Anti mouse cd25

Purified CD4+T cells (CD4⁺CD25⁻CD45RB^hi) from spleens and lymph nodes of Mina KO or WT litter mate control mice were FACS sorted on a Reflection (i-Cyt, Champaign, IL) following staining with anti-mouse CD4, anti-mouse CD45RB and anti-mouse CD25 (eBioscience) were differentiated in to Th1 or Th2 as per previously published conditions [54 (link)]. Briefly, CD4+T cells were stimulated in anti-CD3 coated plates (5ug/ml), anti-CD28 (1ug/ml) in presence of rIL12 (20ng/ml) and anti-IL4(10ng/ml) for Th1 and anti-IFNγ (10ng/ml), rIL4 (100ng/ml) and anti-IL12 (10ng/ml) for Th2 differentiation. Cells were cultured for three days and restimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) in presence of monensin to test for intracellular cytokine IFNγ and IL4.

The antibodies used in this study are as follows: anti-mouse CD30 (clone: mCD30.1) (BD Biosciences, San Jose, CA, USA), anti-mouse CD8α (clone: 53-6.7) (eBioscience, San Diego, CA, USA; BioLegend, San Diego, CA, USA), anti-mouse CD4 (clone: GK1.5) (eBioscience, BioLegend), anti-mouse CD3ε (clone: 145-2C11) (eBioscience, BioLegend), anti-mouse CD44 (clone: IM7) (eBioscience), anti-mouse T-bet (clone: 4B10) (eBioscience), anti-mouse Foxp3 (clone: FJK-16s) (eBioscience), anti-mouse Bcl6 (clone: BCL-DWN) (eBioscience), anti-mouse PD-1 (clone: J43) (eBioscience), anti-mouse CXCR5 (clone:SPRCL5) (eBioscience), anti-mouse CD62L (clone: MEL-14) (eBioscience), anti-mouse IFNγ (clone: XMG1.2) (eBioscience), anti-mouse Tim-3 (clone: RMT3-23) (eBioscience), and anti-mouse CD25 (clone: PC61.5) (eBioscience).

CD4⁺T cells (CD4⁺CD25⁻CD45RB^hi) and B cells (CD19⁺) from spleens and lymph nodes of Mina KO or WT litter mate control mice were FACS sorted on a Reflection (i-Cyt, Champaign, IL) following staining with anti-mouse CD4, anti-mouse CD45RB and anti-mouse CD25 (eBioscience) for T cells and anti-mouse CD19 for B cells. CD4⁺T cells (1x10⁵ cells/ml) were stimulated for 72h with soluble anti-CD28 (1ug/ml, eBioscience) in 96 well flat bottom plates coated with graded amounts of anti-CD3 (eBioscience). B cells (1x10⁶ cells/ml) were stimulated in 96 well flat bottom plates with graded amounts of LPS (O111:B4, Sigma) for 72 h. Cultures were pulsed with 1 μCi of [³H]-thymidine for the final 8h of assay and harvested with a Packard harvester. The counts per minute (cpm) were determined using a Packard Matrix 96 direct counter (Packard Biosciences).

Therapeutic antibodies were as follows: anti-CEACAM1 mAb (Clone MAb-CC1; Cat. #: 134504; Biolegend, Santa Cruz, CA, USA) and Mouse IgG1 isotype control (Clone MOPC-21; Cat. #: 400153; Biolegend). Antibodies for flow cytometry and immunohistochemistry were as follows: anti-mouse CD3e (Clone 145-2C11; Cat. #: 15–0031-82; eBioscience, San Diego, CA, USA), anti-mouse CD4 (Clone GK1.5; Cat. #: 12–0041-82; eBioscience), anti-mouse CD8a (Clone 53–6.7; Cat. #: 11–0081-82; eBioscience), anti-mouse CD25 (Clone PC61.5; Cat. #: 25–0251-82; eBioscience), anti-mouse Foxp3 (Clone FJK-16 s; Cat. #: 11–5773-82; eBioscience), anti-mouse CEACAM1 (Clone 723629; Cat. #: MA5-24338; eBioscience), anti- human CD3 (Clone OKT3; Cat. #: 14–0037-82; eBioscience), anti- human CD4 (Clone RPA-T4; Cat. #: 11–0049-42; eBioscience), anti-mouse CD8a (Clone RPA-T8; Cat. #: 12–0088-42; eBioscience), anti-human CEACAM1 (Clone YTH71.3; Cat. #: MA5-17003; eBioscience),anti-mouse ki-67(SAB5700770; Sigma-Aldrich) and In Situ Cell Death Detection Kit (11684817910;Roche).