Febrile temperature change modulates CD4 T cell differentiation via a TRPV channel-regulated Notch-dependent pathway

Abstract

Fever is a conserved and prominent response to infection. Yet, the issue of how CD4 T cell responses are modulated if they occur at fever temperatures remains poorly addressed. We have examined the priming of naive CD4 T cells in vitro at fever temperatures, and we report notable fever-mediated modulation of their cytokine commitment. When naive CD4 T cells were primed by plate-bound anti-CD3 and anti-CD28 monoclonal antibodies at moderate fever temperature (39 °C), they enhanced commitment to IL4/5/13 (Th2) and away from IFNg (Th1). This was accompanied by up-regulation of the Th2-relevant transcription factor GATA3 and reduction in the Th1-relevant transcription factor Tbet. Fever sensing by CD4 T cells involved transient receptor potential vanilloid cation channels (TRPVs) since TRPV1/TRPV4 antagonism blocked the febrile Th2 switch, while TRPV1 agonists mediated a Th2 switch at 37 °C. The febrile Th2 switch was IL4 independent, but a γ-secretase inhibitor abrogated it, and it was not found in Notch1-null CD4 T cells, identifying the Notch pathway as a major mediator. However, when naive CD4 T cells were primed via antigen and dendritic cells (DCs) at fever temperatures, the Th2 switch was abrogated via increased production of IL12 from DCs at fever temperatures. Thus, immune cells directly sense fever temperatures with likely complex physiological consequences.

Keywords: Fever; Notch; TRPV; Th1/Th2.

Fig. 1.

Effect of fever-range temperatures on effector cytokine programming of naive CD4 T cells (A–C). Levels of IL4 (A), IL13 (B), and IFNg (C) in culture supernatants of primed and restimulated NCD4T cells at indicated recall concentrations of anti-CD3. NCD4 cells were primed with anti-CD3+anti-CD28 at indicated temperatures for 3 d, rested at 37 °C for one day, and restimulated at 37 °C for 24 h. Data representative of three independent experiments. Data shown as mean ± SE unstimulated (unst): cells unstimulated during priming culture and recall. (D) Viable cell proportions after 3 d of activation at temperatures shown. Data representative of three independent experiments. Data shown as mean ± SE. ns, not significant, **P < 0.001, ***P < 0.0001.

Fig. 2.

Alterations in GATA-3 and T-bet expression levels and cytokine levels during NCD4 T cell priming at fever temperature (A and B). MFI levels for GATA3 (A) and T-bet (B) in NCD4 cells primed at indicated temperatures for 3 d, permeabilized and stained. Uns, unstimulated NCD4 cells. Mean ± SE, n = 4. (C and D) Time kinetics of Gata3 (C) and Tbx21 (D) mRNA up-regulation at indicated temperatures in NCD4 T cells stimulated with anti-CD3+anti-CD28. Fold increases over unstimulated NCD4 T cells at 37 °C shown. Mean ± SE, n = 3–5 for different time points. (E–G) Mouse NCD4 cells were primed at 37 °C/39 °C for 1 or 2 d before switching to 39 °C/37 °C, rested for one day, and restimulated. Control groups were primed for 3 d at 37 °C or 39 °C. Culture superntant concentrations of IL13 (E), IL4 (F), and IFNg (G) shown. Mean ± SE, n = 4. **P < 0.001; ***P < 0.0001.

Fig. 3.

TRPV activation phenocopies fever-mediated Th2-skewed programming of responding NCD4 T cells (A–F). Levels of secreted IL13 (A and B), IL4 (C and D), or IFNg (E and F) by NCD4 cells primed at 37 °C or 39 °C in the presence or absence of Caps, panels (A, C, and E) or DkTx (panels [B, D, and F] and recalled with indicated concentrations of anti-CD3. uns, unstimulated NCD4 cells. Mean ± SE, n = 3. (G and H) Fold increase in gata3 mRNA levels induced by fever temperature, Caps, or DkTx in CD4 T cells activated for 6 h with plate-bound anti-CD3+anti-CD28. Data normalized to signals from cells activated at 37 °C. Mean ± SE, n = 3. ***P < 0.0001.

Fig. 4.

TRPV1 and TRPV4 are critical for fever-mediated Th2-skewed reprogramming of responding NCD4 T cells (A). Fold change in Gata3 mRNA levels in the presence or absence of TRPV inhibitors as indicated at 37 °C or 39 °C. NCD4 cells were stimulated for 6 h with plate-bound anti-CD3+anti-CD28. Fold increases in levels above those in cells activated at 37 °C without any TRPV agonists are shown. Unt, untreated with any TRPV agonist. Mean ± SE, n = 3. (B and C) Levels of secreted IL13 (B) and IFNg (C) in 24-h culture supertatants after restimulation of NCD4 cells primed at 37 °C or 39 °C in the presence or absence of TRPV1 or TRPV4 inhibitors (Inhb) for 3 d. Mean ± SE, n = 3. **P < 0.001; ***P < 0.0001.

Fig. 5.

Fever-mediated Th2-skewing of activated CD4 T cell programming is IL4 independent but Notch dependent (A). Up-regulation of Gata3 in wild-type (WT) and IL4^−/− NCD4 cells activated at 37 °C or 39 °C with anti-CD3+anti-CD28 for 6 h. Mean ± SE; n = 3. UNS: unstimulated. (B and C) IL13 (B) and IFNg (C) levels in culture supernatants during recall response of NCD4 T cells from IL4^−/− mice primed at 37 °C or 39 °C. Mean ± SE; n = 3. unst: unstimulated. (D) Change in Gata3, Hes1, and Hey1 mRNA levels of NCD4 T cells activated at 37 °C or 39 °C for 6 h. uns, unstimulated NCD4 T cells. Mean ± SE; n = 3. (E) Change in gata3 and Hes1 mRNA levels of NCD4 T cells activated at 37 °C or 39 °C in the presence or absence of Notch inhibitor DAPT for 6 h. unst, unstimulated NCD4 T cells. Mean ± SE; n = 3. (F and G) IL13 (F) and IFNg (G) levels in culture supernatants during recall response of NCD4 T cells activated at 37 °C or 39 °C in the presence or absence of DAPT. Mean ± SE; n = 3. (H and I). Change in Gata3 (H) and Hes1 (I) mRNA levels of NCD4 T cells from WT and Notch1^−/− (knockout; KO) mice activated at 37 °C or 39 °C in the presence or absence of Caps for 6 h. uns, unstimulated NCD4 T cells. Mean ± SE; n = 3. ns, not significant, *P < 0.05; **P < 0.001; ***P < 0.0001.

Fig. 6.

Fever-mediated Th2-skewed programming of responding CD4 T cells is blocked by fever-mediated IL12 production from APCs (A and B) IL13 (A) and IFNg (B) levels in culture supernatants during recall response of OT-II NCD4 T cells when activated at 37 °C or 39 °C in the presence of BMDCs and Ova2 peptide. Mean ± SE; n = 3. (C) Change in IL12p40 mRNA levels of different cell types either cultured alone or purified from cocultures as indicated after activation at 37 °C or 39 °C for 6 h. mRNA levels for DCs were normalized to unstimulated BMDCs cultured at 37 °C whereas those for T cells were normalized to unstimulated NCD4 T cells. Mean ± SE; n = 3. (D and E) IL13 (D) and IFNg (E) levels in culture supernatants during recall response of OT-II NCD4 T cells when activated at 37 °C or 39 °C with plate-bound anti-CD3+anti-CD28 with or without added rIL12 during priming. Mean ± SE; n = 3. (F and G) IL13 (F) and IFNg (G) levels in culture supernatants during recall response of OT-II NCD4 T cells when activated at 37 °C or 39 °C in the presence of BMDCs and Ova2 peptide with or without anti-IL12 added during priming. Mean ± SE; n = 3. (H) IL12 levels in culture supernatants during recall response of OT-II NCD4 T cells when activated at 37 °C or 39 °C in the presence of either BMDCs or LPS-activated B cells as APCs and Ova2 peptide during priming. Mean ± SE; n = 3. (I and J) IL13 (I) and IFNg (J) levels in culture supernatants during recall response of OT-II NCD4 T cells when activated at 37 °C or 39 °C in the presence of either BMDCs or LPS-activated B cells as APCs and Ova2 peptide during priming cultures. Mean ± SE; n = 3. **P < 0.001; ***P < 0.0001.