Journal: Immunology

Article Title: Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation

doi: 10.1111/imm.12895

Figure Lengend Snippet: (a) Attenuation of cytotoxic T lymphocyte (CTL) responses (51Cr‐release assays performed at day 5) and (b) diminished [3 H]TdR incorporation using splenocytes from skin graft recipients shown in Fig. 1 (killed at 60 days post skin transplantation) as responder cells. CTL in control cultures stimulated with third‐party cells (C3H) were assayed using 72 hr Concanavalin A‐activated C3H splenocyte blasts as target cells. Data are pooled within groups in which mice had accepted/rejected skin grafts. * indicates P < 0·02 for CTL (Fig. 2a) or [3 H]TdR (Fig. 2b) compared with equivalent recipients with rejected grafts. (c) Anti‐donor (BALB/c) IgG levels in naive and immune mice treated with rapamycin and busulphan/cyclophosphamide with and without bone marrow transplantation (n = 15 each). Serum alloantibodies were also quantified for non‐transplanted BL/6 mice as controls (n = 10). Data are reported as median fluorescence intensity (MFI) (mean ± SD). * indicates P < 0·0001 versus mice receiving rapamycin but not bone marrow transplant (BMTx). (d) Attenuation of CTL responses (see legend in Fig. 2a) in mixed lymphocyte co‐cultures (MLCs) using splenocytes pooled from fresh untreated naive BL/6 mice following addition of CD4+‐enriched splenocytes of skin graft recipients shown in Fig. 1. Data were obtained using individual spleen preparations from all mice (n = 15/group) shown in Fig. 1, and were subsequently pooled according to mice that had accepted/rejected skin grafts. As shown, the added cells from grafted recipients were also treated with either rabbit complement alone, or anti‐CD45.1/anti‐CD45.2 and anti‐Thy1.2 with rabbit complement before use in MLCs. * indicates P < 0·05 compared with equivalent recipients with rejected grafts. (e) Similar results were obtained when [3 H]TdR incorporation was measured following addition of CD4+‐enriched splenocytes from transplanted mice to fresh mixed lymphocyte reaction (MLR) cultures. (MLR responses to third‐party C3H antigen were entirely abolished in untreated BL/6 splenocytes after addition of CD4+ suppressor cells from naive BMTx mice with long‐term surviving allografts). **P < 0·05.

Article Snippet: Monoclonal antibodies The following mAbs were used: from BD Pharmingen (San Diego, CA): Fluorescein isothiocyanate‐conjugated anti‐mouse CD4 (RM4‐4); from Biolegend (San Diego, CA): allophycocyanin‐conjugated anti‐mouse CD25 (3C7), allophycocyanin‐conjugated anti‐mouse CD3 ε (145‐2C11), CD45.1 (clone A20), CD45.2 (clone 104); from Cedarlane Laboratories (Hornby, ON), anti‐Thy‐1.2 (Clone 5a‐8) and anti‐B220 (RA3‐6B2); and from Serotec (Mississauga, Canada), fluorescein isothiocyanate‐conjugated anti‐CD3 (clone MCA500F).

Techniques: Transplantation Assay, Fluorescence

Journal: Immunology

Article Title: Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation

doi: 10.1111/imm.12895

Figure Lengend Snippet: Skin allograft survival following autologous bone marrow transplantation in naive and immune (i.e. previously grafted) mice – see text for further details. Fifteen animals/group of naive (▲) or immune (○) CD45.2 BL/6 mice (the latter having rejected a first BALB/c graft 21 days earlier) received BALB/c skin grafts and a sequence of rapamycin treatment, followed by busulphan/cyclophosphamide for 6 days, and a further 21 days of rapamycin treatment beginning 5 days later. Two additional groups of naive (▼) or immune (■) mice also received autologous T‐cell‐depleted CD45.1 BL/6 bone marrow cells (5 × 106 per recipient) 2 days after completion of the course of busulphan/cyclophosphamide, and began 21 days of rapamycin 3 days later. Data show graft survival for all four groups. *P < 0·05 (Mann–Whitney U‐test) for equivalent recipients without bone marrow.

Article Snippet: Monoclonal antibodies The following mAbs were used: from BD Pharmingen (San Diego, CA): Fluorescein isothiocyanate‐conjugated anti‐mouse CD4 (RM4‐4); from Biolegend (San Diego, CA): allophycocyanin‐conjugated anti‐mouse CD25 (3C7), allophycocyanin‐conjugated anti‐mouse CD3 ε (145‐2C11), CD45.1 (clone A20), CD45.2 (clone 104); from Cedarlane Laboratories (Hornby, ON), anti‐Thy‐1.2 (Clone 5a‐8) and anti‐B220 (RA3‐6B2); and from Serotec (Mississauga, Canada), fluorescein isothiocyanate‐conjugated anti‐CD3 (clone MCA500F).

Techniques: Transplantation Assay, Sequencing, MANN-WHITNEY

Journal: Immunology

Article Title: Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation

doi: 10.1111/imm.12895

Figure Lengend Snippet: Evidence for Treg cells of both donor and host origin in bone marrow transplant (BMTx) mice shown in Fig. 3. Cells were pooled within groups from mice with surviving grafts shown in Fig. 3 and as discussed in the legend to Fig. 3 (anti‐CD19 treated: n = 4, no BMTx: n = 8). CD4+ enriched cells (MACS columns) from all groups were then further treated as described in the text and indicated in the axis of Fig. 5, and added as in Fig. 4 to antigen‐stimulated naive splenocytes for attenuation of (a) cytotoxic T lymphocyte (CTL) induction or (b) [3 H]TdR incorporation. All data are mean ± SD of triplicate cultures assayed in triplicate. Anti‐C3H responses in all groups and treatment conditions were observed to be statistically not significant and as such are not shown for clarity. *, ****P < 0·05: untreated splenocytes from each treatment group suppress CTL induction/proliferation compared with equivalent no BMTx control; **P<0·05: anti‐CD45.1 depletion attenuates suppression compared with untreated cells from mice of the same treatment group; ***P < 0·05: anti‐CD45.2 depletion attenuates suppression compared with untreated cells from mice of the same treatment group; ****P < 0·05, decreased suppression in untreated cells, or anti‐CD45.2‐treated cells from the group of BMTx mice receiving anti‐CD19 from day 15, compared with anti‐CD45.2‐treated cells from the groups of mice treated with BMTx + normal rat serum (NRS), or receiving anti‐CD19 from day 5/day 25.

Article Snippet: Monoclonal antibodies The following mAbs were used: from BD Pharmingen (San Diego, CA): Fluorescein isothiocyanate‐conjugated anti‐mouse CD4 (RM4‐4); from Biolegend (San Diego, CA): allophycocyanin‐conjugated anti‐mouse CD25 (3C7), allophycocyanin‐conjugated anti‐mouse CD3 ε (145‐2C11), CD45.1 (clone A20), CD45.2 (clone 104); from Cedarlane Laboratories (Hornby, ON), anti‐Thy‐1.2 (Clone 5a‐8) and anti‐B220 (RA3‐6B2); and from Serotec (Mississauga, Canada), fluorescein isothiocyanate‐conjugated anti‐CD3 (clone MCA500F).

Techniques:

Journal: Cell Reports

Article Title: GM-CSF Calibrates Macrophage Defense and Wound Healing Programs during Intestinal Infection and Inflammation

doi: 10.1016/j.celrep.2020.107857

Figure Lengend Snippet: ILCs and GM-CSF Regulate Intestinal Macrophage Polarization (A) GSEA of top Hallmarks pathways in colonic Ly6C lo MHC-II + MNPs from Rag2 −/− mice treated with anti-CD90.2 IgG or isotype control and 2% DSS. (B) Expression of M2 alternative activation genes in colonic MNPs from (A). Medians indicated. (C) GSEA of top 100 DEGs from human monocyte-derived macrophages (MDMs) treated with various stimuli in colonic MNPs from (A). Data derived from GEO: GSE47189 . (D) Expression of M1 versus M2 genes between flow-sorted intestinal CD45.1 + Csf2rb +/+ and CD45.2 + Csf2rb −/− Ly6C lo MHC-II + MNPs in 80:20 chimeric mice. Data were derived from pooled Ly6C lo MHC-II + macrophages from 5 mice. (E) Correlation between single sample GSEA scores of ILC (left) or CSF2 levels (right) with M1 signatures in ulcerative colitis (healthy control [HC] n = 11, non-inflamed UC n = 23, active UC n = 74) and ileal Crohn’s disease biopsies (HC n = 35, ileal CD n = 210). Data derived from GEO: GSE59071 and GEO: GSE93624 . p values were calculated using the standard DESeq 2 method with multiple comparisons correction using the BH procedure (B), and Spearman’s rank correlation with multiple testing correction (E). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figure S4 .

Article Snippet: Anti-mouse CD45.2 antibody (104) , Thermo Fisher Scientific , Cat#A14736; RRID: AB_2534252.

Techniques: Expressing, Activation Assay, Derivative Assay

Journal: Cell Reports

Article Title: GM-CSF Calibrates Macrophage Defense and Wound Healing Programs during Intestinal Infection and Inflammation

doi: 10.1016/j.celrep.2020.107857

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD45.2 antibody (104) , Thermo Fisher Scientific , Cat#A14736; RRID: AB_2534252.

Techniques: Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Microarray, Software

Journal: eLife

Article Title: Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis

doi: 10.7554/eLife.83004

Figure Lengend Snippet:

Article Snippet: Antibody , violetFluor 450 anti-mouse CD45.2 (104) (mouse monoclonal) , Tonbo Biosciences , Cat# 75-0454; RRID :AB_2621950 , FACS (4 × 10 –2 μL/1 × 10 6 cell).

Techniques: Produced, Sequencing, Recombinant, Protease Inhibitor, Purification, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Software, Immunostaining

Journal: Immunity

Article Title: Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver

doi: 10.1016/j.immuni.2020.08.004

Figure Lengend Snippet:

Article Snippet: Mouse Monoclonal CD45.2 - AF700 (clone 104) , eBioscience , 56-0454-82; RRID: AB_657752.

Techniques: Purification, Recombinant, Staining, Quantitation Assay, cDNA Synthesis, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Software, Microscopy