TGF-β blockade does not improve control of an established persistent viral infection

Abstract

Acute resolving viral infections are often associated with a strong and multi-specific T-cell response, whereas in persistent viral infections T-cell responses are often impaired. It has been suggested that the resuscitation of the antiviral T-cell response could be a powerful tool to target persisting viruses. Several immunoregulatory pathways, such as IL-10 and TGF-β, have been shown to be involved in the induction of T-cell exhaustion and viral persistence. In this study, we sought to investigate whether TGF-β signaling is also relevant in the maintenance of T-cell exhaustion after viral persistence has been established, and whether blockade of TGF-β signaling could improve control of viral replication in a mouse model of persistent virus infection. Using the LCMV clone 13 model, we analyzed the frequency, function, and phenotype of virus-specific CD4 and CD8 T cells following therapeutic TGF-β signaling blockade. We show that in vivo blockade of the TGF-β receptor failed to substantially enhance the antiviral T-cell response, and was insufficient to mediate a therapeutically-relevant reduction of viral titers in different tissues. Thus, although TGF-β signaling has the ability to hamper antiviral immunity, its pharmacological blockade may not be sufficient to tackle persistent viruses.

FIG. 1.

Plasma TGF-β levels increase over time during LCMV clone 13 (cl13) infection, and TGF-βR blockade enhances the frequency of virus-specific CD8 T cells in the spleen. (A) Diagram of experimental design. For selected experiments, C57BL/6 recipient mice were seeded with LCMV-TCR transgenic cells. Starting on day 21 post-LCMV clone 13 infection, mice received 4–5 treatments of either TGF-βR blockade or placebo every 2–3 d. For most experiments, serum and organs were harvested for analysis 7 d after the last treatment. Experiments performed 14 d after completion of the treatment are specifically labeled. (B) Plasma-levels of TGF-β1 in naïve mice (black box), and LCMV clone 13-infected untreated mice (grey boxes), were measured by ELISA on the indicated days post-infection. (C) Pentamer stainings revealed higher frequencies of GP33-specific CD8 T cells after TGF-β blockade. Two representative pseudocolor plots gated on CD8⁺CD19^– T cells are shown. (D) Frequencies of NP396-specific CD8 T cells were not affected by TGF-βR blockade. (E) Overall CD8 T-cell numbers were not affected by TGF-βR blockade (*p<0.05).

FIG. 2.

Virus-specific secretion of IL-2, IFN-γ, and TNF following TGF-βR blockade. (A) Shown is the frequency of CD8⁺ cells that produce IL-2, IFN-γ, or TNF following stimulation with the GP33 peptide. (B) Representative pseudocolor plots from intracellular cytokine stainings gated on CD8 T cells. (C) Pie charts display the cytokine secretion pattern of the cells displayed in A. (D) Frequency of CD8 T cells expressing the degranulation marker CD107a during 5 h incubation with the GP33 peptide. (E) Frequency of CD8⁺ cells that produce IL-2, IFN-γ, or TNF, following stimulation with the GP276-peptide. (F) Frequency of IL-2-, IFN-γ-, or TNF-producing CD4 T cells following stimulation with the GP61 peptide (*p<0.05). Color images available online at www.liebertonline.com/vim

FIG. 3.

Enhancement of CD8 T-cell responses through TGF-β signaling blockade is insufficient to improve control of viral replication. (A) About 2000 naïve GP33–41 transgenic P14 cells were injected into B6 mice 1 d prior to LCMV clone 13 infection. Significantly higher numbers of P14 cells could be recovered in the spleens of treated mice. (B) Percentage of IFN-γ-producing cells among GP33-specific P14 cells. (C) Expression of defined surface molecules on P14 cells. (D) Viral titers were analyzed before and 14 d after completion of the TGF-β blockade in the serum of infected mice. (E) At 7 d after completion of the therapeutic TGF-β blockade, viral titers were analyzed in serum, liver, and kidney, by plaque assay or qPCR (*p<0.05).