FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability

Abstract

Here, we test the role of FoxP3(+) regulatory T cells (Tregs) in controlling T follicular helper (Tfh) and germinal centre (GC) B-cell responses to influenza. In contrast to the idea that Tregs suppress T-cell responses, we find that Treg depletion severely reduces the Tfh cell response to influenza virus. Furthermore, Treg depletion prevents the accumulation of influenza-specific GCs. These effects are not due to alterations in TGFβ availability or a precursor-progeny relationship between Tregs and Tfh cells, but are instead mediated by increased availability of IL-2, which suppresses the differentiation of Tfh cells and as a consequence, compromises the GC B response. Thus, Tregs promote influenza-specific GC responses by preventing excessive IL-2 signalling, which suppresses Tfh cell differentiation.

Figure 1. Treg depletion compromises GC B cell responses to influenza

(a-f) B6 and FoxP3-DTR mice were infected with PR8, administered DT on days 0, 4 and 7 after infection and cells from the mLN were analyzed on day 10. The frequency (a) and number (b) of CD4⁺ T cells that expressed FoxP3 were determined by flow cytometry. The frequency (c) and number (d) of CD19⁺CD138^-PNA⁺CD38^lo GC B cells was determined by flow cytometry. The frequency of NP-specific CD19⁺ B cells (e, top row) and the frequency of NP-specific CD19⁺PNA⁺CD38^lo GC B cells (e, bottom row) were determined by flow cytometry. (f) The number of NP-specific CD19⁺PNA⁺CD38^lo GC B cells was calculated. Data in all panels are representative of five independent experiments (mean ± s.d of 5 mice). P values were determined using a two-tailed Student's t-test.

Figure 2. Influenza-specific Tfh cells are found in mLN but not the lung

(a-d) B6 mice were infected with PR8 and cells were analyzed by flow cytometry on day 10. (a) The frequency of PD-1^hiCXCR5^hi Tfh cells and PD-1^loCXCR5^lo Teff cells in the NP-specific CD4⁺ T cells from the mLN. (b) Bcl6 expression on PD-1^hiCXCR5^hi and PD-1^loCXCR5^lo NP-specific CD4⁺ T cells. (c) The frequency of PD-1^loCXCR5^lo Teff cells in the NP-specific CD4⁺ T cells from the lung. (d) Bcl6 expression on NP-specific CD4⁺ T cells obtained from the lungs and mLNs. (e) B6 mice were infected with PR8 and the number of NP-specific CD4⁺Bcl6^hiPD-1^hiCXCR5^hi cells in the mLN was calculated at the indicated times. Data are shown as the mean ± SD (n=4-5 mice/point). Data are representative of three independent experiments.

Figure 3. Treg depletion compromises influenza-specific Tfh cell responses

(a-e) B6 and FoxP3-DTR mice were infected with PR8, administered DT on days 0, 4 and 7 after infection and the frequency (a) and number (b) of NP-specific CD4⁺ T cells in the mLN was determined by flow cytometry. The frequency of PD-1^hiCXCR5^hi cells (c, top row) and the frequency of Bcl6^hi cells (c, bottom row) in the NP-specific CD4⁺ T cell population in the mLN was determined by flow cytometry and the numbers of NP- specific CD4⁺Bcl6^hiPD-1^hiCXCR5^hi cells (d) and NP-specific CD4⁺Bcl6^loPD-1^loCXCR5^lo cells (e) in the mLN were calculated. (f-g) B6 and FoxP3-DTR mice were infected with PR8, administered DT on days 0, 4 and 7 after infection and the frequency (f) and number (g) of NP-specific CD4⁺ T cells in the lungs on day 10 were determined by flow cytometry. Data are representative of five independent experiments (mean ± s.d of 5 mice per group). P values were determined using a two-tailed Student's t-test. (h-j) B6 and FoxP3-DTR mice were infected with PR8, administered DT on days 6 and 10 and the expression of Bcl6 in NP-specific CD4⁺ T cells (h), the number of NP-specific CD4⁺Bcl6^hiPD-1^hiCXCR5^hi cells (i) and the number of NP-specific CD4⁺Bcl6^loPD-1^loCXCR5^lo cells (j) was determined in the mLN on day 12. Data are representative of two independent experiments (mean ± s.d of 4-5 mice per group). P values were determined using a two-tailed Student's t-test.

Figure 4. Tfh cells do not originate from Foxp3-expressing precursors

(a) B6 mice were infected with PR8 and the expression of FoxP3 on total CD4⁺ T cells NP-specific CD4⁺PD-1^hiCXCR5^hi and NP-specific CD4⁺PD-1^loCXCR5^lo cells from the mLN were analyzed by flow cytometry on day 10. (b-d) B6 mice were irradiated and reconstituted with a 50:50 mix of BM from B6 (CD45.1) and FoxP3-DTR donors (CD45.2). Reconstituted mice were infected with PR8, administered DT on days 0, 4 and 7 after infection the frequency of CD4⁺FoxP3⁺ cells (b), the frequency of NP-specific CD4⁺PD-1^hiCXCR5^hi cells (c) and the frequency of CD4⁺PD-1^hiCXCR5^hi cells (d) from CD45.1 or CD45.2 donors was determined by flow cytometry. Data are representative of three independent experiments (mean ± s.d of 4-5 mice per group). (e-f) B6 mice were infected with PR8 and the frequency of PD-1hiCXCR5hi cells in CD4⁺ FoxP3^- or CD4⁺ FoxP3⁺ T cells (mean ± s.d of 5 mice per group) (e) and the expression of Bcl6 in NP-specific CD4⁺ cells and CD4⁺FoxP3⁺ cells (f) were determined by flow cyometry on day 10. All data were obtained after gatingon CD19- cells. Data are representative of five independent experiments

Figure 5. TGF-β blockade does not affect influenza-specific Tfh or GC B cell responses

(a-g) B6 mice were infected with PR8 and treated with 250 μg of anti-TGF-β or isotype- matched control antibody on days 0 and 5 and the frequency (a) and number (b) of NP- specific CD4⁺ T cells, the expression of Bcl6 on NP-specific CD4⁺ T cells (c), the frequency (d) and number (e) of NP-specific CD4⁺PD-1^hiCXCR5^hi cells was determined by flow cytometry on day 10 in the mLNs. (f-g) B6 mice were infected with PR8 and treated on days 0 and 5 with 250 μg of anti-TGF-β or control antibody and the number of CD19⁺PNA⁺CD38^loCD138^- GC B cells (f) and the number of NP-specific CD19⁺PNA⁺CD38^lo GC B cells (g) in the mLNs was determined by flow cytometry on day 10. Data are representative of three independent experiments (mean ± s.d of 4-5 mice per group).

Figure 6. Increased IL-2 production and CD25 expression in Treg depleted mice

(a-b) B6 and FoxP3-DTR mice were infected with PR8, administered DT on days 0, 4 and 7 and, on day 10, cells from the mLN were re-stimulated in vitro and the frequencies of IFNγ-producing CD4⁺ cells (a) and IL-2-producing CD4⁺ cells (b) were determined by intracellular staining. (c) B6 mice were infected with PR8 and the frequency of NP- specific CD4⁺CD25⁺ T cells was determined by flow cytometry in the mLN on days 7 and 10 after infection. (d-e) B6 and FoxP3-DTR mice were infected with PR8, administered DT on days 0, 4 and 7 and the frequency of NP-specific CD4⁺CD25⁺ cells (d) and the frequency of CD4⁺CD25⁺FoxP3^- cells (e) was determined by flow cytometry in the mLN on 10 after infection. Data are representative of three independent experiments (mean ± s.d of 4-5 mice per group). P values were determined using a two- tailed Student's t-test.

Figure 7. Excessive IL-2 signaling after Treg depletion inhibits Tfh cell responses

(a-c) Irradiated TCRβδ^-/- mice were reconstituted with a 50:50 mix of BM from Cd25^-/- donors (CD45.2) and FoxP3-DTR donors (CD45.1). Reconstituted mice were infected with PR8 and the percentages of CD45.1⁺ and CD45.1^- cells within the CD4⁺FoxP3⁺ cells (b) and the CD4⁺FoxP3^- T cells (c) determined by flow cytometry on day 10. (d-l) Irradiated Tcrbd^-/- mice were reconstituted with a 50:50 mix of BM from Cd25^-/- donors (CD45.2) and FoxP3-DTR donors (CD45.1), infected with PR8, treated with either PBS or DT and the frequency (d) and number (e) of CD4⁺FoxP3⁺ cells derived from either CD45.1 or CD45.2 donors was determined by flow cytometry on day 10. The frequency of CD4⁺CD25⁺ in NP-specific cells (f) derived from either CD45.1 or CD45.2 donors was determined by flow cytometry on day 10. The number of total NP-specific CD4⁺Bcl6^hiPD-1^hiCXCR5^hi cells (g) was calculated on day 10. The frequency of CD4⁺Bcl6^hi in NP-specific cells (h) derived from either the CD45.1 or CD45.2 donors was determined on day 10. The number of NP-specific CD4⁺Bcl6^hiPD-1^hiCXCR5^hi cells (i) derived from either the CD45.1 or CD45.2 donors was determined by flow cytometry. The frequency (j) and number (k) of NP-specific CD19⁺FAS⁺PNA⁺ GC B cells was determined on day 10. The frequency of NP-specific B cells derived from either the CD45.1 or CD45.2 donors (l) was determined by flow cytometry on day 10. Data are representative of four independent experiments (mean ± s.d of 5-7 mice per group). P values were determined using a two-tailed Student's t-test.