3, filled red symbols) mAbs. 5 days after transfer, mice were analyzed as described above and the percentages of donor T cells CellTrace Violet+ t-STAT1high, CellTrace Violet− t-STAT1high and CellTrace Violet− t-STAT1low are indicated. (d) The MFI of t-STAT1 in donor T cells were compared between groups using a nonparametric Mann-Whitney test. Data are from one representative experiments out of three, including 3 mice per group.

Figure 7. Continuous exposure to Type-I IFN under lymphopenic conditions leads to CD4 T cell depletion and CD8 T cell expansion.

Lymphopenic RAG−/− mice reconstituted with CellTrace Violet-labeled LN cells from congenic B6 CD45.2 mice were treated with either rmIFN-α (10,000 U per mouse, daily; n = 8) or PBS (n = 7). The numbers of CD4 and CD8 donor T cells in LNs and spleen were determined on day 35 after transfer. (a) Percentages of highly dividing CD45.2+ CD3+ CD4+ and CD8+ donor lymphocytes in LNs and spleen are shown. (b) Absolute numbers of CD4+ and CD8+ donor T cells in the lymphoid organs were enumerated following the treatment with rmIFN-α (black symbols) and PBS (open symbols). A nonparametric Mann-Whitney test was performed for comparisons of CD4 and CD8 T cell counts between groups. Data are from two representative experiments out of four, including an average of 4 mice per group.