Partial Regulatory T Cell Depletion Prior to Acute Feline Immunodeficiency Virus Infection Does Not Alter Disease Pathogenesis

Feline immunodeficiency virus (FIV) infection in cats follows a disease course similar to HIV-1, including a short acute phase characterized by high viremia, and a prolonged asymptomatic phase characterized by low viremia and generalized immune dysfunction. CD4+CD25hiFoxP3+ immunosuppressive regulatory T (Treg) cells have been implicated as a possible cause of immune dysfunction during FIV and HIV-1 infection, as they are capable of modulating virus-specific and inflammatory immune responses. Additionally, the immunosuppressive capacity of feline Treg cells has been shown to be increased during FIV infection. We have previously shown that transient in vivo Treg cell depletion during asymptomatic FIV infection reveals FIV-specific immune responses suppressed by Treg cells. In this study, we sought to determine the immunological influence of Treg cells during acute FIV infection. We asked whether Treg cell depletion prior to infection with the highly pathogenic molecular clone FIV-C36 in cats could alter FIV pathogenesis. We report here that partial Treg cell depletion prior to FIV infection does not significantly change provirus, viremia, or CD4+ T cell levels in blood and lymphoid tissues during the acute phase of disease. The effects of anti-CD25 mAb treatment are truncated in cats acutely infected with FIV-C36 as compared to chronically infected cats or FIV-naïve cats, as Treg cell levels were heightened in all treatment groups included in the study within two weeks post-FIV infection. Our findings suggest that the influence of Treg cell suppression during FIV pathogenesis is most prominent after Treg cells are activated in the environment of established FIV infection

Anti-CD25 mAb treatment does not significantly alter FIV levels in the blood.

<p>Cats were infected with 2×10^5.2 TCID₅₀ units molecular clone FIV-C36 by intravaginal and intravenous routes on day 0. (A) To detect viremia in plasma samples, one-step quantitative real-time RT-PCR was performed on extracted RNA samples. FIV RNA copy number was quantified relative to an FIV C <i>gag</i> RNA standard curve. (B) Provirus in PBMCs was detected via quantitative real-time RT-PCR on extracted DNA samples. FIV copy number was quantified relative to an FIV C <i>gag</i> DNA standard curve, and then normalized relative to CCR5 copy number. Mean ± SEM is shown (n = 8/group).</p

T cell proliferation due to FIV infection is not enhanced by anti-CD25 mAb treatment.

<p>Percent (A) CD4⁺ Ki67⁺ T cells and (B) CD8⁺ Ki67⁺ T cells in circulation were quantified by flow cytometry on days −12, 0, 7, 14, 35, and 54 p.i. (n = 8/group). Mean ± SEM is shown. Significance is relative to day 0 values for each group. No significant differences were found between treatment groups. * indicates <i>p</i><0.05.</p

Anti-FIV p24, but not anti-FIV gp95, IgG titers are significantly lower after anti-CD25 mAb treatment during acute FIV infection.

<p>(A) Anti-FIV gp95 and (B) anti-FIV p24 IgG titers were quantified in serum samples on days 0, 7, 14, 35, and 54 post-FIV infection. Positive antibody titers were calculated based on a 3-fold higher optical density than day 0 pre-inoculation controls. Mean ± SEM is shown. Statistical significance was determined between treatment groups for each time point (n = 8/group). * indicates <i>p</i><0.05.</p

FIV-specific IFN-γ responses during acute infection are not altered by anti-CD25 mAb treatment.

<p>Popliteal lymph node cells from day 14 and 35 p.i. and mesenteric and retropharyngeal lymph node cells and spleen cells from day 54 p.i. were incubated for 48 hours with 100 µg/mL FIV p24 peptides or 1% DMSO/media as a background control. IFN-γ spot forming cells (SFCs) per million cells from CD25 depleted, isotype control, and vehicle control treated groups in response to FIV p24 minus background were determined by ELISpot (n = 8/group).</p

Anti-CD25 monoclonal antibody treatment depletes Treg cells in cats.

<p>Cats received 9 mg/kg anti-CD25 mAb (9F23) (n = 8) or 9 mg/kg isotype control mAb (CRL-1689) (n = 8) i.p. on day −12. A third group of cats received 3 mL/kg saline on day −12 (n = 8). (A) PBMCs were labeled with antibodies for CD4 and CD25 and analyzed by flow cytometry for CD4⁺CD25^hi percentages within the lymphocyte population on days −12, 0, 7, 14, 35, and 54 post-FIV infection. Percentages were multiplied by lymphocyte absolute number to quantify absolute numbers of CD4⁺CD25^hi cells. (B) CD4⁺CD25⁺FoxP3⁺ cell percentages were determined by flow cytometry. CD4⁺CD25⁺FoxP3⁺ absolute number was calculated. Mean ± SEM is shown. * indicates <i>p</i><0.05.</p

Peripheral lymphocyte dynamics during acute FIV-C36 infection are not altered by anti-CD25 mAb treatment.

<p>Absolute numbers of (A) CD3⁺CD4⁺, (B) CD3⁺CD8⁺, (C) CD3⁻B220⁺ (B cells), and (D) CD3⁻CD56⁺ (NK cells) lymphocytes in the periphery were quantified on days −12, 0, 7, 14, 35, and 54 p.i. based on percentages obtained by flow cytometry and absolute number of peripheral lymphocytes. Mean ± SEM is shown (n = 8/group).</p

FIV proviral load in tissues is unaffected by anti-CD25 mAb treatment.

<p>(A–B) Provirus in popliteal lymph nodes was quantified on days 14 and 35 p.i. (C–E) Provirus in mesenteric and retropharyngeal lymph nodes and spleen was quantified on day 54 p.i. * indicates <i>p</i><0.05.</p

Peripheral Treg cells are induced during acute FIV infection.

<p>(A) CD4⁺CD25^hi percentages within the PBMC population were determined by flow cytometry on days −12, 0, 7, 14, 35, and 54 post-FIV infection. Percentages were multiplied by lymphocyte absolute number to quantify absolute numbers of CD4⁺CD25^hi cells. Absolute numbers were then expressed as a percentage of day −12 values and averaged. (B) CD4⁺CD25⁺FoxP3⁺ percentages within the PBMC population were determined by flow cytometry, then absolute numbers were expressed as a percentage of day −12 values and averaged. (A, B) Significance was determined relative to day −12 values. * indicates <i>p</i><0.05. (C) FoxP3 mean fluorescence intensity (MFI) within CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁻FoxP3⁺ PBMCs from vehicle control-treated cats was determined by flow cytometry. Levels and kinetics did not significantly differ between treatment groups. (D) FoxP3 and TGF-β MFI within CD4⁺CD25⁺ PBMCs from vehicle control-treated cats was determined by flow cytometry. Levels and kinetics did not significantly differ between treatment groups. (E) Percent of CD4⁺CD25⁺FoxP3⁺ PBMCs expressing CD62L was determined by flow cytometry. (F) Percent CD4⁺Ki67⁺ PBMCs from vehicle control-treated cats was determined by flow cytometry and is compared to CD4⁺CD25⁺FoxP3⁺ PBMC absolute number. Levels and kinetics did not significantly differ between treatment groups. (C–F) Significance was determined relative to day 0 values. * indicates <i>p</i><0.05. (G) Percent of CD4⁺ T cells expressing CD25 and FoxP3 in lymphoid tissues was determined by flow cytometry on days 14, 35, or 54 post-FIV infection. (A–G) Mean ± SEM is shown.</p