Blockade of virus infection by human CD4+ T cells via a cytokine relay network

Abstract

CD4(+) T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8(+) T cell functions, we sought to determine whether human CD4(+) T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-gamma and TNF-alpha, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-gamma and TNF-alpha acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-gamma and TNF-alpha also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-gamma and TNF-alpha secreted by human Th1 cells acted indirectly through the IFN-alpha/beta receptor. These results highlight the importance of CD4(+) T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.

FIGURE 1

Soluble factors secreted by human CD4⁺ T cells inhibit VSV infection. A, THP-1 cells were infected for 16 hours with VSV-GFP. GFP expression was analyzed by flow cytometry. Representative FACS plots showing THP-1 cells left uninfected or infected with VSV-GFP in the presence or absence of 100 U/ml rhIFN-αA and/or a neutralizing anti-hIFNAR2 antibody. B, THP-1 cells were left uninfected (square) or were infected for 16 hours with VSV-GFP in the absence (triangle) or presence (circles) of increasing concentrations of rhIFN-αA. GFP expression was analyzed by flow cytometry. C, THP-1 cells were left uninfected (■) or were infected for 16 hours with VSV-GFP. Cells were treated at the time of infection with either media alone (▲) or with increasing doses of conditioned media from T cells polarized with the following conditions: Neutralized (anti-IFN-γ, anti-IL-4, anti-IL-12, and anti-IFNAR2), □; IL-12, (rhIL-12, anti-IFN-γ, anti-IL-4, and anti-IFNAR2) ;▵ IFN-αA, rhIFN-αA, anti-IFN-γ, anti-IL-4, and anti-IL-12) ∇; or IL-12 + IFN-αA, (rhIL-12 and rhIFN-αA in the presence of anti-IFN-γ and anti-IL-4) ○. GFP expression was analyzed by flow cytometry. *, p < 0.05 versus neutralized. D, THP-1 cells were infected for 16 hours with VSV-GFP in the absence or presence of T cell conditioned media from T cells which were left unstimulated (black bars) or were restimulated on day 7 for 24 hours with plate-bound anti-CD3 (hatched bars). GFP expression was analyzed by flow cytometry. E, THP-1 cells were cultured for 24 hours in the absence or presence of 100 U/ml rhIFN-αA or T cell conditioned media derived from cells polarized as indicated in the figure. Cells were then washed and then infected for an additional 24 hours with VSV-GFP, and viral replication was quantified by plaque assay.

FIGURE 2

Inhibition of RSV and HCV replication factors secreted by human CD4⁺ T cells. A, HeLa cells were infected for 72 hours with RSV-GFP in the absence or presence of 100 U/ml rhIFN-αA or 10% (v/v) T cell conditioned media as indicated. GFP expression was analyzed by flow cytometry. Data are expressed as mean +/− SEM of three replicates. B, A7 replicon cells were incubated for 24 hours in the absence (lane 1) or presence of 100 U/ml rhIFN-αA (lane 2) or 5% (v/v) T cell conditioned media (lanes 3–10) as indicated. Cell lysates were prepared, and Western blotting was performed using antibodies against HCV NS5A and human GAPDH.

FIGURE 3

IL-12 promotes IFN-γ and TNF-α secretion by human CD4⁺ T cells. (A – D) Naïve (CD45RA⁺ CD4⁺) T cells were sorted from peripheral blood of healthy donors and activated for 7 (A, C – D) or 14 (B) days with plate-bound anti-CD3 and anti-CD28 in the presence of cytokines and neutralizing antibodies as indicated. (A – B) Cells were restimulated for 4 hours with PMA and ionomycin, and flow cytometry was performed using antibodies against human IFN-γ and human TNF-α. Black bars, percent IFN-γ single positive cells; hatched bars, percent TNF-α single positive cells; open bars, percent IFN-γ/TNF-α double positive cells. Data are expressed as mean +/− SEM of three replicates. C and D, Cells were washed and left unstimulated (open bars) or restimulated for 24 hours with plate-bound anti-CD3 (black bars). Supernatants were harvested, and ELISAs were performed for human IFN-γ (C) or human TNF-α (D). Data are expressed as mean +/− SEM of three replicates.

FIGURE 4

Inhibition of Listeria replication by human CD4⁺ T cells secreting IFN-γ and TNF-α. THP-1 cells were infected with Listeria innocua in the absence or presence of recombinant cytokines (A) or 50% (v/v) T cell conditioned media (B) for 16 hours. Cell monolayers were harvested, and L. innocua was quantified by colony-forming assay from cell lysates. Data are expressed as mean +/− SEM of three replicates.

FIGURE 5

IFN-γ and TNF-α secreted by human CD4⁺ T cells exert antiviral activity against HCV infection. A, A7 replicon cells were incubated for 24 hours in the absence (lane 1) or presence (lanes 2–11) of cytokines, neutralizing anti-cytokine antibodies, and 5% (v/v) T cell conditioned media from IL-12 + IFN-α activated T cells as indicated. Cell lysates were prepared, and Western blotting was performed using antibodies against HCV NS5A, human ISG56, and human GAPDH. B, A7 replicon cells were incubated for 24 hours in the absence (lane 1) or presence of 100 U/ml rhIFN-αA (lanes 2–3) or 5% (v/v) T cell conditioned media (lanes 4–12), in the absence or presence of 5 μg/ml anti-hIFNAR2 antibody, as indicated. Cell lysates were prepared, and Western blotting was performed using antibodies against HCV NS5A, human ISG56, and human GAPDH.

FIGURE 6

IFN-γ and TNF-α secreted by CD4⁺ T cells inhibit VSV infection. THP-1 cells were infected for 16 hours with VSV-GFP. GFP expression was analyzed by flow cytometry. Data are expressed as mean +/− SEM of three replicates. (A) THP-1 cells were infected in the absence (■) or presence of 100 U/ml rhIFN-αA (▴) or increasing concentrations of rhIFN-γ ( ), rhTNF-α (◇), or rhIFN-γ + rhTNF-α (○) as indicated. (B) THP-1 cells were infected in the absence (1) or presence (–7) of 10% (v/v) T cell conditioned media from IL-12 + IFN-α activated T cells in the absence (2) or presence of 5 μg/ml mouse IgG₁ isotype control antibody (3), 5 μg/ml anti-hIFNAR2 (4), 10 μg/ml anti-hIFNγR1 (5), 5 μg/ml anti-hTNF-α (6), or a combination of anti-hIFNγR1 and anti-hTNF-α (7).

FIGURE 7

IFN-γ- and TNF-α-mediated antiviral activity requires availability of the IFNAR. THP-1 cells were infected for 16 hours with VSV-GFP. GFP expression was analyzed by flow cytometry. Data are expressed as mean +/− SEM of three replicates. A, THP-1 cells were infected in the absence or presence of 100 U/ml rhIFN-αA or a combination of 2.5 ng/ml rhIFN-γ and 2.5 ng/ml rhTNF-α in the absence (open bars) or presence (black bars) of 5 μg/ml anti-hIFNAR2 antibody. *, p < 0.05, two-way ANOVA. B, THP-1 cells were infected in the absence or presence of 10% (v/v) T cell conditioned media as indicated in the absence (black bars) or presence (hatched bars) of 5 μg/ml anti-hIFNAR2 antibody.

FIGURE 8

Neutralization of IFN-α, IFN-β, and IFN-ω fails to inhibit the antiviral activity of human CD4⁺ T cells. THP-1 cells were infected for 16 hours with VSV-GFP. GFP expression was analyzed by flow cytometry. Data are expressed as mean +/− SEM of three replicates. (A) THP-1 cells were infected in the absence (1) or presence (–8) of 10% (v/v) T cell conditioned media from IL-12 + IFN-α activated T cells in the absence (2) or presence of 5 μg/ml mouse IgG₁ isotype control antibody (3), 5 μg/ml anti-hIFNAR2 (4), 5 μg/ml anti-hIFN-α (5), 1700 NU/ml anti-hIFN-β (6), 5 μg/ml anti-hIFN-ω (7), or a combination of anti-hIFN-α, anti-hIFN-β, and anti-hIFN-ω (8). (B) THP-1 cells were infected in the absence or presence of 100 U/ml rhIFN-αA or 100 U/ml rhIFN-β or 10 U/ml rhIFN-ω in the absence (open bars) or presence of 5 μg/ml mouse IgG₁ isotype control antibody (hatched bars), 5 μg/ml anti-hIFNAR2 antibody (black bars), or 5 μg/ml anti-hIFN-α antibody or 1700 NU/ml anti-hIFN-β antibody or 5 μg/ml anti-hIFN-ω antibody (double hatched bars) as indicated.

FIGURE 9

IFN-γ and TNF-α signal through a cytokine relay network involving the type I interferon receptor. Wild-type 2fTGH cells and hIFNAR2-deficient U5A cells were infected for 16 hours with VSV-GFP. GFP expression was analyzed by flow cytometry. Data are expressed as mean +/− SEM of three replicates. A, 2fTGH and U5A cells were infected in the absence (open bars) or presence of 100 U/ml rhIFN-αA (hatched bars), 2.5 ng/ml rhIFN-γ (black bars), 2.5 ng/ml rhTNF-α (double hatched bars), or a combination of 2.5 ng/ml rhIFN-γ and 2.5 ng/ml rhTNF-α (gray bars). B, 2fTGH and U5A cells were infected in the absence (black bars) or presence of 100 U/ml rhIFN-αA (hatched bars) or 10% (v/v) T cell conditioned media generated from IL-12 + IFN-α activated hCD4⁺ T cells (white bars). *, p < 0.05, one-way ANOVA.