Functional comparison of thymic B cells and dendritic cells in vivo by Petra Kleindienst, Isabelle Chretien, Thomas Winkler, and Thomas Brocker Blood Volume 95(8):2610-2616 April 15, 2000 ©2000 by American Society of Hematology

Restriction map of the transgenic construct CD19-IE Restriction map of the transgenic construct CD19-IE.MHC class II I-Eα d cDNA (striped box) was placed under the control of a human CD19 promoter containing DNA segment (gray box). Restriction map of the transgenic construct CD19-IE.MHC class II I-Eα d cDNA (striped box) was placed under the control of a human CD19 promoter containing DNA segment (gray box). The rabbit β-globin gene fragment providing the cDNA with an intron and a polyadenylation signal (A(n)) is displayed as a white box. X, XhoI; N, NotI; B,BamHI; E, EcoRI; S, SpeI; restriction sites in parenthesis () have been destroyed by blunt-end cloning. Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Flow cytometric analysis of cell suspensions from (pooled axillary, inguinal, and brachial) lymph nodes of CD19-IE transgenic mice (B6 CD19-IE) and their nontransgenic littermates (B6).The double stainings were performed with mAbs specific for the B-lineage... Flow cytometric analysis of cell suspensions from (pooled axillary, inguinal, and brachial) lymph nodes of CD19-IE transgenic mice (B6 CD19-IE) and their nontransgenic littermates (B6).The double stainings were performed with mAbs specific for the B-lineage markers B220 (FITC) or CD19 (FITC) and an MHC class II I-E specific mAb (PE). FITC, fluorescein isothiocyanate; PE, phycoerythrin. Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Flow cytometric analysis of spleen cell suspensionsfrom nontransgenic B6 animals (Figure 3B), transgenic B6 CD19-IE mice (Figure 3D), nontransgenic I-A–deficient B6/I-A−/− littermates (Figure 3C) and their transgenic B6 CD19-IE/I-A−/− counterparts (Figure 3... Flow cytometric analysis of spleen cell suspensionsfrom nontransgenic B6 animals (Figure 3B), transgenic B6 CD19-IE mice (Figure 3D), nontransgenic I-A–deficient B6/I-A−/− littermates (Figure 3C) and their transgenic B6 CD19-IE/I-A−/− counterparts (Figure 3E). Suspensions were stained with mAbs specific for CD19 (FITC), I-E (PE), and I-A–biotin plus Streptavidin APC. In all 3 color analyses, living cells were first gated according to their FSC/SSC properties and then for their expression of CD19 (Figure 3A). CD19+ cells were then further analyzed for expression of I-A and I-E (Figures 3B–E). Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Flow cytometry analysis of nonadherent cells from bone marrow GM-CSF cultures derived from either nontransgenic mice (B6) or mice expressing transgenic MHC class II I-E under the control of the MHC class II promoter (B6-IE56) or the CD19 promoter (B6 CD19-I... Flow cytometry analysis of nonadherent cells from bone marrow GM-CSF cultures derived from either nontransgenic mice (B6) or mice expressing transgenic MHC class II I-E under the control of the MHC class II promoter (B6-IE56) or the CD19 promoter (B6 CD19-IE).Gates were set on large cells according to FSC/SSC criteria (top panel), and cells fulfilling these criteria were further analyzed for expression of the DC marker CD11c (PE) and MHC class II I-E (FITC). Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Expression of the CD19-IE transgene leads to I-E expression on thymic B cells but not on thymic dendritic cells.(A) B cells. Expression of the CD19-IE transgene leads to I-E expression on thymic B cells but not on thymic dendritic cells.(A) B cells. Thymi of B6 and B6 CD19-IE mice were teased, and cell suspensions were stained with mAbs specific for the pan B-cell antigen CD19 (FITC) (data not shown) and further analyzed for expression of I-E or I-A (PE), respectively (upper panels). (B) Dendritic cells. Thymi of B6 and B6 CD19-IE mice were collagenase digested, and DC were analyzed according to their FSC/SSC criteria (data not shown). Cells were stained with mAbs specific for CD11c (FITC) (data not shown), and CD11c-positive cells were further analyzed for I-E or I-A (PE) expression (lower panels). Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Functional expression of the IE transgene on B cells from B6 CD19-IE mice induces antigen-specific proliferation in I-E restricted CD4 T cells.B220-positive splenocytes from B6 and B6 CD19-IE animals were sorted, irradiated, and used as APC for preactivated... Functional expression of the IE transgene on B cells from B6 CD19-IE mice induces antigen-specific proliferation in I-E restricted CD4 T cells.B220-positive splenocytes from B6 and B6 CD19-IE animals were sorted, irradiated, and used as APC for preactivated T-cell blasts from AD10 TCR transgenic mice. The coculture was performed in the presence of graded amounts of antigenic peptide (MCC88-103), and proliferation was measured by [3H] thymidine incorporation. Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Influence of the different I-E transgenes on the number of peripheral (top row) and thymic (bottom row) CD4+ T cells.Lymph node and thymus cell suspensions were stained with mAbs specific for CD4 (PE) and CD8 (FITC). Influence of the different I-E transgenes on the number of peripheral (top row) and thymic (bottom row) CD4+ T cells.Lymph node and thymus cell suspensions were stained with mAbs specific for CD4 (PE) and CD8 (FITC). The CD4/CD8 ratios in the LN T lymphocyte populations were determined from 4 mice per group, and the following values were obtained: B6, 8.2; B6/I-A−/−, 0.09; B6-IE/I-A−/−, 4.8; B6 CD19IE/I-A−/−, 0.06. The percentages of CD4+CD8− thymocytes in the thymi of the same animals were B6, 6.08%; B6/I-A−/−, 1.26%; B6-Eα dI-A−/−, 8.16%; B6CD19-IE/I-A−/−, 1.26%. Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology

Petra Kleindienst et al. Blood 2000;95:2610-2616 ©2000 by American Society of Hematology