This study tested the hypothesis that B cells from salivary tissue are distinct in terms of proliferative capacity, immunoglobulin M secretion, repertoire, and autoantibody enrichment in Sj?grens syndrome. these data suggest salivary B cells have unique repertoire characteristics that likely influence autoantigen binding and contribute to Sj?grens syndrome disease in a tissue-specific manner. = 10; cLN, = 9; SMG, = 10) and C57BL/6 B cells (spleen, = 9; cLN, = 7). Data are shown as the percentage of total B cells secreting IgM. (B) Mean spot size of Id3?/? and C57BL/6 B cell data in (A). Data from 2 independent experiments are shown. To determine whether antibody secretion per cell is enhanced in the SMG population, we examined the mean spot size for each sample. Analysis of Id3?/? and C57BL/6 B cells isolated from the spleen, cLN, and SMG revealed no differences in the amount of IgM secreted (Fig. 1B). Together, these findings suggest salivary B cells are similar to those from other immune sites in the percentage of cells that secrete IgM and also in the amount of antibody secreted per B cell in pSS. B cells from salivary tissue are not as hyperproliferative in response to LPS as compared to those from other immune sites To test whether salivary gland B cells are hyperproliferative, we activated B cells from Identification3?/? pets and assessed proliferation (Fig. 2). B cells produced from splenic cells showed higher proliferation pursuing LPS excitement than do splenic B cells from C57BL/6 pets ( 0.0001) and Identification3?/? cLNs (= 0.01). Oddly enough, Identification3?/? B cells produced from salivary cells proliferated just like B cells produced from cLNs (= 0.6) but showed reduced proliferation in comparison to those through the spleen (= 0.04). B cell proliferation was identical in cLN B cells produced from Identification3?/? and C57BL/6 control pets (= 0.2). Therefore, outcomes from our proliferation studies also show salivary gland B cells usually do not screen a hyperproliferative phenotype in response to LPS excitement and, thus, aren’t distinguishable from B cells isolated from additional sites predicated on Tlr4-mediated proliferation. Open up in another window Shape 2. Proliferative capability of SMG B cells is comparable or reduced in comparison with B cells isolated from supplementary lymphoid organs.B cells were sort-purified from the indicated site and stimulated with LPS (25 g/ml) for 72 h before proliferation assay. Cells were incubated with tritium or BrdU for 6 h or before harvesting. Results from 3 experiments are pooled and normalized to SMG data from each experiment; Id3?/? (spleen, = 13; cLN, = 13; pooled SMG, = 3) and C57BL/6 B cells (spleen, = 11; cLN, = 12). (N.S., not significant; * 0.05, ** 0.01, and **** 0.001). The IgM heavy-chain repertoire differs in B cells derived from SMG tissue, cLNs, and spleen To determine whether B cells in salivary tissue have unique repertoire characteristics, we single-cell sorted B cells from Id3?/? SMG, cLN, and spleen. Splenic and cLN B cell sequences were generated from 4 independent experiments, each with spleens pooled from 4 C 5 animals. SMG B cell sequences were also generated from 4 independent experiments, with SMG tissue from 8C10 animals pooled for each experiment. We performed sequence analysis on IgM from Id3?/? B cells derived from spleen (= 265), cLN (= 59), and SMG tissue (= 63). VH usage was BI6727 inhibitor similar among B cells isolated from the 3 sites, with the exception of skewed VH14 usage by Id3?/? splenocytes and cLNs (= 0.02 and = 0.01, respectively) (Fig. 3A). Differences were also seen in DH usage. Interestingly, IgM derived from Id3?/? SMG B cells and cLN cells had increased DH4 use as compared with spleen (= 0.006 and = 0.01, respectively) (Fig. 3C). JH usage was also skewed BI6727 inhibitor because IgM from Id3?/? spleen showed more frequent usage of JH4 than that from salivary tissue (= 0.03) Moreover, salivary IgM showed increased JH1 usage BI6727 inhibitor as compared with cLN (= 0.05) (Fig. 3B). Of note, the CDR-H3 length of IgM derived from Id3?/? SMG B cells was shorter than that of spleen (average lengths = 11.6 vs. 12.4 amino acids, respectively (= 0.02) (Fig. 4A). However, CDR-H3 hydrophobicity and charge were similar in each of the sites we examined hSPRY2 (Fig. 4B and C, respectively). Open in a separate window Figure 3..