Partial depletion of natural CD4⁺CD25⁺ regulatory T cells with anti-CD25 antibody does not alter the course of acute influenza A virus infection

Abstract

Foxp3⁺CD4⁺ regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8⁺ T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25⁺Foxp3⁺ regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4⁺CD25⁺ regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection.

Figure 1. Influenza A virus infection results in the robust induction of a CD25⁺Foxp3⁺ Treg response.

Mice were inoculated with 10pfu influenza A/PR8/34 and sacrificed at various timepoints and the induction of CD25⁺Foxp3⁺ Treg cells determined across the course of infection. (A) Representative FACS plots of the proportion of T cells collected from BAL expressing CD25 and Foxp3, gated on CD3 and CD4 positive cells. (B) Proportion of CD3⁺CD4⁺ T cells expressing Foxp3⁺ across a timecourse in cells collected from BAL, lung, and mediastinal lymph node. (C) Proportion of Foxp3⁺ Tregs that co-express CD25⁺ across a timecourse in cells collected from BAL, lung, and mediastinal lymph node. (D) Total numbers of CD25⁺Foxp3⁺ and CD25-Foxp3⁺ T cells across a timecourse in cells collected from BAL, lung, and mediastinal lymph node. Data represents mean ± S.E.M, n = 4–7 for all.

Figure 2. PC61 α-CD25 antibody administration results in partial depletion of Foxp3⁺ regulatory T cells.

Mice were inoculated with 10pfu influenza A/PR8/34 and administered PBS or 400 µg of PC61 antibody i.v. at days -2 and 4 post-inoculation, and expression of CD25⁺ and Foxp3⁺ within CD3⁺CD4⁺ T cells examined at days 7 and 12 post-inoculation within lung, BAL and mLN samples. (A) Representative FACS plots of CD25 and Foxp3 expression on BAL CD3⁺CD4⁺ T cells at days 7 and 12 post-inoculation in PC61 and PBS-injected mice. (B) Percentage of CD4⁺ T cells expressing Foxp3⁺ in cells collected from lung, BAL and mLN of PBS and PC61 treated mice at days 7 and 12 post-inoculation. (C) Percentage of CD4⁺Fopx3⁺ T cells co-expressing CD25 in PBS and PC61 treated mice from lung, BAL and mLN of PBS and PC61 treated mice at days 7 and 12 post-inoculation. * denotes t<0.05, ** t<0.01, Student' t-test. Data represents mean ± S.E.M, n = 6–8 for all.

Figure 3. PC61 α-CD25 antibody administration does not alter influenza A virus-induced weight loss of influenza A-virus induced mortality.

Mice were inoculated with influenza A/PR8/34 and administered PBS or 400 µg of PC61 antibody i.v. at days -2 and 4 post-inoculation. (A) Influenza-induced weight loss in mice inoculated with a sub-lethal dose of 10pfu influenza A virus and administered PBS or 400 µg PC61 α-CD25 antibody. (B) Influenza A virus-induced mortality in mice inoculated with lethal dose of 250pfu Influenza A virus and administered PBS or 400 µg PC61 α-CD25 antibody. Data represents mean ± S.E.M, n = 6 for all.

Figure 4. PC61 α-CD25 antibody administration does not alter influenza A virus-induced BAL cellularity, antigen-specific CD8⁺ T cell numbers, CD4⁺Foxp3^- T cell numbers, NK cell activation, IFN-γ levels, immunopathology and pulmonary viral load. alter influenza A virus-induced weight loss of influenza A-virus induced mortality.

Mice were inoculated with influenza A/PR8/34 and administered PBS or 400 µg of PC61 antibody i.v. at days -2 and 4 post-inoculation. (A) BAL cellularity as determined by flow cytometry at day 7 (A) or day 12 (B) post-inoculation in mice administered PBS or PC61 α-CD25 antibody. Antigen-specific CD8⁺ T cell numbers in lung (C) or mLN (D) at days 7 and 12 post-inoculation in mice administered PBS or PC61 antibody. (E) Percentage of CD4⁺Foxp3^- T cells expressing CD25 and (F) total CD4⁺Foxp3^- T cell numbers at days 7 and 12 post-inoculation in mice treated with PBS or PC61 antibody. (G) Percentage NK cells expressing activation marker CD107α at day 2 post-inoculation in mice administered 400 µg of PC61 antibody i.v. at days -2 post-inoculation. (H) IFN-γ levels in BAL fluid at days 7 and 12 post-inoculation in mice administered PBS or 400 µg of PC61 antibody i.v. at days -2 and 4 post-inoculation. (I) BAL albumin at days 7 and 12 post-inoculation in mice administered PBS or 400 µg of PC61 antibody. (J) Pulmonary viral titre at day 7 post-inoculation in mice administered PBS or PC61 antibody. Data represents mean ± S.E.M, n = 5–7 for all.