AntiCD43-APC was obtained from eBioscience. in lupus patients express more CD86 and have increased T cellstimulating activity in disease. This work distinguishes a novel, T cellinteracting B1 cell population whose abundance and activity 6-Carboxyfluorescein may be a reflection of, and a therapeutic target in, autoimmune disease. The immune response against foreign pathogens must arise promptly, develop effectively, and end appropriately, to counteract infection and avoid injury to normal tissue. In autoimmunity, however, the immune response is misdirected against self, resulting in tissue damage. Understanding of these processes has been advanced by recognition of individual cell types that carry out specific effector functions. Although the principal mission of B lymphocytes is considered to reside in immunoglobulin production, an effector role for these cells in regulating immune activity has been repeatedly noted (Zouali, 2008). With the recent success of B cell depletion therapy in autoimmune diseases, there is growing evidence that a population of cells contained within the B cell pool expresses immunostimulatory activity and is involved in clinical autoimmunity (Jacob and Stohl, 2010;Perosa et al., 2010;Sanz and Lee, 2010). Yet clear identification of the B cells that possess this function has remained a mystery. B1 cells are a small innate B cell population that is responsible for constitutively producing ZPK protective natural immunoglobulin (Baumgarth, 2011). In the mouse system B1 cells have 6-Carboxyfluorescein been shown to arise early and to derive from a distinct progenitor, lending support to their status as a separate lineage in a layered immune system (Herzenberg and Tung, 2006;Montecino-Rodriguez et al., 2006). Current studies have revealed novel activities of B1 cells including, in particular, high level stimulation of T cell expansion (Zhong et al., 2007). The recent identification of human B1 cells (Griffin et al., 2011) provides an opportunity to elucidate activities of this B cell population that may contribute to immune function and autoimmune disease. We found that human B1 cells are divisible into two readily separable, distinct populations and that T cellstimulatory activity is a property of one population and not the other, which is instead characterized by heightened immunoglobulin secretion. The former, T cellinteracting population is markedly increased in patients with lupus. == RESULTS AND DISCUSSION == Human B1 cells obtained from both umbilical cord and adult peripheral blood express index functional features not expressed by other mature B cells and phenotype as CD20+CD27+CD43+CD70(Griffin et al., 2011). In further study we have now found that B1 cells can be divided into two distinct populations by phenotypic criteria. Immunofluorescent staining reveals that some CD20+CD27+CD43+B1 cells express CD11b, whereas the bulk of B1 cells do not (Fig. 1, A and B). In view of 6-Carboxyfluorescein evidence in the mouse system that B1 cells readily 6-Carboxyfluorescein form aggregates (Ghosn et al., 2008), and the association of CD11b with the monocyte lineage, we verified that CD11b expression is an intrinsic property of some human B1 cells. We stained DNA with Hoechst 33342 and then analyzed only those cells that were singlets (Fig. 1 A). With 6-Carboxyfluorescein this approach, we established that among cells defined on the basis of having only one nucleus, a specific fraction coexpressed CD20, CD27, CD43, and CD11b. Separately, we exerted strict FSC-H by FSC-A doublet gating (Fig. 1 A) but, regardless of the level of restriction, CD11b+B1 cells were readily identified in similar proportions within the B1 population. Overall, we found that CD11b+B1 cells represent 1 out of every 810 B1 cells for both adult peripheral blood (n= 67) and umbilical cord blood (n= 6) samples, with the.