TH17 cells require ongoing classic IL-6 receptor signaling to retain transcriptional and functional identity

Acting in concert with TGF-b, IL-6 signaling induces Th17 cell development by programming Th17-related genes via STAT3. A role for IL-6 signaling beyond the inductive phase of Th17 cell development has not been defined, as IL-23 signaling downstream of Th17 cell induction also activates STAT3 and is thought responsible for Th17 cell maintenance. Here, we find that IL-6 signaling is required for both induction and maintenance of Th17 cells; IL-6Ra–deficient Th17 cells rapidly lost their Th17 phenotype and did not cause disease in two models of colitis. Cotransfer of WT Th17 cells with IL-6Ra–deficient Th17 cells induced colitis but was unable to rescue phenotype loss of the latter. High IL-6 in the colon promoted classic, or cis, rather than trans receptor signaling that was required for maintenance of Th17 cells. Thus, ongoing classic IL6 signaling underpins the Th17 program and is required for Th17 cell maintenance and function

IL-21 and IL-6 Are Critical for Different Aspects of B Cell Immunity and Redundantly Induce Optimal Follicular Helper CD4 T Cell (Tfh) Differentiation

Cytokines are important modulators of lymphocytes, and both interleukin-21 (IL-21) and IL-6 have proposed roles in T follicular helper (Tfh) differentiation, and directly act on B cells. Here we investigated the absence of IL-6 alone, IL-21 alone, or the combined lack of IL-6 and IL-21 on Tfh differentiation and the development of B cell immunity in vivo. C57BL/6 or IL-21−/− mice were treated with a neutralizing monoclonal antibody against IL-6 throughout the course of an acute viral infection (lymphocytic choriomeningitis virus, LCMV). The combined absence of IL-6 and IL-21 resulted in reduced Tfh differentiation and reduced Bcl6 protein expression. In addition, we observed that these cytokines had a large impact on antigen-specific B cell responses. IL-6 and IL-21 collaborate in the acute T-dependent antiviral antibody response (90% loss of circulating antiviral IgG in the absence of both cytokines). In contrast, we observed reduced germinal center formation only in the absence of IL-21. Absence of IL-6 had no impact on germinal centers, and combined absence of both IL-21 and IL-6 revealed no synergistic effect on germinal center B cell development. Studying CD4 T cells in vitro, we found that high IL-21 production was not associated with high Bcl6 or CXCR5 expression. TCR stimulation of purified naïve CD4 T cells in the presence of IL-6 also did not result in Tfh differentiation, as determined by Bcl6 or CXCR5 protein expression. Cumulatively, our data indicates that optimal Tfh formation requires IL-21 and IL-6, and that cytokines alone are insufficient to drive Tfh differentiation

In vitro stimulation of purified naïve CD4 T cells with IL-6 or IL-21 drives high IL-21 expression.

<p>(<b>A–D</b>) Naïve sorted CD4 T cells (CD4⁺CD44⁻NK1.1⁻CD25⁻) were stimulated for 3 days on αCD3/αCD28 coated plates in the presence of αIFNγ, αIL-4, and αTGFβ (Th0) ± IL-6 or IL-21. Reagents to block IL-21 (5 µg/ml) or IL-6 (10 µg/ml) signaling were also added at this time where indicated. IL-21R-Fc intracellular staining was used to identify IL-21 producing cells after 4 h of PMA/ionomycin stimulation. (<b>A</b>) Flow cytometry analysis of IL-21 and IFNγ production in day 7 Th0, Th0 + IL-6, and Th0 + IL-6 + αIL6 polarized CD4 T cell cultures. Cells were gated on B220⁻CD44^hi CD4 T cells. *** P<0.0005. (<b>B–D</b>) FACS analysis of IL-21 and IFNγ production in day 3 Th0 + IL-21± αIL-21 or IL-21R-Fc differentiated CD4 T cells. (<b>B</b>) Representative plots (B220⁻CD44^hi CD4 T cells). (<b>C</b>) Quantitation of IL-21 production or (<b>D</b>) IL-21 MFI. ** P<0.003, *** P<0.0003. Data are representative of ≥2 independent experiments for each condition with duplicate samples. MFI, mean fluorescence intensity.</p

IL-6 or IL-21 is required for optimal Tfh differentiation.

<p>(<b>A–G</b>) Splenocytes were analyzed 8 days following LCMV infection in C57BL/6 (B6) or IL-21^−/− mice ± αIL6. (<b>A</b>) Follicular helper T (Tfh) cells (CXCR5⁺SLAM^lo, boxed) gated on activated (CD44^hi) CD4 T cells. (<b>B</b>) Percentage of Tfh differentiation (from gated Tfh population). **, P = 0.0016. (<b>C</b>) Quantitation of ΔCXCR5 mean fluorescence intensity (MFI) gated on Tfh (normalized against CD44^lo CD4 T cells). **, P = 0.003. ***, P<0.0001. (<b>D</b>) Left, quantitation of Bcl6 protein ΔMFI in Tfh (normalized against CD44^lo CD4 T cells). ***, P = 0.0009. Right, histogram overlay shows Bcl6 expression in a representative B6 vs. IL-21^−/− + αIL-6 Tfh population (B6, black; IL-21^−/− + αIL-6, red; B6 stained with isotype mAb, gray). (<b>E</b>) FACS analysis of germinal center CD4 T (GC Tfh) cells (CXCR5⁺GL7⁺, boxed). Gated CD44^hi CD4 T cells shown. (<b>F</b>) GC Tfh quantitation. **, P = 0.003. (<b>G</b>) ΔCXCR5 MFI, gated on GC Tfh and normalized against CD44^lo CD4 T cells. *, P = 0.0405. Flow cytometric analysis of intracellular cytokine staining for IFNγ production by CD4 T cells after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A. (<b>H</b>) Frequency of IFNγ⁺CD44^hi CD4 T cells. Data are representative of ≥2 independent experiments; n = 5–6 mice per group. MFI, mean fluorescence intensity; ns, not significant.</p

Cooperation of IL-6 and IL-21 for plasma cell generation and antibody responses.

<p>C57BL/6 (B6) or IL-21^−/− mice treated with αIL-6 or isotype mAb throughout the acute LCMV infection. (<b>A</b>) Titers of anti-LCMV IgG in the serum of B6 or IL-21^−/− mice ± αIL-6 mAb 8 days following LCMV infection: B6 vs. B6 + αIL-6 (**, P = 0.0016), B6 vs. IL-21^−/− (*, P = 0.025), B6 vs. IL-21^−/− + αIL-6 (**, P = 0.0016), and IL-21^−/− vs. IL-21^−/− + αIL-6 (**, P = 0.0012). (<b>B</b>) Quantitation of plasma cell development by FACS: B6 vs. IL-21^−/− (***, P = 0.0008), B6 vs. IL-21^−/− + αIL-6 (***, P<0.0001), and IL-21^−/− ± anti-IL-6 (*, P = 0.0143). (<b>C</b>) Plasma cell (CD138⁺IgD⁻) development 8 days post-infection, gated on CD19⁺ splenocytes. Data are representative of 3 independent experiments; n = 5–6 mice per group. ns, not significant.</p

High IL-21 production in CD4 T cell cultures does not instruct Tfh differentiation.

<p>Naïve sorted CD4 T cells were stimulated for 72 h on αCD3 plus αCD28 coated plates in the presence of αIFNγ, αIL-4, and αTGFβ (Th0) ± IL-6 or IL-21. (<b>A</b>) CXCR5 mRNA expression normalized to a housekeeping gene, G6PDH, in naïve (CD4⁺CD44^lo), day 3 in vitro polarized CD4 T cell cultures (Th0 or Th0 + IL-6), or OTII CD4 T cells from day 8 NP-OVA plus alum in vivo immunized mice (CD44^hiCXCR5⁺GL7⁻ Tfh or CD44^hiCXCR5⁺GL7⁺ GC Tfh). (<b>B</b>) Quantitation of CXCR5 mean fluorescence intensity (MFI) in day 3 cultures (Th0 or Th0 + IL-6) or CD4 splenocytes (naïve, CD44^lo; CXCR5⁺, CD44^hi), and histogram. (<b>C</b>) CXCR5 analysis in 72 h Th0 (black) or Th0 + IL-21 (red) cultures. Th0 and Th0 + IL-21 cultures stained with isotype mAb (blue). (<b>D–E</b>) Timecourse of Bcl6 (<b>D</b>) and Blimp-1 (<b>E</b>) mRNA expression normalized to a housekeeping gene, G6PDH, in Th0 or Th0 + IL-6 CD4 T cell cultures. (<b>F</b>) FACS analysis of Bcl6 protein expression in germinal center B cells (GL7⁺B220⁺) versus non GC B cells (GL7⁻B220⁺) in splenocytes 8 days following LCMV infection. (<b>G</b>) Bcl6 protein expression in day 3 in vitro differentiated CD4 cultures (Th0 or Th0 + IL-6) versus freshly isolated naïve CD4 splenocytes (CD44^lo). Left, quantitation of Bcl6 MFI. Right, Bcl6 histogram. (<b>H</b>) Bcl6 analysis in 72 h Th0 (black) or Th0 + IL-21 (red) cultures. Naïve splenocytes are stained for comparison (CD44^lo, gray). Data are representative of ≥2 independent experiments with duplicate samples.</p

IL-21 can be produced by Th1, Th2 or Th17 cell in vitro.

<p>(<b>A–D</b>) Magnetic bead purified total CD4 T cells were stimulated with plate-bound αCD3 plus αCD28 without cytokine blocking mAbs (unbiased, U), or in the presence of αIFNγ + αIL-4 (Th0), IL-12 + αIL-4 (Th1), IL-4 + αIFNγ (Th2), or IL-6/TGFβ + αIFNγ & αIL-4 (Th17). Day 7 CD4⁺B220⁻ gated in vitro cultured cells were stained by ICS after 4 h stimulation in PMA/ionomycin. Representative dot plots of IL-21 and the respective cytokine associated with each subset, (<b>A</b>) IFNγ (Th1), (<b>B</b>) IL-4 (Th2), or (<b>C</b>) IL-17 (Th17). (<b>D</b>) IL-21 was measured by ELISA from day 7 cells that had been washed, and then restimulated overnight in the presence of PMA/ionomycin. IL-21 was normalized to cell number per well. (<b>E</b>) Sorted naïve CD4 T cells (CD4⁺CD44⁻NK1.1⁻CD25⁻) were cultured under unbiased (U), Th0 or Th1 conditions. ELISA analysis of IL-21 production from day 7 cultured CD4 T cells restimulated overnight with PMA/ionomycin (normalized to cell number per well). “—”  =  no stim. Data are representative of ≥2 independent experiments.</p

IL-21, but not IL-6, is needed for optimal germinal center B cell development.

<p>(<b>A</b>) Germinal center B cells (GL7⁺Fas⁺ gated, total B cell gate shown) in C57BL/6 (B6) or IL-21^−/− mice treated with anti-IL-6 (αIL6) or isotype mAb. Splenocytes were analyzed 8 days following acute LCMV infection. (<b>B</b>) Frequency of germinal center B cells of total B cells. *, P<0.02. (<b>C</b>) Quantitation of the PNA mean fluorescence intensity (MFI) in germinal center B cells. ***, P = 0.0003. Data are representative of 3 independent experiments; n = 5–6 mice per group. MFI, mean fluorescence intensity; ns, not significant.</p

Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled