Characterization of murine CEACAM1 in vivo reveals low expression on CD8(+) T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1

McLeod, Robbie L.; Angagaw, Minilik H.; Baral, Toya Nath; Liu, Liming; Moniz, Raymond Joseph; Laskey, Jason; Hsieh, SuChun; Lee, Mike; Han, Jin-Hwan; Issafras, Hassan; Javaid, Sarah; Loboda, Andrey; Sadekova, Svetlana; O'Connor, Joann A.; Tse, Archie; Punnonen, Juha

Immunophenotyping of tumor cells from the CT26, MC38, MB49, and MBT2 models conducted by flow cytometry. Tumor cells were excised at the indicated time point, minced by mechanical digestion, and subjected to enzymatic digestion using a gentleMACS and the mouse tumor digestion kit per the manufacturer's instructions (Miltenyi Biotec). Following digestion, single cell suspensions were obtained after filtration and multiple washes, from which the absolute live cell count was determined using a ChemoMetec NucleoCounter. Cells suspension were then stained with a fixable LIVE/ DEAD stain (Life Technologies), washed, and exposed to mouse Fc-Blok (BD Biosciences). Immune cell profiling was conducted using anti-mouse antibodies (clone) purchased from either BD Biosciences, BioLegend, or eBioscience/Thermo: CD45 (30-F11); CD8 (53-6.7); CD3 (145-2C11); TCRβ (h57597); CD4 (GK1.5); CD11b (M1/70); Ly6G (1A8); Ly6C (AL-21); CD45R (RA3-6B2); CD11c (N418); I-A/I-E (M5/114.15.2); or CD66a (clone CC1). For intracellular staining, cells were permeabilized using Foxp3/Transcription Factor Staining Buffer Set and incubated with anti-mouse Foxp3 (FJK-16S) and HELIOS (22F6) (eBioscience). Stained cells were fixed in 4% formaldehyde, washed with PBS, and stored at 4° C until analysis on a BD LSRFortessa (BD Bioscience). Data analysis was performed using FCS Express (De Novo Software) or FlowJo (FlowJo LLC). 10 mice per treatment group were included in all flow cytometry analyses. Immune subset discrimination was determined by gating on live, CD45+ cells in the leukocyte gate (FSC vs SSC size discrimination) via the following gating schemes; T cells (TCRβ+CD3+CD4 or CD8+), Treg (FOXP3+HELIOS+ in the CD4+ T cell gate), G-MDSC (CD11b+I/A-I/ElowLy6G+Ly6Clow), M-MDSC (CD11b+ I/A-I/ElowLy6G-Ly6C+), cDC (I/A-I/EhighCD11blow/-CD11c+CD45R−), pDC (I/A-I/ EhiCD11b-CD11clowCD45R+), macrophages (CD11b+ I/A-I/EhighLy6C+F4/80+), monocytes (CD11b+ I/A-I/ EhighLy6C+F4/80low), B cells (I/A-I/EhiCD11b-CD11c-CD45R+), and "NK-like" cells (CD45+CD49b+). For each population, a minimum of 250 events were acquired.