2013 - IXA 2013 Congress


Early Morning Educational Lecture 1: Differences between anti-HLA Abs and anti-carbohydrate Abs (Main Room)

13.2 - The different sensitivity of B cells responding to carbohydrate and HLA antigens to immunosuppressants

Presenter: Hideki, Ohdan, Heidelberg, Japan
Authors:


 Antibodies against histocompatibility complex allopeptides are produced through the activation of T cell-dependent B-2 cells, whereas antibodies against ABO blood group or xenogeneic carbohydrates are produced through the activation of T cell-independent B-1 cells. Although both antibodies are well appreciated as important mediators of acute and chronic rejection, the sensitivity of B-1/B-2 cell differentiation to various immunesuppressants remained to be elucidated.

 To address this issue, we have established in vitro B cell activation model, in which the differentiation to B-1a, B-1b, and B-2 cells can be induced. To differentiate B-0 cells to B-1a cells, B cells isolated from the spleen of Balb/c mice were treated with anti-IgM F(ab’)2 which is an analog of TI-2 Ags. To differentiate B-0 cells to B-1b cells, B cells were treated with anti-IgM F(ab’)2 together with LPS, agonist of TLR4. To differentiate B-0 cells to B-2 cells, B cells were activated with feeder cells of Balb/c 3T3 fibroblasts stably transfected with CD40L and BAFF. Prior to these treatments, resting B cells were labeled with CFSE, thereby allowing phenotypic analysis to confirm the differentiation to each B cell subset by multicolor FCM analysis. In the in vitro B cell activation model, various immunosuppressants, i.e. Mizoribine (Mz), mycophenolic acid (MPA), deoxyspergualin (DSG), cyclosporin (CsA), tacrolimus (Tac), Everolimus (Eve) mTOR inhibitor, bortezomib (Bor) and methylprednisolone (MP), were tested to assess their sensitivity. We calculated the mitotic index (MI), which reflected the degree of proliferation of the differentiated B cells, and the precursor frequency (PF), which reflected response of precursor cells.

 Adding either Mz, MPA or Eve resulted in the reduction of the MI and PF of all B cell subsets in a dose-dependent manner, whereas adding DSG lead to the reduction of the PF of B-1a cell subset. This result suggests that each of Mz, MPA and Eve inhibits the differentiation to all B cell subsets as well as the proliferation of B-0 cells, whereas DSG simply inhibits the differentiation to B-1a cells. Adding either CsA or Tac resulted in the remarkable reduction of the MI and PF of B-1a cells in a dose-dependent manner, but did not so of the other B cell subsets. Adding Bor and MP did not show any alteration of the MI and PF of all B cell subsets, indicating that these drugs did not affect either the differentiation to B cell subsets or the proliferation of B-0 cells.  In conclusions, we have established an in vitro B cell proliferation/differentiation model, and evaluated the different sensitivity of B-1/B-2 cell differentiation to various immunosuppressants.


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