Article Title: Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity
Figure Lengend Snippet: IgA + plasmocytes regulate tumour killing by CD8 + T cells a , Dih10 and dihXY HCC cells were transfected with an inducible ovalbumin (Ova) expression vector, and Ova expression and presentation were confirmed by flow cytometry, using an antibody that recognized the SIINFEKL peptide on the MHCI molecule H-2Kb. b – h , Ova-expressing dih cells or controls (dih–RFP) were starved overnight (5% cell death), after which their medium was changed and B cells from WT, Iga −/− , Pdl1/2 −/− , or SW-HEL mice were added in the presence of TGFβ (5 ng ml −1 ) and CTGF (3 ng ml −1 ), for an additional 24 h. Thereafter, the medium was replaced and CFSE-labelled OT-I T cells were added to the cultures that either contained or did not contain the B cells described above. After 4–6 days, the co-cultured cells were analysed by flow cytometry, while the secretory IgA was analysed by ELISA ( n = 2–4 wells per group per day). a – j , Experiments were repeated with two different Ova-expressing HCC and one prostate cancer cell lines. Shown are the representative flow cytometry histograms or plots depicting ( b ) OT-I CD8 + T-cell proliferation, ( c ) PD-L1 and SIINFEKL/H-2Kb expression on cancer cells, ( d ) PD-L1 expression on B cells, ( e ) cancer cell death. f , Relative dih-Ova–RFP killing by OT-I CD8 + T cells in the presence or absence of the indicated B cells. g , Total secretory IgA and anti-OVA-IgA antibody amounts in culture supernatants. h , Percentages of OT-I CD8 + cells in each culture, as indicated. i , j , TRC2-Ova–RFP cells or its control cell line (TRC2–RFP) were co-cultured with OT-I cells and splenic B cells (WT and Il10 −/− ), as described for a – h. i , Proliferation of OT-I cells was analysed using CFSE ( n = 3, 7, 5, 4). j , The amounts of secretory IgA were analysed using ELISA as indicated ( n = 3 per group). k – o , Liver cells from indicated 3-month-old mice were stained and analysed by flow cytometry. Experiments were repeated at least two or three times. Each dot represents one mouse. Shown are the percentage of CD8 + T cells among CD45 + cells ( n = 6, 5, 3, 7, 4) ( k ), absolute CD8 + T-cell number per gram of liver ( n = 3, 3, 7, 4) ( l ), the percentage of CD8 + CD44 + Ki-67 + T cells with representative scatter plots ( n = 3 or 4 per group) ( m , n ), and the representative scatter plots of perforin and GrzB among CD8 + CD44 + Ki-67 + T cells ( o ). p , q , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the absolute CD8 + T-cell number in both STAM-BL6 and STAM-FVB mice ( n = 8, 4, 5, 3, 5, 5, 6, 7) ( p ), and the percentage of T EM cells using CD8, CD44, and CD62L ( n = 4, 4, 6, 9) ( q ). r , Liver cells from indicated 3-, 6-, and 11-month-old mice kept on HFD ( n = 4–10) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + IFNγ + CD107a + T cells. Detailed n values are shown in . s – y , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the percentage of Th17 cells using CD4 and IL-17a ( n = 4, 7, 9, 10, 4, 9, 11) ( s ), the percentage of regulatory T cells using CD4 and Foxp3 ( n = 4, 3, 8, 11, 7, 13) ( t ), the percentage of Tfh-like cells using CXCR5, PD1 and CD4 (3, 5, 6, 5) ( u ), the percentage of B220 + CD19 + B cells ( n = 4, 7, 7, 4, 5, 3, 9, 3, 11) ( v ), absolute B220 + CD19 + B-cell number per gram of liver ( n = 5, 7, 12, 27, 15, 7) ( w ), the percentage of IgG + cells ( n = 4, 3, 14, 11) ( x ), and CD138 + plasma cells ( n = 4, 7, 4, 7, 7, 8, 5, 8, 6, 6, 13, 27, 17) ( y ). Two-sided t -test (means ± s.e.m.; f – m ) and Mann–Whitney test (median; p – y ) were used to determine significance. * P
Article Snippet: Antibodies specific for the following antigens were used (anti-mouse antigens were abbreviated as ‘m’, and anti-human as ‘h’): Tim-3 (mB8.2C12- AlexaF647; hF38-2E2-PerCP-eF710); CD138 (m281-2-PE/APC; hDL-101- APC); IgA (mA-6E1-PE/FITC; m11-44-2-Biotin; mRMA-1; hIS11-8E10- FITC; DAKO hA0262); CD8α (m53-6.7-PerCp-Cy5.5/PE-Cy7; hRPA-T8-PE; hC8/144B); CD45 (m30-F11-V500; hHI30-eF450); CD20 (mAISB12-PE); CD44 (mIM7-PE/FITC/eF450); CD4 (mRM4-5-eF450; mGk1.5-FITC/PE/PE-Cy7; hRPA-T4-APC); B220 (mRA3-6B2-eF450/FITC); CD19 (meBio1D3-eF660/PE-Cy7; hHIB19-PE); IgM (mII/41-PE-Cy7; hSA-DA4-eF450); IgD (m11- 26c.2a-Pacific-Blue; m11-26c-PerCP-eF710); TNF (mMP6-XT22-FITC/PE); IFNγ (mXMG1.2-eF660); GrzB (mNGZB-eF660); CD107a (meBio1D4B-PE); PD-1 (mJ43-eF450/FITC; mRMP1-30-APC; hMIH4-FITC); PD-L1 (mMIH5- PE-Cy7/PE; hMIH1-PE-Cy7); FAS-L1 (mMFL3-FITC); Ki-67 (mSolA15-eF450); IgG2a (m2a-15F8-FITC/APC; mRMG2a-62-PE); IgG1 (mM1-14D12-eF450; mRMG1-1-FITC); IL-10 (mJES5-16E3-PE); CD11c (mN418-eF450/PE); CD11b (mM1/70-eF450/eF660); MHCII (mM5/114.15.2-FITC/PE); Gr-1 (m1A8-Ly6G-PerCP- eF710); IL-17 (meBio17B7-PE-Cy7); Foxp3 (mFJK-16s-eF450); Perforin (meBioOMAK-D-FITC); CXCR5 (mSPRCL5-PE/APC/Biotin); CD62L (mMEL- 14-PE/APC); SIINFEKL/H-2Kb (meBio25-D1.16-APC); F4/80 (mBM8-PE/FITC); anti-Biotin (BK-1/39-PE); CD5 (m53-7.3-FITC/eF450) and NK1.1 (mPK136- APC); CD31 (m390-eF450) (most from eBioscience and Biolegend); pAb rabbit to CD3 (DAKO, IS503); and alpha-SMA (m-ab5694; DAKO, h1A4).
Techniques: Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY