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

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