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

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.

A role for cell-autocrine interleukin-2 in regulatory T-cell homeostasis.

Activated T-cells make both interleukin-2 (IL2) and its high-affinity receptor component CD25. Regulatory CD4 T-cells (Treg cells) do not make IL2, and the IL2-CD25 circuit is considered a paracrine circuit crucial in their generation and maintenance. Yet, all T-cells are capable of making IL2 at some stage during differentiation, making a cell-intrinsic autocrine circuit additionally possible. When we re-visited experiments with mixed bone marrow chimeras using a wide range of ratios of wild-type (WT) and IL2-/- genotype progenitors, we found that, as expected, thymic Treg cells were almost equivalent between WT and IL2-/- genotypes at ratios with WT prominence. However, at WT-limiting ratios, the IL2-/- genotype showed lower thymic Treg frequencies, indicating a role for cell-intrinsic autocrine IL2 in thymic Treg generation under IL2-limiting conditions. Further, peripheral IL2-/- naive CD4 T-cells showed poor conversion to inducible Tregs (pTregs) both in vivo and in vitro, again indicating a significant role for cell-intrinsic autocrine IL2 in their generation. Peripherally, the IL2-/- genotype was less prominent at all WT:IL2-/- ratios among both thymic Tregs (tTregs) and pTregs, adoptively transferred IL2-/- Tregs showed poorer survival than WT Tregs did, and RNA-seq analysis of WT and IL2-/- Tregs showed interesting differences in the T-cell receptor and transforming growth factor-beta-bone morphogenetic protein-JNK pathways between them, suggesting a non-titrating role for cell-intrinsic autocrine IL2 in Treg programming. These data indicate that cell-intrinsic autocrine IL2 plays significant roles in Treg generation and maintenance.

Role of NF-kappaB2-p100 in regulatory T cell homeostasis and activation.

The immunological roles of the nuclear factor-kappaB (NF-κB) pathway are mediated via the canonical components in immune responses and via non-canonical components in immune organogenesis and homeostasis, although the two components are capable of crosstalk. Regulatory CD4 T cells (Tregs) are homeostatically functional and represent an interesting potential meeting point of these two NF-κB components. We show that mice deficient in the non-canonical NF-κB component gene Nfkb2 (p100) had normal thymic development and suppressive function of Tregs. However, they had enhanced frequencies of peripheral 'effector-phenotype' Tregs (eTregs). In bi-parental chimeras of wild-type (WT) and Nfkb2-/- mice, the Nfkb2-/- genotype was over-represented in Tregs, with a further increase in the relative prominence of eTregs. Consistent with distinct properties of eTregs, the Nfkb2-/- genotype was more prominent in Tregs in extra-lymphoid tissues such as liver in the bi-parental chimeras. The Nfkb2-/- Tregs also displayed greater survival, activation and proliferation in vivo. These Nfkb2-/- Tregs showed higher nuclear NF-κB activity mainly comprising of RelB-containing dimers, in contrast to the prominence of cRel- and RelA-containing dimers in WT Tregs. Since p100 is an inhibitor of RelB activation as well as a participant as cleaved p52 in RelB nuclear activity, we tested bi-parental chimeras of WT and Relb-/- mice, and found normal frequencies of Relb-/- Tregs and eTregs in these chimeric mice. Our findings confirm and extend recent data, and indicate that p100 normally restrains RelB-mediated Treg activation, and in the absence of p100, p50-RelB dimers can contribute to Treg activation.

Effector/memory CD4 T cells making either Th1 or Th2 cytokines commonly co-express T-bet and GATA-3.

Naïve CD4 T (NCD4T) cells post-activation undergo programming for inducible production of cytokines leading to generation of memory cells with various functions. Based on cytokine based polarization of NCD4T cells in vitro, programming for either 'Th1' (interferon-gamma [IFNg]) or 'Th2' (interleukin [IL]-4/5/13) cytokines is thought to occur via mutually exclusive expression and functioning of T-bet or GATA-3 transcription factors (TFs). However, we show that a high proportion of mouse and human memory-phenotype CD4 T (MCD4T) cells generated in vivo which expressed either Th1 or Th2 cytokines commonly co-expressed T-bet and GATA-3. While T-bet levels did not differ between IFNg-expressing and IL-4/5/13-expressing MCD4T cells, GATA-3 levels were higher in the latter. These observations were also confirmed in MCD4T cells from FVB/NJ or aged C57BL/6 or IFNg-deficient mice. While MCD4T cells from these strains showed greater Th2 commitment than those from young C57BL/6 mice, pattern of co-expression of TF was similar. Effector T cells generated in vivo following immunization also showed TF co-expression in Th1 or Th2 cytokine producing cells. We speculated that the difference in TF expression pattern of MCD4T cells generated in vivo and those generated in cytokine polarized cultures in vitro could be due to relative absence of polarizing conditions during activation in vivo. We tested this by NCD4T cell activation in non-polarizing conditions in vitro. Anti-CD3 and anti-CD28-mediated priming of polyclonal NCD4T cells in vitro without polarizing milieu generated cells that expressed either IFNg or IL-4/5/13 but not both, yet both IFNg- and IL-4/5/13-expressing cells showed upregulation of both TFs. We also tested monoclonal T cell populations activated in non-polarizing conditions. TCR-transgenic NCD4T cells primed in vitro by cognate peptide in non-polarizing conditions which expressed either IFNg or IL-4/5/13 also showed a high proportion of cells co-expressing TFs, and their cytokine commitment varied depending on genetic background or priming conditions, without altering pattern of TF co-expression. Thus, the model of mutually antagonistic differentiation programs driven by mutually exclusively expressed T-bet or GATA-3 does not completely explain natural CD4 T cell priming outcomes.