Upregulated Expression of the IL-9 Receptor on TRAF3-Deficient B Lymphocytes Confers Ig Isotype Switching Responsiveness to IL-9 in the Presence of Antigen Receptor Engagement and IL-4

Abstract

The pleiotropic cytokine IL-9 signals to target cells by binding to a heterodimeric receptor consisting of the unique subunit IL-9R and the common subunit γ-chain shared by multiple cytokines of the γ-chain family. In the current study, we found that the expression of IL-9R was strikingly upregulated in mouse naive follicular B cells genetically deficient in TNFR-associated factor 3 (TRAF3), a critical regulator of B cell survival and function. The highly upregulated IL-9R on Traf3−/− follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3−/− B cells, which was not observed in littermate control B cells. We further demonstrated that blocking the JAK-STAT3 signaling pathway abrogated the enhancing effect of IL-9 on class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3−/− B cells. Our study thus revealed, to our knowledge, a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Taken together, our findings provide (to our knowledge) new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation such as autoimmune disorders.

The adaptor protein TRAF3 is an immune checkpoint that inhibits myeloid-derived suppressor cell expansion

“…Single cell suspensions were made from the spleen, draining lymph node (DLN), BM, peripheral blood cells (PBL), and tumor. Immunofluorescence staining and FACS analyses were performed as previously described ( 14 , 16 , 21 ). Erythrocytes from the spleen were depleted with ACK lysis buffer.…”

“…FACS data were acquired on a Northern Lights spectral flow cytometer (Cytek, Fremont, CA) or a FACSCalibur (Becton Dickinson, Mountain View, CA). The results were analyzed using the FlowJo software (TreeStar, San Carlos, CA) ( 21 ).…”

“…Each reaction also included the primers and probe (VIC-labeled) specific for mouse Actb , which served as an endogenous control. Reactions were performed on a QuantStudio™ 3 Real-Time PCR System (96-well, 0.1ml Block, Applied Biosystems) ( 21 ). Real-time PCR data were analyzed using the QuantStudio™ Design and Analysis Software v1.5.2 (Applied Biosystems) ( 21 ).…”

“…Reactions were performed on a QuantStudio™ 3 Real-Time PCR System (96-well, 0.1ml Block, Applied Biosystems) ( 21 ). Real-time PCR data were analyzed using the QuantStudio™ Design and Analysis Software v1.5.2 (Applied Biosystems) ( 21 ). Relative mRNA expression levels of each gene were calculated using the comparative Ct method (ΔΔCt) as described ( 27 ).…”

“…Being a member of the TRAF family of adaptor proteins and E3 ubiquitin ligases, TRAF3 is utilized by a variety of immune receptors for signaling, including the TNF-R superfamily, pattern recognition receptors (PRRs), and cytokine receptors, among others ( 11 – 13 ). TRAF3 has been recognized as a pivotal regulator of the adaptive immune system, including B cell survival and homeostasis ( 14 – 19 ), antibody class switching and B cell anergy ( 20 , 21 ), T cell activation and memory responses ( 22 , 23 ), and regulatory T cell (Treg) development and effector functions ( 24 , 25 ). In particular, increasing evidence indicates that TRAF3 is a tumor suppressor in B lymphocytes and that its absence in B cells is frequently associated with the development of B cell neoplasms, including non-Hodgkin lymphoma and multiple myeloma ( 11 , 13 , 26 ).…”