Induction of IgG2a Class Switching in B Cells by IL-27

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b−/− T cells transferred to Rag1−/− recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b−/− B cells. In vitro, STAT5a/b−/− T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b−/− T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b−/− T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-γ mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-γ-producing T cells in both STAT5a/b+/+ and STAT5a/b−/− splenic populations. Sera from STAT5a/b+/+ and STAT5a/b−/− rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b−/− recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b−/− T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.

Section: Discussionsupporting

confidence: 55%

Allograft Rejection Requires STAT5a/b-Regulated Antiapoptotic Activity in T Cells but Not B Cells

Zhang

Kirken

Furian

et al. 2006

“…Purified mouse IL-27 was prepared as a FLAG-tagged single chain protein by flexibly linking EBV-induced gene 3 to p28 using HEK293-F cells (Invitrogen Life Technologies) as described before (26). Mouse IL-6R and IL-6 cDNAs were isolated by RT-PCR using total RNA prepared from liver and Con A-activated spleen cells, respectively.…”

Section: Preparation Of Purified Il-27 and Soluble Il-6r/il-6 Proteinsmentioning

confidence: 99%

“…A single-chain soluble (s)IL-6R/IL-6 expression vector, in which the extracellular domain of IL-6R is followed by the (Gly 4 Ser) 3 linker (27) and by IL-6, was then generated by standard PCR methods and cloned into a p3xFLAG-CMV-9 vector (Sigma-Aldrich), which has preprotrypsin signal peptide and 3xFLAG-epitope-tag sequences at the N-terminal. Purified mouse soluble IL-6R (sIL-6R)/IL-6 was prepared using HEK293-F cells similarly as described before (26).…”

Section: Preparation Of Purified Il-27 and Soluble Il-6r/il-6 Proteinsmentioning

confidence: 99%

STAT3 Is Indispensable to IL-27-Mediated Cell Proliferation but Not to IL-27-Induced Th1 Differentiation and Suppression of Proinflammatory Cytokine Production

Owaki

Asakawa

Morishima

et al. 2008

Self Cite

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rβ2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.

“…rIL-27 was prepared as a soluble tagged fusion protein by flexibly linking EBV-induced gene 3 to p28 using HEK293-F cells, as described before (16).…”

Section: Preparation Of Purified Ril-27 Proteinmentioning

confidence: 99%

Augmentation of Effector CD8+ T Cell Generation with Enhanced Granzyme B Expression by IL-27

Morishima

Owaki

Asakawa

et al. 2005

Self Cite

170

132

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rβ2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-γ production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-γ production with IL-12 was diminished with decreased expression of T-bet, IL-12Rβ2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-γ production with IL-12 was diminished with decreased expression of IL-12Rβ2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.