Project description:Plasma cell gene expression is driven both by isotype and tissue location. In this series we examine gene expression of bone marrow IgA, IgM and IgG plasma cells as well as IgA plasma cells from small intestine lamina propria. To validate tissue specific gene expression we also include gene expression from lamina propria IgA-/- plasma cells. All plasma cell samples are from Blimp1+/GFP reporter animals and splenic follicular and marginal zone B cell gene expression have been added as reference populations.
Project description:B cell and plasma cell fates are controlled by different transcriptional networks, as exemplified by the mutually exclusive expression and cross-antagonism of the B cell identity factor Pax5 and the plasma cell regulator Blimp1. It has been postulated that the repression of Pax5 by Blimp1 is essential for plasma cell development. Here, we challenged this hypothesis by analyzing the IghPax5/+ mouse, which expressed a Pax5 minigene from the immunoglobulin heavy-chain locus. Despite high Pax5 expression, plasma cells efficiently developed in young IghPax5/+ mice at steady state and upon immunization, while their number moderately declined in older mice. Although Pax5 significantly deregulated the plasma cell expression program, key plasma cell regulators were normally expressed in IghPax5/+ plasma cells. While IgM secretion by IghPax5/+ plasma cells was normal, IgG secretion was modestly decreased. Hence, Pax5 repression is not essential for robust plasma cell development and IgM secretion, although it is required for efficient IgG secretion and the accumulation of long-lived plasma cells.
Project description:Plasma cells are key components of humoral immunity by secreting antibodies and providing protection against pathogens. These cells can be of IgM, IgA, or IgG subclass and migrate to class-specific niches. Localization and rareness of plasma cells make it challenge to define subclass-specific molecular hallmarks. Here, we describe how in-vitro differentiation of peripheral B-cells results in antibody-secreting plasma cells. Using a single-cell multi-modal sequencing approach (RAID) we find subclass-specific hallmark transcriptional profiles, surface protein expression and signaling pathway activation.
Project description:Antibody secretion by plasma cells provides acute and long-term protection against pathogens. The high secretion potential of plasma cells depends on the unfolded protein response, which is controlled by the transcription factor Xbp1. Here, we analyzed the Xbp1-dependent gene expression program of plasma cells and identified Bhlha15 (Mist1) as the most strongly activated Xbp1 target gene. As Mist1 plays an important role in other secretory cell types, we analyzed in detail the phenotype of Mist1-deficient plasma cells in Cd23-Cre Bhlha15(fl/fl) mice under steady-state condition or upon NP-KLH immunization. Under both conditions, Mist1-deficient plasma cells were 1.4-fold reduced in number and exhibited increased IgM production and antibody secretion compared to control plasma cells. At the molecular level, Mist1 regulated a largely different set of target genes compared to Xbp1. Notably, expression of the Blimp1 protein, which is known to activate immunoglobulin gene expression and to contribute to antibody secretion, was 1.3-fold upregulated in Mist1-deficient plasma cells, which led to a moderate downregulation of most Blimp1-repressed target genes in the absence of Mist1. Importantly, a 2-fold reduction of Blimp1 (Prdm1) expression was sufficient to restore the cell number and antibody expression of plasma cells in Prdm1(Gfp/+) Cd23-Cre Bhlha15(fl/fl) mice to the same level seen in control mice. Together, these data indicate that Mist1 restricts antibody secretion by restraining Blimp1 expression, which likely contributes to the viability of plasma cells.
Project description:The fasX gene encodes a small regulatory RNA in the group A Streptococcus (GAS). To determine the FasX regulon during GAS exposure to human plasma we compared parental strain MGAS2221 with isogenic fasX mutant strain 2221ΔFasX. Gene expression was analyzed 0, 15, and 60 minutes post-plasma exposure. GAS strains were grown in THY broth to mid-exponential phase, corresponding to an O.D.600 of 0.5. Two 16ml aliquots of each GAS culture were pelleted by centrifugation and the supernatant removed. Each GAS cell pellet was resuspended in 16ml of pre-warmed human plasma. Thus, four GAS-plasma suspensions were created, two each for each of the GAS strains, with one of the two using plasma from person A, and the other two using plasma from person B. Aliquots were recovered from each of the four GAS-plasma suspensions at T = 0, 15, and 60 mins. For the first two time-points 4.5ml of GAS-plasma suspension were added to 9ml of RNAprotect (Qiagen Inc.). For the final time-point, 4ml was added to 8ml of RNAprotect. Following addition of RNAprotect, samples were incubated at room temperature for 5 mins before centrifuging. Supernatant was removed and the pellets quick frozen in liquid nitrogen and stored at -80 °C until ready to use. RNA isolation, cDNA synthesis, and use of our custom Affymetrix microarray was as previously described (Perez et al., 2009)
Project description:Primary outcome(s): Overall percent agreement (OPA) of both KRAS and NRAS gene exon 2, 3 and 4 mutations between Plasma RAS testing and Reference method
Project description:IgM monoclonal gammopathy of undetermined significance (IgM MGUS) is an early precursor stage of the rare lymphoma Waldenström Macroglobulinemia (WM). Although comparative gene expression studies on WM, IgM MGUS, and normal B cells identified several genes differently expressed, reliable predictors of progression from IgM MGUS to WM have not yet been identified. We performed a microarray study on CD19+ and CD138+ cells of WM vs. IgM MGUS vs. CTRLs to determine gene signatures for both cell populations. We demonstrated that hematopoietic antigens, cell-adhesion molecules, Wnt signaling, BCR signaling, calcium signaling, coagulation cascade, and pathways responsible for cell cycle and proliferation were significantly enriched for genes expressed in B cells of WM vs. IgM MGUS vs. CTRLs. Notably, we showed nine genes which displayed the same expression levels in both WM and IgM MGUS compared to CTRLs, suggesting their possible role in the risk of transformation in IgM MGUS to WM.
Project description:Dogma holds that plasma cells (PC), as opposed to B cells, cannot bind antigen (Ag) because they have switched from expression of membrane-bound immunoglobulins that constitute the B cell Ag receptor (BCR) to production of the secreted form of immunoglobulins. We have compared the phenotypical and functional attributes of PC generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We unexpectedly found that the nature of the secreted Ig isotype rather than the chemical structure of the immunizing Ag defines two functionally distinct populations of PC. Fully mature IgM-expressing bone marrow PC retain expression of a functional BCR while their IgG+ counterparts do not. We show that Ag boost modifies the gene expression profile of IgM+ PC and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing PC can sense Ag and acquire competence for cytokine production upon antigenic challenge.