Induction of Multiple Immune Regulatory Pathways with Differential Impact in HCV/HIV Coinfection

Abstract

Persistent viral infections including HCV, HBV, and HIV are associated with increased immune regulatory pathways including the extrinsic FoxP3+CD4+ regulatory T cells (Tregs) and intrinsic inhibitory pathways such as programed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) with potentially reversible suppression of antiviral effector T cells (1-12). Immunological consequences of viral coinfections relative to these immune regulatory pathways and their interplay are not well-defined. In this study, we examined the frequency, phenotype, and effector function of circulating T cell subsets in patients with chronic HCV and/or HIV infection, hypothesizing that HCV/HIV coinfection will result in greater immune dysregulation with pathogenetic consequences (13, 14). We show that multiple T cell inhibitory pathways are induced in HCV/HIV coinfection including FoxP3+ Tregs, PD-1, and CTLA-4 in inverse association with overall CD4 T cell frequency but not with liver function or HCV RNA titers. The inverse association between CD4 T cell frequency and their FoxP3, PD-1, or CTLA-4 expression remained significant in all subjects combined regardless of HCV and/or HIV infection, suggesting a global homeostatic mechanism to maintain immune regulation relative to CD4 T cell frequency. PD-1 blockade rescued T cell responses to HIV but not HCV without significant impact by CTLA-4 blockade in vitro. Collectively, these findings highlight complex immune interactions in viral coinfections and differential regulatory pathways influencing virus-specific T cells that are relevant in immunotherapeutic development.

Keywords: CTLA-4; FoxP3; HCV; HIV; PD-1; Tregs; coinfection; immune pathogenesis.

Figure 1

FoxP3+ Tregs are enriched in CD4 T cells from HCV/HIV-coinfected patients. (A) Frequency of CD4 T cells among viable lymphocytes is compared between 20 HCV-monoinfected (HCV), 23 HCV/HIV-coinfected (HCV/HIV), 21 HIV-monoinfected (HIV), and 17 seronegative control (NC) subjects. Horizontal bars indicate median values. P-values are calculated with non-parametric Kruskal–Wallis for comparison between all four groups and Mann–Whitney U for pair-wise comparisons. Two-way comparison of HCV/HIV coinfection group with HCV or HIV-monoinfection group was made using non-parametric Mann–Whitney U and corrected for multiple comparisons. (B) Frequency of FoxP3+CD4 T cells (FoxP3+ Tregs) in CD4 T cells is compared between 20 HCV-monoinfected (HCV), 23 HCV/HIV-coinfected (HCV/HIV), 21 HIV-monoinfected (HIV), and 17 seronegative control (NC) subjects. Horizontal bars indicate median values. P-values are calculated with non-parametric Kruskal–Wallis for comparison between all four groups and Mann–Whitney U for pair-wise comparisons. Two-way comparison of HCV/HIV coinfection group with HCV or HIV monoinfection group was made using non-parametric Mann–Whitney U and corrected for multiple comparisons. (C) Left panel: representative FACS plots are shown for FoxP3 and CD4 expression in live lymphocytes: Right panel: frequency of FoxP3+CD4+ T cells in total live lymphoid cells is compared between 20 HCV-monoinfected (HCV), 23 HCV/HIV-coinfected (HCV/HIV), 21 HIV-monoinfected (HIV), and 17 seronegative control (NC) subjects. Horizontal bars indicate median values. P-values are calculated with non-parametric Kruskal–Wallis for comparison between all four groups and Mann–Whitney U for pair-wise comparisons. Two-way comparison of HCV/HIV coinfection group with HCV or HIV monoinfection group was made using non-parametric Mann–Whitney U and corrected for multiple comparisons. (D) Phenotype of FoxP3+CD4 T cells (filled black bars) and FoxP3− CD4 T cells (unfilled bars) from HCV/HIV-coinfected patients are compared using percentage of cells expressing CD25, CTLA-4, CD45RO, CD127, and CD69. (E) Percent suppression of proliferation in AutoMACS-sorted CD4+CD25− “responder” T cells alone and with increasing proportion of autologous CD4+CD25+ “suppressor” T cells enriched for FoxP3+CD4 T cells upon in vitro stimulation with anti-CD3/CD28 or phytohemagglutinin (PHA) followed by 3H thymidine uptake. Proliferation was expressed as a stimulation index (SI): the mean cpm in stimulated wells divided by the mean cpm in unstimulated wells. T cell proliferation in each coculture was normalized by proliferation in CD4+CD25− T cells alone and compared to the calculated percentage of FoxP3+CD4 T cells in each coculture condition, based on %FoxP3+ cells in CD4+CD25− and CD4+CD25+ cell subsets determined by FACS.

Figure 2

T cell expression levels of PD-1 and/or CTLA-4 associate with FoxP3+ Tregs in patients with HCV and/or HIV infection. (A) Frequency of CD4 and CD8 T cells expressing CTLA-4, PD-1, CD28, or CD127 are compared between 10 HCV-monoinfected (C), 9 HCV/HIV-coinfected (C/I), 10 HIV-monoinfected (I), and 10 seronegative control subjects (N). Horizontal bars indicate median values. P-values are calculated with non-parametric Kruskal–Wallis for comparison between all four groups. Two-way comparison of HCV/HIV coinfection group with HCV or HIV monoinfection group was made using non-parametric Mann–Whitney U and corrected for multiple comparisons. P-values above 0.05 before correction are shown as “ns” or “not significant.” (B) Frequency of CD4 and CD8 T cells expressing CTLA-4, PD-1, CD28, or CD127 are correlated with FoxP3+ Treg frequency in the CD4 T cell compartment, with non-parametric Spearman rank-order correlation and P-values.

Figure 3

FoxP3+ Treg expression levels of PD-1 and/or CTLA-4 in patients with HCV and/or HIV infection. (A) Frequency of FoxP3+ Tregs expressing CTLA-4 and PD-1 are compared between 10 HCV-monoinfected (C), 9 HCV/HIV-coinfected (C/I), 10 HIV-monoinfected (I), and 10 seronegative control subjects (N). Horizontal bars indicate median values. P-values are calculated with non-parametric Kruskal–Wallis for comparison between all four groups. Two-way comparison of HCV/HIV coinfection group with HCV or HIV monoinfection group was made using non-parametric Mann–Whitney U and corrected for multiple comparisons. (B) Relative frequencies of cells expressing PD-1 in FoxP3+CD4 T cells and FoxP3−CD4 T cells are compared in a representative FACS overlay on the left panel (FoxP3+CD4 T cells as red dot plot, FoxP3− CD4 T cells in blue density plot) and in pair-wise comparison in 10 HCV, 9 HCV/HIV, 10 HIV, and 10 seronegative control. P-value is calculated by pairedt-test.

Figure 4

Circulating CD4 T cell frequency is associated with FoxP3, PD-1, CTLA-4, and CD28 expression on T cell subsets. (A) Frequency of FoxP3+ Tregs in CD4 T cell compartment is compared with overall CD4 T cell frequency in live lymphocytes for each patient group and with all groups combined with 20 HCV-monoinfected (HCV), 23 HCV/HIV-coinfected (HCV/HIV), 21 HIV-monoinfected (HIV), and 17 seronegative control (NC) subjects. Spearman rank-order correlation and P-values are shown. (B) FoxP3 mean fluorescence intensity (MFI) is compared with overall CD4 T cell frequency in live lymphocytes. Spearman rank-order correlation and P-values are shown. (C) Frequency of FoxP3+ Tregs in CD4 T cell compartment is compared between in HCV/HIV-coinfected and HIV-monoinfected patients with CD4 T cell frequency below or higher than a cutoff of 31% (median CD4 T cell frequency). P-values are calculated by non-parametric Mann–Whitney U test. (D) Frequency of FoxP3+CD4+ T cells in live lymphocyte compartment is compared with CD4 T cell frequency with Spearman rank-order correlation and P-values. (E) CTLA-4, PD-1, CD28, and CD127 mean fluorescence intensities (MFI) in CD4 and CD8 T cells are compared with overall CD4 T cell frequency in live lymphocyte compartment. Spearman rank-order correlation and P-values are shown.

Figure 5

T cell cytokine responses to HCV, HIV, and Flu peptides. (A) Median T cell IFNγ responses and percent positive responders to HCV, HIV, and Flu peptides in HCV, HCV/HIV, and HIV-infected patient groups are shown in log scale (log spot forming units per million PBMC) based on IFNγ ELISPOT assay using PBMCs stimulated ex vivo with overlapping 15mer peptide pools spanning HCV NS3 (pHCV), HIV Gag (pHIV), and influenza matrix (pFlu). Unfilled bar (HCV-monoinfected, n = 6), black bar (HCV/HIV-coinfected, n = 6), gray bar (HIV-monoinfected, n = 5). Positive responses were defined by cutoff values for each assay (>average background + 3 SDs). P-values comparing three groups were calculated by non-parametric Kruskal–Wallis with only significant difference seen for HIV-specific T cell response (both by SFU and % responders) due to lack of HIV-specific T cell response in HCV-monoinfected patients. (B) Frequency of CD8+ T cells with antigen-specific IFNγ production in short term PBMC cultures is shown from HCV/HIV-coinfected patients, HCV-monoinfected, and HIV-monoinfected patients. PBMC cultures were stimulated for 7 days with overlapping peptide pools (2 μM) and rIL-2 followed by further 5.5 h peptide stimulation in the presence of Brefeldin A, intracellular cytokine staining, and FACS analysis. Total of 8 HCV/HIV-coinfected patients, 11 HCV-monoinfected, and 9 HIV-monoinfected patients were included, with sample size for each stimulation condition shown as defined by available lymphocytes. Red dotted line indicates the 0.27%, which was the cutoff value for a positive response based on average background + 3 SDs. Positive responses above 0.27% are further highlighted as dark shaded diamonds. P-values comparing two groups were calculated by non-parametric Mann–Whitney U without significant differences between the groups. (C) Representative FACS plots show TNFα and IFNγ expression in CD8-gated cells from an HCV/HIV-coinfected (C423) and HIV-monoinfected (I026) subjects. (D) Peptide-specific IFNγ response (in spot forming units per million PBMC) is compared with the frequency of FoxP3+ Tregs in CD4 T cell compartment from HCV/HIV-coinfected patients. Spearman rank-order correlation and P-values are shown without significant associations.

Figure 6

Effect of in vitro PD-1 blockade on virus-specific T cell cytokine responses in HCV/HIV-coinfected patients. T cell cytokine responses (combining IFNγ and/or TNFα responses) to HCV, HIV, and Flu peptides were examined in HCV/HIV-coinfected patients with and without PD-1 and/or CTLA-4 blockade in vitro. PBMCs from HCV/HIV-coinfected patients were cultured for 7 days in vitro with pHCV NS3, pHIV GAG, or pFlu Matrix in the presence of isotype control or blocking antibodies. The 7-day cultures were further stimulated for 6 h with media alone or with each peptide before intracellular IFN-γ and TNF-α staining. Virus-specific responses were calculated by subtracting the cytokine responses in media control samples from peptide-stimulated samples during the intracellular cytokine staining. A positive response to blockade was defined as antigen-specific response that was greater than the isotype control condition by at least a standard deviation of the all background responses (0.36%). (A) Percentage of virus-specific cytokine+ cells in CD8 (left panel) and CD4 T cells (right panel) are shown from HCV/HIV-coinfected patients, comparing responses with isotype antibody and with anti-PDL1 blocking Ab (10 μg/ml). Significant P-value per paired t-test is shown for HIV Gag. Assay pairs with augmented responses to PD-1 blockade above isotype control condition are highlighted as red lines. Significant increase in cytokine response by PD-1 blockade is shown for HIV-specific CD8 T cells (P = 0.047) via paired t-test. (B) Percentage of positive responders (augmentations above 0.36%) to anti-PDL1 or anti-CTLA-4 is shown for virus-specific CD8 (left panel) and CD4 T cells (right panel) from HCV/HIV-coinfected patients. Significant anti-PDL1-mediated augmentation is shown for HIV-specific CD4 T cell response by Kruskal–Wallis (P = 0.037). (C) Representative FACS plots are shown for HIV Gag-specific IFN-γ and TNF-α production in CD8 (left) and CD4 T cells (right) in short term PBMC culture from HCV/HIV-coinfected patients, following 7 day culture with HIV peptides in the presence of either isotype antibody or anti-PDL1. Marked induction in IFNγ and TNFα responses is apparent for CD4 T cells following PD-1 blockade.