Coordination of early protective immunity to viral infection by regulatory T cells

Abstract

Suppression of immune responses by regulatory T cells (Tregs) is thought to limit late stages of pathogen-specific immunity as a means of minimizing associated tissue damage. We examined a role for Tregs during mucosal herpes simplex virus infection in mice, and observed an accelerated fatal infection with increased viral loads in the mucosa and central nervous system after ablation of Tregs. Although augmented interferon production was detected in the draining lymph nodes (dLNs) in Treg-deprived mice, it was profoundly reduced at the infection site. This was associated with a delay in the arrival of natural killer cells, dendritic cells, and T cells to the site of infection and a sharp increase in proinflammatory chemokine levels in the dLNs. Our results suggest that Tregs facilitate early protective responses to local viral infection by allowing a timely entry of immune cells into infected tissue.

Fig. 1

Tregs respond to local HSV-2 infection with kinetics similar to those of effector CD4⁺ T cells. (A to F) Foxp3^gfp mice were infected with HSV-2, and vaginas and dLNs were prepared for analysis by flow cytometry. CD4⁺ Tcells are CD4⁺Foxp3^- and Tregs are CD4⁺Foxp3⁺. CD4⁺ Tcells (C) and Tregs (D) in the dLNs were stained for inducible costimulatory molecule expression. Mice were administered BrdU in the drinking water throughout the course of infection (E and F), and BrdU incorporation by CD4⁺ T cells and Tregs was detected by flow cytometry. (G) Effector T cells and antigen-pulsed APCs were cocultured with Foxp3⁺ suppressor cells as indicated, ³H was added, and cells were cultured for an additional 20 hours before harvest and measurement of proliferation with a beta-counter.

Fig. 2

Tregs are required to prevent early death from HSV-2 infection. Foxp3^WT or Foxp3^DTR mice were infected with HSV-2 and treated with DT. Survival (A) and diseasescore (B) were monitored daily and vaginal washes were collected daily to assess vaginal viral titer (C) by plaque assay on Vero cells. (D) Spinal cords were collected 4 days after infection and plaque assays were performed on homogenates.

Fig. 3

Tregs are required to mount a protective immune response at the site of infection subsequent to genital HSV-2 infection. Foxp3^WT or Foxp3^DTR mice were infected with HSV-2 and treated with DT. (A) For ex vivo cultures, CD4⁺ T cells were isolated from the dLNs of naïve or infected Foxp3^WT or Foxp3^DTR mice and cultured for 3 days with irradiated, heat-inactivated HSV-2-pulsed APCs. IFN-γ was detected by enzyme-linked immunosorbent assay (ELISA). (B) Mice were infected and treated with DT as in Fig. 2, dLNs were collected 2 days after infection, and extracts were prepared for IFN-α detection by ELISA. (C and D) IFN-γ present in vaginal washes collected at various times after infection (C) and IFN-α from vaginal washes collected 2 days after infection (D) were measured by ELISA. (E) At the indicated times after infection, dLNs and vaginal tracts were subjected to flow cytometric analysis. Naïve mice received DT according to the same schedule as infected mice.

Fig. 4

Tregs modulate the chemokine gradient that controls proper effector cell homing to the dLNs and site of infection. (A) NK cells, CD11b⁺ DCs, and pDCs from naïve donors or CD4⁺ T cells isolated from the dLNs of Foxp3^gfp donor mice infected with HSV-2 for 6 days were labeled with CFSE and injected into recipient mice. Recipients were Foxp3^WT or Foxp3^DTR mice treated with DT and infected with HSV-2, and mice received CFSE-labeled cells 2 days after infection. Twenty-four hours after transfer, dLNs and vaginal tracts from recipient mice were examined for percentage of the indicated population that was CFSE⁺ by flow cytometry. (B) Foxp3^WT or Foxp3^DTR mice were infected with HSV-2 and treated with DT. Two days after infection, dLNs and vaginal tract extracts were prepared for chemokine detection by Luminex bead assay. (C) Chemokine mRNA amounts in DCs and CD45^- stromal cells were measured by real-time PCR and are shown as the mean of three independent experiments relative to hypoxanthine-guanine phosphoribosyl transferase expression. (D) Foxp3^DTR mice were treated with DT and infected with LCMV. Five days after infection, livers were collected and assayed for viral titer, and LNs were analyzed for chemokines as in (B).