These findings are in keeping with previous observations by others that CD73 is not expressed by splenic myeloid cells  and that CD73 mRNA is inducible in neutrophils with LPS stimulation . Loss of BM PCs is dependent on CD73 expression by BM-derived cells CD73 is expressed on stromal cells, such as endothelia , , as well as hematopoietically-derived cells. NP-CGG in alum i.p. Cells were stained with reagents to identify expression of TCRbeta, CD4, CD44, PD1, and ICOS as detailed in Material and Methods.(TIF) pone.0092009.s001.tif (558K) GUID:?B75DAD05-2ABB-4A20-89AD-842AF995FB0A Physique S2: CD73 is not expressed by BM eosinophils or basophils. BM cells from WT mice immunized with NP-CGG in alum i.p. 28-days previously were stained and analyzed by circulation cytometry. Representative FACS histograms are shown. Live, single cells were first gated by EMA exclusion. (A) Basophil profiles. Basophils were recognized by high surface expression of Siglec-F and F4/80 and intermediate expression of CD11b. Shown are CD73 (heavy collection) and isotype control (heavy shading) stained basophils. (B) Eosinophil profiles. Eosinophils were recognized by high surface expression of CD49b and IgE. Shown are CD73 (heavy collection) and isotype control (heavy shading) stained eosinophils.(TIF) pone.0092009.s002.tif (313K) GUID:?1990D585-6CDF-487F-9B0B-4A58ECDFE9B9 Figure S3: Splenic myeloid compartments are relatively unaffected by the absence of CD73. At the indicated days pre or post i.p. immunization with NP-CGG in alum, spleens from B6 WT and CD73KO control mice were stained and analyzed by circulation cytometry. (A) CD73 expression around the indicated cell types from unimmunized spleens of WT (solid collection) and CD73KO (shaded gray) mice. (B) Complete figures cDCs, pDCs, neutrophils and macrophages per spleen. Macrophages were identified as Gr1int/low F4/80+ CD11b+ CD19?, cDCs as CD11c+ IA/IE+ CD19?, pDCs as SiglecH+ CD317(BST2)+ CD19? and neutrophils as CD11b+ Ly6g+ CD19? live cells. Each point represents the average of 5C10 individual spleens. Error bars depict standard deviations. * and ** indicate Student’s (39). IL-21 and beta-actin products were amplified from Larotaxel SPRY1 identical cDNA cell equivalents. Shown is relative amplification of IL-21 cDNA normalized to beta-Actin expression, expressed as beta-Actin threshold cycle (Ct) minus Larotaxel IL-21 Ct (Student’s t-test p?=?0.9236). Shown is one of two comparable experimental replicates with 4C5 individual mice per group. (B) For circulation cytometric analysis of IL-21 protein expression, splenocytes were stimulated in vitro for 5 hours with phorbol-12-myristate-13-acetate (PMA; 20 Larotaxel ng/ml; EMD Millipore, Billerica, MA) and ionomycin (750 ng/mL; EMD Millipore, Billerica, MA). After 1-hour, transport out the endoplasmic reticulum was inhibited by the addition of Brefeldin A (Biolegend, San Diego, CA), per the manufacture’s instructions. Post activation, splenocytes were stained for surface markers, permeabilized with Perm/Wash Buffer (BD Biosciences), incubated with 10% goat and rat serum followed with recombinant Mouse IL-21R Fc Chimera (R&D Systems, Minneapolis, MN) and finally PE goat-F(ab)2 -anti-human IgG-Fc (Jackson ImmunoResearch, West Grove, PA). TFH cells were gated as Larotaxel EMA?TCRbeta+ CD4+ CD44+ PD1+ ICOS+. Shown are the percent of TFH cells that express IL-21 protein among stimulated, unstimulated and secondary staining-only control mice (10, 2 and 10 replicates per group, respectively). Student’s t-test of CD73KO and WT stimulated samples yielded p-value of 0.7971. (C) Median fluorescence intensity (MFI) of IL-21 expression among IL21+ TFH cells, recognized in (B). Student’s t-test p-value of 0.4150.(TIF) pone.0092009.s007.tif (118K) GUID:?5CAB3AEA-F5F5-4519-879A-DE5F25E5DA84 Abstract CD73 catalyzes the conversion of extracellular nucleosides to adenosine, modulating inflammatory and T cell responses. Elevated expression of CD73 marks subpopulations of murine memory B cells (MBC), but its role in memory development or function is usually unknown. Here, we demonstrate that CD73 is progressively upregulated on germinal center (GC) B cells following immunization, is usually expressed at even higher levels among T follicular helper cells, but is usually absent among plasma cells (PC) and plasmablasts (PB). We analyzed the T-dependent B cell response in CD73 knockout mice (CD73KO). During the early response, CD73KO and wild type (WT) mice created GCs, MBCs and splenic PBs and PCs similarly, and MBCs functioned similarly in the early secondary response. Late in the primary response, however, bone marrow (BM) PCs were markedly decreased in CD73KO animals. Tracking this phenotype, we found that CD73 expression was required on BM-derived cells for optimal BM PC responses. However, deletion of CD73 from either B or T lymphocytes alone did not recapitulate the phenotype. This suggests that CD73 expression is sufficient on either cell type, consistent with its function as an ectoenzyme. Together, these findings suggest that CD73-dependent adenosine signaling is usually prominent in the mature GC and required for establishment of the long-lived PC compartment, Larotaxel thus identifying a novel role for CD73 in humoral immunity. Introduction CD73, ecto-5 – nucleotidase, is usually a glycosylphosphatidylinositol-linked surface glycoprotein that plays a rate-limiting role regulating extracellular ATP and adenosine levels C. ATP is usually released into the extracellular space after tissue injury or inflammation and functions as a danger transmission. CD39.
These findings are in keeping with previous observations by others that CD73 is not expressed by splenic myeloid cells  and that CD73 mRNA is inducible in neutrophils with LPS stimulation