B cells are known to offer different contributions to immune system including regulatory function of the immune responses. The regulatory role of B cell "subset" (Bregs) have been identified in different autoimmune models. Our hypothesis for this project is that Bregs play a regulatory role in inducing transplant tolerance. The aims of this project are: i) to identify the conditions under which B cells with regulatory functions are expanded in transplanted animals; ii) to define the different characteristics of these cells and whether antigen specificity plays a role in their function; iii) whether the mechanism of action of B cells is carried through cytokines production, such as IL-10, and/or through their cross-talk with other cells such as regulatory T (Treg) cells and Natural Killer T (NKT) cells; iv) whether they express Galectin-1 (Gal-1) and if Gal-1 plays a role in the mechanism of tolerance as demonstrated in Tregs.
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In this study we have so far i) shown that regulatory B (Breg) cells, which are defined as CD19+, CD21+, CD23+ and CD24+, in naive C57BL/6 (B6) mice are 0.5-0.6 % of the total splenocytes, while during the course of skin graft rejection this number doubled (11 days following the graft). However, this sharp increase was followed by a drop in the Breg numbers at day 15. These results suggest that cells with a Breg phenotype are expanded but then decreased to the initial number during rejection. These experiments will be followed by studying Breg numbers in mice rendered tolerance with different strategies. ii) We documented that by injecting small numbers of Bregs (obtained by FACS sorting) into B6 mice in order to prevent skin rejection, very little effect was observed. However, this result needs to be confirmed by injecting higher number of cells. Regarding the importance of B cell specificity for regulatory function to occur we have detected antibodies specific for the graft in the sera of B6 mice, which have received skin grafts, suggesting that allo-specific B cells are contributed during transplantation, but whether these allo-antibodies assist in the survival or the rejection of the graft will further be addressed with in vivo experiments. iii) Our results have also shown that co-culturing of T cells with different subsets of LPS/CPG activated B cells showed that activated B cells stimulated T cells to produce TNF-Î± and IFN-Î³. However, co-culture of T cells with Bregs resulted in the lowest amount of T cells TNF-Î± and IFN-Î³ production when compared to co-culturing with other B cell subsets. Moreover, we found that under these conditions Marginal zone B cells produced higher amounts of IL-10 than Bregs, while IL-10 was produced by neither Transional-1 nor Folicular cells. Also, we have found that Bregs express CD1d molecule and that the levels of expression increased in animals rejecting skin allo-graft (11 days after transplantation). This expression might be relevant to their cross-talk with NKT cells. The relationship between Bregs, NKT cells as well as Tregs will be further examined in vivo and in vitro studies. iv) Using western blot, B cells were found to express Gal-1. Furthermore, we have found that sera obtained from Gal-/- mice express more allo-specific IgG2a antibodies than B6 mice, while the level of allo-specific IgG1 antibodies were almost the same in B6 and Gal-/- mice. These findings suggest that Gal-1 might play a role in B cells function, and this will be further confirmed by in vitro studies. Collectively, this project will reveal the role of B cells in inducing tolerance to the allograft and their mechanism of action in transplantation.
Transplantation is currently the only treatment for organ failure and has become commonplace with the use of immunosuppressive drugs. Although immunosuppressive treatment help in increasing the degree of success in transplantation it has many side effects, such as infections and malignancies . As a result, many approaches have attempted to replace the use of immunosuppressive drugs such as B cells depletion by rituximab, which is humonized anti-CD20 that deplete B cells . However, depleting B cells provided mixed results ranges from complete respond, which rarely was observed, to the death of the patients. This results suggest that B cells can play both pathogenic as well as protective role in transplantation . Therefor, the aim of this project is to identify the regulatory role of B cells in transplant models.
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The regulatory B (Breg) cells, have been identified differently in various murine models of chronic inflammation by several group of scientists with particular respect to their phenotype, mechanism of action and stimuli. For example, the study by Mauri et al. characterized Breg cells as CD19+, CD21+, CD23+, CD24+, IgM+, BCR+ and CD1d+, which represent the phenotype of the Transitional 2-Marginal Zone Precursor (T2-MZP) cells. In This study, T2-MZP cells were documented to be regulatory upon stimulation with collagen and anti-CD40 in Collagen induced arthritis (CIA), mainly through Interleukin-10 (IL-10) production . On the other hand, Tedder and colleagues showed that spleenic Breg cells are CD5+, CD1d+ and CD19+ B cells, and they labeled them as B10 cells because they only produce IL-10 . Also, in Diabetes model, Tian et al. showed that Lipopolysaccharides (LPS) activated B cells produce high level of Transforming growth factor beta (TGF-Î²) and express Fas ligand. The adaptive transfer of these cells inhibit both T helper 1 (Th1) immunity against Î² cells antigen and disease progress in Non-obese diabetic (NOD) mice . While in inflammatory Bowel Disease (IBD), Mizoguchi and his group found that the transfer of CD1dhi B cells with regulatory function were capable of downregulating the inflammation associated with IL-1Î² again through IL-10 (7). All the latter studies, suggested that B cells with regulatory functions might vary in their mechanism and phenotype depending on the type of inflammation. Moreover, they emphases that Breg cells mainly act through IL-10 or TGF-Î² production. However, whether Breg cells interact with other cells such as regulatory T (Treg) cells, Invanient Natural Killer T (iNKT) cells and their role in transplantation still to be addressed (Figure 1).
NKT cells, characterized by their unique TCR-Î± chain molecule that recognize CD1d (MHC class I like molecule), which present glycolipid instead of peptides .
NKT cells can be divided into three groups according to the TCR molecule that they express . The study of NKT cells type II and type III was limited as the reagent to identify them is not available . However, NKT cells type I, has been investigated extensively during the last 5 years. They are known as iNKT cells because they express invariant TCR-Î± chain (VÎ±24-JÎ±18 in human and VÎ±14-JÎ±18 in mouse) .
The most well known CD1d ligand to stimulate iNKT cells is Î±-GalactosylCeramide (Î±-GalCer) that trigger them to produce variety of cytokines such as TGF-Î±, IL-10, Th1 and Th2 cytokines, therefor; they have the ability to effect different cells including Dendritic cells (DCs), NK cells, Macrophages (MO), T and B lymphocytes (Figure 2) . Moreover, t
Figure 1: Schematic view of the possible regulatory mechanisms mediated by the regulatory B (Breg) cells.
Mauri et al.  hey are divided into two subpopulations, the immunoregulatory one (CD4+), and the CD8-/CD4-(Double negative (DN)) subpopulation that has cytolytic function . As discussed earlier, Breg cells express high level of CD1d that could play a role in their crosstalk with iNKT cells to downregulate the immune system (Figure 2). As wermeling group demonstrated, in Systemic lupus erythematosus (SLE) murine model that iNKT cells play a protective role and that autoreactive B cells are regulated by their CD1d expression . That is in agreement with what has been found in several other murine autoimmune models such as Diabetes that iNKT cells has a regulatory role and that their number is decreased in autoimmune patients .
Figure 2: NKT cells function.
Godfrey et al. 
In addition, to the possible cellular interaction between iNKT cells and Breg cells several studies, have suggested a link between Breg cells and Treg cells. Treg cells, can be divided into at least two groups. The first group known as natural CD4+ Treg cells they are CD25+ and Foxp3+ (Forkhead box P3). The latter group develop in the thymus and act through IL-10 and TGF-Î² production. The inducible Treg cells represent the second group they arise from conventional CD4+ T cells under the effect of IL-10, TGF-Î², IL-2 or co-stimulatory signals, and they are CD25+ and Foxp3+ and known as Tr1 or Th3 . The fact that Breg cells produce IL-10 and TGF-Î² may explain the possible link between those two cells.
Moreover, we will be focusing in this project on the regulatory role of galactin-1 (Gal-1) on the function of B cells. Gal-1, is defined as carbohydrate binding protein that recognize carbohydrate bind to lipid and proteins on the extracellular and intracellular levels. Also, it has been documented to be involved in several homeostatic functions, for instant; mediating cells adhesion, cell-cell interaction, protein-protein interaction, mitosis and apoptosis . Furthermore, in Treg cells, Garin et al. showed that upon Treg cells activation through their TCR receptor Gal-1 expression is upregulated. Moreover, they found that blocking Gal-1 influence the suppressive function of the Treg cells in human . These data could hint that Gal-1 may play a role in B cells function as well.
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Project hypothesis: Bregs play a regulatory role in inducing transplant tolerance.
i) To identify the conditions under which B cells with regulatory functions are expanded in transplanted animals.
ii) To define the functional characteristics of these cells and whether antigen specificity plays a role in their function.
iii) To study whether the mechanism of action of B cells is carried through cytokines production, such as IL-10, and/or through their cross-talk with other cells such as regulatory T (Treg) cells and Natural Killer T (NKT) cells.
iv) To determine whether they express Galectin-1 (Gal-1) and if Gal-1 plays a role in the mechanism of tolerance as demonstrated in Tregs.