LCMV induced downregulation of HVEM on antiviral T cells is critical for an efficient effector response

T-cell responses against tumors and pathogens are critically shaped by cosignaling molecules providing a second signal. Interaction of herpes virus entry mediator (HVEM, CD270, TNFRSF14) with multiple ligands has been proposed to promote or inhibit T-cell responses and inflammation, dependent on the context. In this study, we show that absence of HVEM did neither affect generation of effector nor maintenance of memory antiviral T cells and accordingly viral clearance upon acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, due to potent HVEM downregulation during infection. Notably, overexpression of HVEM on virus-specific CD8(+) T cells resulted in a reduction of effector cells, whereas numbers of memory cells were increased. Overall, this study indicates that downregulation of HVEM driven by LCMV infection ensures an efficient acute response at the price of impaired formation of T-cell memory.ISSN:0014-2980ISSN:1521-414

IL-21 antagonizes virus-induced Treg cell expansion during chronic infection.

<p>(A) Frequencies of splenic Foxp3⁺CD25⁺CD4⁺ T cells from WT and IL-21R^−/− mice at indicated days after infection with indicated doses of LCMV-DOC. (B) Total numbers of Foxp3⁺ cells (2000 PFU LCMV-DOC, 15 dpi). (C) IL-21R expression on GFP⁺CD4⁺Treg cells of naïve and LCMV-DOC infected DEREG mice (2×10⁴ PFU, 15 dpi). The mean fluorescent intensity (MFI) of single mice (left panel) and averages ± SEM of groups (n = 4) (right panel) are shown. (D–G) Analysis of WT (CD45.1⁺):IL-21R^−/−(CD45.2⁺) mixed BM chimeras. (D) Blood frequencies of CD4⁺, CD8⁺ and Foxp3⁺CD25⁺CD4⁺ T cell populations derived from WT and IL-21R^−/− BM 8 weeks after BM transfer prior LCMV infection. Reconstitution efficiency of WT and IL-21R^−/− lymphocytes (lym) were in average 51%:49% and values for the CD4⁺ and CD8⁺ T cell populations were corrected for chimerism. (E) FACS plots show CD25 and Foxp3 expression of CD4⁺ T cells, (F) graphs show frequencies and (G) total numbers of splenic Foxp3⁺CD25⁺ Treg cells 35 dpi with 2000 PFU LCMV-DOC. (H) Suppressive activity of FACS-sorted CD4⁺CD25⁺ Treg cells from infected DEREG or DEREGxIL-21R^−/− mice was assessed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003362#s4" target="_blank">Materials and Methods</a>. (I) Cell surface expression of putative Treg markers and (J) IL-10 production by Foxp3⁺CD4⁺ T cells isolated from WT or IL-21R^−/− mice infected with 2×10⁴ PFU LCMV-DOC 15 dpi. (J) Dot plots depict IL-10 production of splenic Foxp3⁺ T cells of WT or IL-21R^−/− mice after restimulation with gp61. Shown are representative individuals of groups of mice (n = 4). (K–N) IL-6^−/− and WT mice (n = 5/group) were infected with 2×10⁴ PFU LCMV-DOC and analyzed 30 dpi. Shown are frequencies of Foxp3⁺ cells (K), gp33-specific CD8⁺ T cells (L), IFN-γ-producing CD8⁺ and CD4⁺ T cells (M), and virus titers in organs indicated (N). (A–N) Data represent one of two independent experiments.</p

Virus dose-dependent expansion of the Treg cell population in response to chronic viral infection.

<p>Characterization of the Foxp3⁺ Treg cell population in spleen (A–C), liver and kidney (B) of C57BL/6 WT mice at days 0 and 7 (A) and at day 15 (B, C) post infection with indicated doses of LCMV-DOC. (A, B) Bar graphs show the mean ±SEM of groups (n = 4–9) of mice. (C) Dot plots gated on CD4⁺ T cells from spleens of individual mice representative for the group. (D) Viral titer in organs of individual mice as determined by plaque forming assay. Dotted horizontal lines indicate the detection limit (DTL). (E) Percentage of gp33-specific CD8⁺ T cells in spleen, liver and kidney at 15 days post infection (dpi). Pooled data from 2–4 independent experiments are shown. Data represent the mean ±SEM of 4–9 mice/group and was compared to mice infected with 200 PFU LCMV-DOC by one-way ANOVA using the Bonferroni correction. (F–H) <i>Il2</i>-emGFP-<i>Il21</i>-mCherry dual reporter transgenic mice were infected with low dose (i.e. 200 PFU) LCMV-WE or high dose (i.e. 2×10⁵ PFU) LCMV-DOC. (F) Dot plots show IL-2-(emGFP) and IL-21 (mCherry) expression of CD4⁺ T cells in blood of naïve and day 8 LCMV infected mice without or with gp61 restimulation. (G–H) Shown are frequencies of (G) IL-2- or (H) IL-21- expressing CD4⁺ and CD8⁺ T cells without (w/o) or with gp61 or gp33 peptide restimulation at days indicated. Values indicate averages of groups (n = 2–3/group).</p

IL-21 delivery impairs IL-2ic driven expansion of Treg cells.

<p>Expression vectors encoding the IL-21 gene fused with the hIgG1 (IL-21pDNA) or a control hIgG1 gene (ctrl pDNA) alone were delivered to C57BL/6 mice by hydrodynamic injection 24 h prior injection of IL-2ic. Alternatively, IL-2ic treated C57BL/6 WT mice were injected with 2 µg of IL-21-hIgG1 fusion protein consecutively every 12 h from days 0–5. Foxp3⁺ Treg cells were measured (A) in the blood at days indicated and (B) in spleen and liver at day 6 by flow cytometry. (C) Marginal zone B cells in the spleen at day 6. Values show percentages of Foxp3⁺ T cells of CD4⁺ T cells (A, B) and CD21⁺CD23^low of CD19⁺ cells (C). Dots represent individual mice and lines indicate averages (*<i>p</i>≤0.05).</p

Depletion of Treg cells partially restores CD8⁺ T cell functionality in WT and IL-21R^−/− mice.

<p>DEREG, C57BL/6 (WT), DEREGxIL-21R^−/− and IL-21R^−/− mice were infected with 2000 PFU LCMV-DOC and treated with diphtheria toxin (DT) at indicated days before analysis at day 15 as illustrated in (A). (B) Percentages of Foxp3⁺ Treg cells and (C) gp33-specific CD8⁺ T cells in the spleen. (D, E) Frequency of IFN-γ-producing CD8⁺ (D) and CD4⁺ (E) T cells as assessed by intracellular cytokine staining after <i>in vitro</i> restimulation with specific peptide or PMA/Ionomycin. (F) Shown are virus titers in organs of individual mice 15 dpi. Dotted lines indicate the detection limit (DTL). Dots represent individual mice, the lines the averages (*<i>p</i>≤0.05).</p

Depletion of Treg cells partially restores CD8⁺ T cell functionality and increases disease severity.

<p>DEREG and C57BL/6 WT mice were infected with 2×10⁵ PFU LCMV-DOC and treated with diphtheria toxin (DT) for depletion of Foxp3⁺ cells as illustrated in (A, I). (B) Dot plot of GFP and Foxp3⁺ expressing cells gated on CD4⁺ T cells from naïve and infected mice treated or untreated with DT. (C) Morbidity indicated by weight loss and (D) percentage of surviving mice for groups of 8 mice. (E) Expansion of virus-specific CD8⁺ T cells and cytokine production of splenic (F) CD8⁺ and (G) CD4⁺ T cells as detected with gp33-specific tetramers or intracellular cytokine staining after restimulation with specific peptide or PMA/Ionomycin. (H) Virus titers in blood (left panel) and organs (right panel) of individual mice at 15 dpi. Dotted lines indicate the detection limit (DTL). (J) Percentages of gp33-specific CD8⁺ T cells in spleen, liver, and lungs 10 dpi. Frequencies of splenic IFN-γ-producing CD8⁺ T cells (K) and CD4⁺ T cells (L) after restimulation with specific peptide (left graph) or PMA/Ionomycin (right graph). (M) Virus titers in blood and organs of individual mice 10 dpi. Dotted lines indicate the detection limit (DTL). Data are representative of two independent experiments including n = 8 (A–H) or n = 4 (I–H) mice per group. Symbols depict individual mice and lines indicate averages of groups.</p

IL-21 restricts virus-driven Treg cell expansion in chronic LCMV infection

Foxp3+ regulatory T (Treg) cells are essential for the maintenance of immune homeostasis and tolerance. During viral infections, Treg cells can limit the immunopathology resulting from excessive inflammation, yet potentially inhibit effective antiviral T cell responses and promote virus persistence. We report here that the fast-replicating LCMV strain Docile triggers a massive expansion of the Treg population that directly correlates with the size of the virus inoculum and its tendency to establish a chronic, persistent infection. This Treg cell proliferation was greatly enhanced in IL-21R-/- mice and depletion of Treg cells partially rescued defective CD8+ T cell cytokine responses and improved viral clearance in some but not all organs. Notably, IL-21 inhibited Treg cell expansion in a cell intrinsic manner. Moreover, experimental augmentation of Treg cells driven by injection of IL-2/anti-IL-2 immune complexes drastically impaired the functionality of the antiviral T cell response and impeded virus clearance. As a consequence, mice became highly susceptible to chronic infection following exposure to low virus doses. These findings reveal virus-driven Treg cell proliferation as potential evasion strategy that facilitates T cell exhaustion and virus persistence. Furthermore, they suggest that besides its primary function as a direct survival signal for antiviral CD8+ T cells during chronic infections, IL-21 may also indirectly promote CD8+ T cell poly-functionality by restricting the suppressive activity of infection-induced Treg cells