PMC

2 × 10⁶ TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO) Stg cells were adoptively transferred into C57BL/6 congenic recipients that were infected with WSN-GP33/66 the following day. On day 4–5 p.i., CD44^hi Stg cells in the MLN were assessed for the indicated proteins (CD25, Ly6C, T-bet, IFN-γ, IL-2, Bcl6) by flow cytometry. Cells were stimulated with PMA and ionomycin for 4 hr to assess IFN-γ and IL-2. Bar graphs are representative of three independent experiments (n = 3–4 mice/group/experiment). *p < 0.05, **p < 0.005, ***p < 0.0005.
DOI: http://dx.doi.org/10.7554/eLife.04851.010

1 × 10⁴ Stg CD4 T cells were adoptively transferred into congenic C57BL/6 recipient mice, which were infected with LCMV Armstrong the following day. Eight days p.i., splenocytes were assessed for intracellular FoxP3 expression and the other indicated proteins. Top row depicts LCMV-specific Stg CD4 T cells, while the bottom row is gated on the host-derived CD4 T cells. Data depict an individual mouse representative of more than three independent experiments with 10⁺ total mice.
DOI: http://dx.doi.org/10.7554/eLife.04851.004

(A) Stg chimeric mice (1 × 10⁶ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO)) were infected with LCMV Armstrong and 3 days p.i., spleens were collected and analyzed by flow cytometry for the indicated proteins. (B–D) Stg chimeric mice (1 × 10⁴ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO)) were infected with LCMV Armstrong and the phenotype of Stg cells was assessed 8 days p.i. Data are representatives of five independent experiments with 15–20 total mice/group. *p < 0.05.
DOI: http://dx.doi.org/10.7554/eLife.04851.006

2 × 10⁵ Stg CD4 T cells were adoptively transferred into congenic C57BL/6 recipient mice, which were infected with influenza WSN-GP33/66 i.n. the following day. 8 days p.i., lymphocytes in the MLN were assessed for the indicated proteins. (A and C) Representative example of Stg cells in the MLN. In the histograms, red lines denote PSGL1^hi Ly6C^hi Stg cells, blue is PSGL1^hi Ly6C^lo Stg cells, and green is gated on PSGL1^lo Ly6C^lo Stg cells. (B) Stg cells from the MLN, spleen, lung, and airways. (C) includes FoxP3⁺ Tregs in the shaded gray histograms. Data depict an individual mouse representative of more than 30 total mice from 10⁺ independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.04851.007

5 × 10⁶ CD44^lo Thy1.2⁺ CD4 T cells from TGF-βRII^f/f Lck-cre⁻ (WT) or TGF-βRII^f/f Lck-cre⁺ (KO) mice were adoptively transferred into congenic Thy1.1⁺ OT-II TCR transgenic mice and infected with WSN-GP33/66 the following day. 14 days p.i., host OT-II (Thy1.1⁺) and donor (Thy1.2⁺) CD4 T cells in the MLN (A) and spleen (B) were assessed for expression of CD44 (to distinguish activated T cells, see histogram plots left) and CXCR5, PSGL1, and Ly6C to distinguish Tfh and Th1 attributes. FACS plots are from representative mice and bar graphs are representative of one of four independent experiments (n = 4–5 mice/group/ experiment). *p < 0.05 and colored asterisks correspond to the color in the stacked graphs.
DOI: http://dx.doi.org/10.7554/eLife.04851.008

This is a great point raised by the reviewers. Because our in vivo analysis of TGF-βR KO CD4 T cells showed heightened CD25, pSTAT5, and pS6 (new addition to ), we conclude that TGF-β is directly modulating CD25 expression on virus-specific CD4 T cells during infection in vivo. However, in vitro, it is possible that TGF-β was acting on APCs or other cells in the culture to regulate CD25 on the activated CD4 T cells; two situations that are also not mutually exclusive. To this end, we performed the experiment suggested by the reviewers and first purified the CD4 T cells prior to stimulation with anti-CD3+ anti-CD28^+/- TGF-β for 2-3 days. In agreement with our in vivo findings, this experiment showed that TGF-β could suppress CD25 and IL-2 responsiveness (i.e., pSTAT5) over time (). Thus, we believe that TGF-β is acting directly on the CD4 T cells in vitro to modulate CD25 expression.

2 × 10⁵ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO) Stg cells were adoptively transferred into C57BL/6 congenic recipients infected with WSN-GP33/66 the following day. (A–D) On day 8 p.i, Stg cells in the MLN, spleen, and lung were stained with antibodies against the indicated proteins to distinguish Th1 and Tfh cells. Cells were also stimulated with GP₆₆-peptide for 6 hr to assess IFN-γ and IL-2 production by intracellular cytokine staining and flow cytometry. (E) Stg cells (blue, highlighted by white arrows) located within MLN PNA⁺ GCs (green) were assessed using immunofluorescent microscopy and their numbers were enumerated using Imaris software. Graphs in A–D are representative of one of five independent experiments (n = 4–5 mice/group/experiment). Panel E shows representative microscopy images and the cumulative data from two independent experiments with 8 total mice/group were graphed. *p < 0.05, **p < 0.005, and colored asterisks correspond to the color in the stacked graphs.
DOI: http://dx.doi.org/10.7554/eLife.04851.005

(A) Stg CD4 T cells were labelled with Cell Trace dye and cultured in vitro with 0.1 μM GP₆₆ peptide ± 10 ng/ml TGF-β and stained for surface expression of CD25 (top) or restimulated with IL-2 to assess pSTAT5 (bottom). Data are representative of four independent experiments. (B) Stg chimeric mice (1 × 10⁶ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO)) were infected with LCMV Armstrong and 3 days p.i., spleens were fixed immediately in 2% PFA and stained with antibodies to measure surface expression of CD25, Ly6C, and intracellular phosphorylation of STAT5 (pSTAT5) and pS6 directly ex vivo. Data are representative of three independent experiments including 3–5 total mice/group. (C–D) 2 × 10⁵ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO) Stg cells were adoptively transferred into congenic C57BL/6 recipients infected with WSN-GP33/66 the following day. Mice were treated with PBS or 75 mg/kg rapamycin i.p. daily. On day 8 p.i, Stg cells in the MLN were assessed for expression of PSGL1 and Ly6C (C) or T-bet or GzmB (D). Data are representative of three independent experiments encompassing a total of 9–15 mice/group. *p < 0.05, **p < 0.005 and colored asterisks correspond to the color in the stacked graphs.
DOI: http://dx.doi.org/10.7554/eLife.04851.011

(A) Bar graph shows a selected set of genes upregulated in d8 LCMV-specific Stg PSGL1^lo Ly6C^lo Tfh cells relative to PSGL1^hi Ly6C^hi Th1 cells isolated and sorted from the spleen as measured using Illumina DNA microarrays () that have been described to be induced by TGF-β or associated with Treg cells (). (B) Representative histogram plot (top) shows amount of intracellular FoxP3 in total host splenic CD4 T cells (shaded gray) and LCMV-specific Th1 (hatched line) and Tfh (black line) Stg CD4 T cells from the spleen at day 8 p.i. Region gated identifies FoxP3⁺ nTregs. Bar graphs (bottom) depict the cumulative frequency (left) of FoxP3⁺ CD4 T cells or gMFI averages (right) of the indicated CD4 T cell populations. (C) Expression of the indicated Treg-associated proteins in (A) was compared between LCMV-specific Th1 (hatched line) and Tfh cells (black line), and FoxP3⁺ Treg cells gated on total host CD4 T cells (shaded gray) from the spleen at day 8 p.i. Histogram plots (top) are representative examples of individual mice and bar graphs (bottom) depict the gMFI averages of each protein in the indicated CD4 T cell populations. Graphs in B and C are representative of one of five independent experiments (n = 4–5 mice/group/experiment). *p < 0.05, ***p < 0.0005.
DOI: http://dx.doi.org/10.7554/eLife.04851.003

5 × 10⁶ CD44^lo Thy1.2⁺ CD4 T cells from TGF-βRII^f/f Lck-cre⁻ (WT) or TGF-βRII^f/f Lck-cre⁺ (KO) mice were adoptively transferred into congenic Thy1.1⁺ OT-II TCR transgenic mice and infected with WSN-GP33/66 the following day. 14 days p.i., GC B cells (Fas⁺, GL7⁺ IgM⁻) (A) and IgG1⁺ GC B cells (B) in the MLN and spleen (SPL) were assessed by flow cytometry (left plots) and enumerated in bar graphs (right). (C) Splenic PNA⁺ GCs (green, highlighted by red ellipses) were assessed by immunofluorescent microscopy of frozen sections and the numbers of GC/tissue section was calculated using Imaris software. (D–E) Influenza-specific IgG and IgA were measured from bronchoaviolar lavage fluid (BAL) by ELISA at day 10 p.i. (D) or longitudinally at the indicated time points (E). Data in panels A–B are representative of four independent experiments (n = 3–5 mice/group/experiment). The bar graphs in panels C–D show cumulative data from two independent experiments (n = 3–5 mice/group/experiment), the images in panel C are from representative mice of these cohorts. *p < 0.05, **p < 0.005, ***p < 0.0005.
DOI: http://dx.doi.org/10.7554/eLife.04851.009

We’ve also thought about this issue at length and cannot fully rationalize how TGF-β can sustain (i.e., not suppress) CD25 in one setting, but restrict its expression in another. The most likely explanation is that it depends on the different combinations and doses of cytokines involved in specifying CD4 T cell fates. For example, it is known that T cell differentiation is highly sensitive to TGF-β concentrations and that different types of T cells form according to the concentration of TGF-β (e.g., Tregs vs T_H17 cells, and Tfh cells) (Zhou, Nature 2008: Schmitt, Nat Immunol, 2014). Additionally, distinct signaling cascades downstream of TGF-βR (e.g., Smad vs. MAPK) may differ based on cell type or TGF-β concentrations (Park, Mol. Immunol., 2007; Giroux, Blood, 2011; Gu, PNAS 2012, among others), which could impact the expression of TGF-β target genes. Furthermore, the specific activity the commercially available recombinant TGF-β is likely to differ considerably between vendors and/or lots, and therefore it is difficult to directly compare one study to another simply based on amount of TGF-β used. Finally, rTGF-β requires activation (cleavage) in a 10mM citric acid solution and the efficiency of activation can be variable from lot-to-lot, not to mention lab-to-lab. For instance, the concentration of TGF-β (10ng/ml) used in our studies was insufficient on its own to maintain FoxP3 protein expression in the absence of exogenous IL-2 in vitro (), though it has been demonstrated to do so in other studies (Fantini, Nat Protocols, 2007).

We thank the reviewers for allowing us to address this point. We designed the experiments using OT-II recipients to specifically ask whether T cell-directed TGF-β was required for GC reactions and influenza-specific antibody, but we understand the desire to also assess the pathological outcomes of infection. We felt the experimental approach, while being suitable for comparing the ability of WT and Tgf-βRII KO CD4 T cells to help WT B cells, may present an unphysiological setting for comparing more general aspects of anti-influenza immunity, such as viral control. However, it is still worth examining if a general reduction overall in anti-influenza antibodies may affect the rates of viral clearance. Because our previous experiments went out to day 14 to measure influenza antibodies (and would likely be devoid of virus as influenza is typically cleared within 10 days), we repeated the experiment to examine viral titers at an earlier time point, day 6 p.i. At this relatively early time point, we found that the OT-II mice engrafted with WT T cells had lower amounts (∼10 fold) of influenza viral RNA in their lungs (). As this experiment was only done once, we hesitate to make any strong conclusions, and quite honestly, we were surprised to see any effects at all at day 6 because this is relatively early to see the effects of a primary adaptive immune response on control of an influenza A infection (Kamperschroer, J Immunol, 2006; Mozdzanowska, J Immunol, 2000). Nonetheless, this result supports the model that the Tgf-βRII KO cells are less efficient at eliciting anti-viral Ab responses and this correlates with delayed viral control.