Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Heterogeneity in the Memory B Cell Compartment in the Bone Marrow in Human Rheumatoid Arthritis.
Anolik, Jennifer H., Owen, Teresa, Hossler, Jennifer, Cistrone, Christopher A., Palanichamy, Arumugam
In addition to being the site of B cell development, the bone marrow (BM) contains mature lymphocytes that may participate in secondary immune responses including formation of germinal centers. However, the detailed phenotype, function, and regulation of mature B cell subsets in the BM microenvironment in humans is largely unexplored, particularly in the setting of autoimmunity.
BM aspirates and paired peripheral blood (PB) samples were collected from RA patients and age-matched normal controls (NC) (n=10). B cell subsets and effector molecules were defined using multiparameter flow cytometry by the expression of markers including CD19, IgD, CD27, Mitotracker G, CD10, CD38, CD24, B220, CD95, CXCR3 and CD21. In select experiments, B cells were enriched by rosette separation and single memory B cells from the switched (SM: IgD-, CD27+), unswitched (USM: IgD+, CD27+) and double negative memory (DN: IgD-, CD27-) subsets were sorted. Sorted naïve (IgD+, CD27-) B cells served as controls. B cell mRNA was reverse transcribed, immunoglobulin (Ig) genes were amplified by a nested PCR and products sequenced. Rearranged Ig gene products were compared to the closest germline fit on JoinSolver and a detailed mutational analysis was performed.
Both NC and RA contained diverse mature memory B cell populations in the BM. The USM population in RA BM was decreased compared to NC BM ( P=0.02 ) Analysis of activated, effector phenotype molecules on BM memory subsets showed significant levels of B220+, CD95+ and CXCR3+, with some RA subjects displaying a profound expansion of effector memory. Interestingly, the expression level of these molecules between BM and PBL subsets were closely comparable. However, in the RA DN compartment, the CD24- effector subset was larger in the BM ( P=0.04, BM Vs. PB ) and CXCR3+, CD95+ subsets represented in lower levels ( P=0.001, BM Vs. PB ). On the molecular level, the overall mutational frequencies in the SM, USM and DN Ig genes were similar between RA and NC BM. However, detailed mutational analysis revealed that all memory subsets in RA BM showed increased transversions compared to NC counterparts (P=0.001). Of note, SM and DN fractions from RA PB showed an elevated mutational frequency compared to NC PB (SM: 7.7% vs. 5.8%, DN: 4.5% vs. 2.8%). Moreover, similar to BM, RA PB also had elevated levels of transversions compared to NC PB (P<0.05 in all cases). Further analysis revealed a distinct pattern of nucleotide substitutions in RA BM and PB memory B cells compared to NC counterparts (e.g. predominant T/A to G substitutions in the RA BM SM B cells and T to C substitutions in RA PB SM B cells).
Tissue specific differences in the effector molecule expression on B cell subsets in RA suggests preferential homing or in-situ generation of these B cells in the BM. Elevated transversions in RA memory B cells may indicate increased activity of Uracil DNA glycosylase and warrants further investigation to understand the precise mechanisms underlying the mutational machinery in an autoimmune setting.
To cite this abstract, please use the following information:
Anolik, Jennifer H., Owen, Teresa, Hossler, Jennifer, Cistrone, Christopher A., Palanichamy, Arumugam; Heterogeneity in the Memory B Cell Compartment in the Bone Marrow in Human Rheumatoid Arthritis. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :2186