Immunoprep reagent system

Immediately after blood collection, 100ul of EDTA whole blood was labelled with CD45-ECD, CD18-FITC, CD62L-PE (Beckman), and CD181-PC5 (BD Biosciences) or with CD45-ECD, CD64-FITC, and CD11b-PE for 10 min at room temperature,. Samples underwent red cell lysis and fixation using the ImmunoPrepTM reagent system (Beckman-Coulter) according to the manufacturer’s instructions and were analysed using a 3 laser Beckman-Coulter Navios Flow Cytometer. Cell surface marker expression was measured by gating on CD45+ granulocyte and monocyte populations and collecting the mean fluorescence intensity (MFI) values for the CD18, CD62L, CD181, CD64, and CD11b markers on granulocytes and the CD62L, CD64, and CD11b markers on monocytes.

After oral administration of MT-1303 at 0.3 mg/kg once daily for 3 days, blood and mesenteric lymph nodes were collected from C57BL/6 mice under isoflurane anesthesia. Single-cell suspensions were prepared from the mesenteric lymph nodes. Expression of the S1P₁ receptor on lymphocytes was examined by incubating the cell suspensions with rat anti-mouse S1P₁ mAb or rat IgG2a isotype control (eBR2a; eBioscience) at 40 μg/mL in flow cytometry staining buffer solution (FCS buffer; eBioscience) containing 1 mM EDTA and 2% normal mouse serum on ice in the dark for 1 h. The cells were then washed with PBS and incubated with biotin-conjugated anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) at 7 μg/mL for 20 min, followed by PE-conjugated streptavidin (eBioscience) at 1 μg/mL for 20 min. Cell suspensions were subsequently incubated with 2% normal rat serum in FCS buffer for 20 min, followed by FITC-conjugated anti-mouse CD4 mAb for 20 min. After washing, the cells were resuspended in FCS buffer for flow cytometric analyses (LSR, BD Biosciences). Collected peripheral blood (0.1 mL) was hemolyzed and fixed using the IMMUNOPREP^TM Reagent System (Beckman Coulter). The number of lymphocytes, T cells, and CD4⁺ T cells was measured using a Cytomics^TM FC500 flow cytometer (Beckman Coulter) with Flow-Count^TM (Beckman Coulter) as the internal standard.

One peripheral blood EDTA tube was drawn per patient (n=70) prior to ICI therapy initiation, at an early (after one to two cycles, n=51) and late time-point (after three to five cycles, n=47) during therapy. Freshly isolated cells were lysed using the Immunoprep Reagent System (Beckman Coulter) and staining was performed with two different flow cytometry panels. Panel 1 was stained with the antibody mix CD45-FITC/CD56-PE/CD19-ECD/CD3-PC5, to which the antibody CD-16 PE was added, and panel 2 was stained with the antibody mix CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5, to which the antibody HLA-DR-PC7 was added (all antibodies from Beckman Coulter, Krefeld, Germany), according to manufacturer´s instructions. Flow-cytometry analysis was carried out and analyzed using NAVIOS cytometer and analysis software (Beckman Coulter). These analyses were performed within the clinical routine diagnostics of immune status by the hematological laboratory of the department of medicine IV of the University Medical Center Aachen, which includes standardized gating strategy to distinguish B cells (CD19+), NK cells (CD3-CD56+CD16+), and T cell subsets (CD3+CD4+, CD3+CD8+, CD3+CD56+CD16+, CD3+HLA-DR+) (Supplementary Figures 1A–D).