Blocking Specific CD44 Splicing Variant Isoform Inhibits the Differentiation of Th-2 Cells through Interfering with IL-4/IL-4R Signaling Pathway
Authors: Yang Chun , Lin Jianhong , Liang Hongyan , Kwart Ariel , Jiang Meng , Zhao Jianjun , Jiang Xiaofeng , Munshi Nikhil C. 
Published: October 2019

The polarization of naïve CD4+ T cells may initiate multiple reactions in immune system. The balance between Th1 and Th2 cells is critical for innate and acquired immune reactions. But the exact mechanism of its polarization is still unclear. IL-4 is specifically produced by Th2 cells, and regulates Th2 differentiation. Once IL-4 binds to IL-4 receptor (IL-4R), the Th2 polarization signal is activated by phosphorylation of STAT6 (recruited by IL-4Rα), its relocation to nucleus, activation of STAT6 downstream genes (gata3, il-4 and il-4rα etc) and consequent Th2 polarization. CD44 an important T cell activation and T helper cell differentiation gene participates in the regulation of Th1 and Th2 differentiation. Furthermore, CD44 variant isoforms produced by alternative RNA splicing, have different physiological and pathological functions including tumor metastasis, drug resistance and anti-apoptosis effect in tumor cells. Here we report hitherto unknown specific CD44 variant isoforms involved in T helper cell differentiation and functional regulator of CD4+ T cell polarization.

We developed various PCR primer sets able to distinguish different CD44 isoforms in human and mouse Th2 cells. We found higher expression of CD44 variant 4 (CD44v4) and CD44v5 in both human and mouse Th2 cells compared with Th1 cells, indicating their role in Th2 cell differentiation. In order to investigate the role of CD44v4 and CD44v5 in Th2 cells polarization, we treated human naïve CD4+ T cells with CD44v4 or CD44v5 antibody separately for 3 days in polarizing condition. We observed that CD44v5 antibody treatment dramatically decreased the level of phospho-JAK1 and pSTAT6 compared to control cells treated with same amount of normal mouse IgG. At the same time, the expression of GATA3 detected by western blot and the secretion of IL-4 measured by ELISA decreased. Notably, the phosphorylation of STAT1 in Th1 cells was not inhibited by CD44v5 blocking. There is a significant decrease in GATA3 and IL4 expression with CD44v5 antibody but not in CD44v4 antibody treated group, which indicated that Th2 polarization was mainly influenced by CD44v5. In order to verify our finding, CD44v5 specific siRNA were used and we observed similar result in CD4+ T cells. Interestingly, we found that the degradation of IL-4Rα increased after treatment of Th2 cells with CD44v5 antibody compared with control group. Using confocal microscopy of single cell, we observed that CD44v5 co-localized with IL-4Rα. Importantly, CD44v5 antibody treatment could interrupt the CD44v5 and IL-4Rα interaction and also the co-localization of T cell receptor (TCR). On Th1 cells, we didn't find the co-localization between IFNgR and CD44v5. The IFN-γ secretion in Th1 cells were not influenced by either CD44v5 blocking or CD44v5-deficient CD4+ T cells indicating that CD44v5 only influenced the differentiation of Th2 cell, but not Th1 cells.

In conclusion, CD44v5 plays an important role in naive T cell differentiates into Th2 cells. We hypothesis that CD44v5 can bind to IL-4Rα through CD44v5 variant domain and stabilize the IL-4Rα, blocking CD44v5 induced IL-4R degradation and reduce the Th2 cell differentiation. CD44v5 antibody treatment inhibiting Th2 differentiation without affecting Th1 development provides a potential novel immuno-therapy target.

Disclosures

No relevant conflicts of interest to declare.