Although IL-21R- and IL-21-deficiency each prevent mortality in BXSB.Yaa mice [31], a detailed description of BXSB.Yaa.IL21–/– mice has not been reported. Together, these studies indicate that neither IL-21 overexpression nor expansion of Tfh

or extrafollicular T helper cells can predict a requirement for IL-21 in autoimmune pathology. This suggests that only certain subsets of patients would benefit from therapeutic inhibition Ixazomib cost of IL-21. In contrast, IL-6, which acts upstream of and more broadly than IL-21, may be a more widely effective target [11-13]. lyn–/– [6], lyn–/–Btklo [61, 40], and lyn–/–IL-6–/– [11] mice were described previously. All mice used in lyn–/–Btklo and lyn–/–IL-6–/– studies were backcrossed onto the C57BL/6 background. IL-21–/– (B6.129S-Il21tm1Lex/Mmcd) mice were obtained from the Mutant Mouse Regional Resource Center and crossed with lyn–/– mice to generate lyn–/–IL-21–/– mice. Mice used in the lyn–/–IL-21–/– studies were of mixed C57BL/6 × 129 background; WT and lyn–/– littermates were

used as controls. All animals were housed in a specific pathogen free barrier facility, and all procedures were approved by the UT Southwestern Institutional Animal Care and Use Committee. Single-cell suspensions of spleens or collagenase-digested (30 min at 37°C) kidneys were Fc-blocked with anti-mouse CD16/CD32 prior to incubation with some combination of the following monoclonal antibodies: FITC-conjugated anti-CD21, anti-PD1, anti-CD11b, or anti-CD45; PE-conjugated GSI-IX supplier anti-CD23, anti-ICOS, anti-PSGL-1, anti-CD8, or anti-CD11b; PerCP-conjugated eltoprazine anti-B220 or anti-CD4; and biotin-conjugated anti-CD138, anti-CXCR5, anti-CD11c, or anti-CD69. Biotinylated antibodies were detected with strepavidin-allophycocyanin. Intracellular cytokine staining as described in [62] was adapted for murine cells. Briefly, splenocytes were resuspended at 106/mL and stimulated for 5 h with PMA and ionomycin. Cytokine secretion was blocked by incubating with brefeldin

A. Cells were stained extracellularly with PacBlue-conjugated anti-CD4. Permeabilization and fixation were performed using a Foxp3 Staining Kit (Miltenyi Biotec) per the manufacturer’s instructions. Cells were then washed and incubated with anti-mouse cytokine antibodies: PE-Cy7-conjugated anti-IFN-γ, PE-conjugated anti-IL4, and allophycocyanin-conjugated anti-IL17. All antibodies were from BD Biosciences. Samples were acquired on a FACSCalibur or LSRII cytometer and analyzed using CellQuest (all BD Biosciences) or FlowJo (Tree Star) software. Total Ig and autoantibody ELISAs were performed as in [11] and [40] with the following modifications. ssDNA was prepared by boiling dsDNA and promptly chilling on ice. For experiments with dsDNA plus histones, dsDNA-coated plates were subsequently incubated with total histones (Roche) in 0.06 M HCO3−. Autoantibodies were measured on an autoantigen proteomic array as in [43].