Cd45 til microbeads

Panc02-SIY or MC38 tumors were treated ±12 Gy radiation as described above. Tumors were harvested 24 h posttreatment (n = 3 animals/group), processed into a single-cell suspension as described above and magnetically enriched using CD45⁺ TIL MicroBeads (Miltenyi Biotec). Enriched cells were labeled with viability dye and CD45-APC. Live CD45⁺ cells were sorted using a 100-μM nozzle on a BD Biosciences Aria cell sorter, and cells were processed according to the manufacturers protocol for the Chromium Single Cell 3′ Reagent Kit (v3.0) from 10X Genomics. Libraries were sequenced using an Illumina NovaSeq 6000 using the NovaSeq 6000 S2 Reagent Kit (v1.0). Data were processed using the Cell Ranger pipeline (v3.1) and subsequently analyzed with the Loupe Browser from 10X Genomics (v5.0). Using the Loupe Browser, differentially expressed genes between groups were considered significant if fold change of gene expression was > 1.3, and the Benjamini–Hochberg adjusted P-value was < 0.1. IPA software was from QIAGEN (v01-19-00), using default settings for Core Analysis. IPA canonical pathways related to “cellular immune response” with an absolute z-score greater than 2.0 and −log(P-value) greater than 1.3 were considered significant.

Tumor samples were processed using the mouse tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, No. 130-096-730). CD45⁺ tumor-infiltrating leucocytes were enriched from the cell suspension using CD45 (TIL) MicroBeads (Miltenyi Biotec, No. 130-110-618) according to the manufacture’s instruction. The single-cell suspension of tumor cells or isolated CD45⁺ cells with high cell viability was loaded and single cells were captured by the BD Rhapsody Single-cell Analysis System. Single-cell cDNA was prepared using BD Rhapsody cDNA Kit (No. 633773). The constructed cDNA libraries were sequenced with the Illumina NovaSeq PE150 platform and each unique molecular identifier was captured at least six times. For analysis, the sequencing data were processed using Rhapsody pipeline (V.1.8). The expression matrix of each sample was used for further analysis using the R package Seurat (V.3.1.4)28 (link) in R (V.3.5.1). As a quality-control step, low-quality cells (gene number <100, mitochondrial gene percentage >80%) and duplicated cells were excluded. Gene set variation analysis implemented in the GSVA package (V.4.0)29 (link) was used for gene set enrichment analysis (GSEA). The cell cycle analysis was performed using Seurat package according to the gene lists of cell cycles integrated in Seurat.

For separating of intratumoral CD45⁺ cells, single-cell suspensions of digested tumor were resuspended in a buffer (PBS, 0.5% bovine serum albumin, and 2 mm thylenediaminetetraacetic acid) and labeled with CD45 (TIL) MicroBeads (Order no. 130‐110‐618, Miltenyi Biotec Inc.) for 15 min in the dark at 4 °C. CD45⁺ cells were separated whit LS columns (order no. 130‐042–401, Miltenyi Biotec Inc.). The purity of the separated cells was used for scRNA-seq analysis.

Flow cytometry (FC) and fluorescence-activated cell sorting (FACS) were performed on a FACSCanto II and a FACS Aria IIIu (both BD Biosciences, Heidelberg, Germany), respectively. For FC and FACS, cells underwent a washing step by centrifugation at 310× g for 5 min at RT and were resuspended in approx. 250 μL PBS. Cells were stained with FITC- or VioBlue-labelled anti-human monoclonal REAfinity CD45 antibodies (Miltenyi Biotec) according to the manufacturer’s protocol. Unstained cells were used as controls. FC data were analyzed using FlowJo 10 (BD Biosciences).
Magnetic cell sorting (MACS) was used to separate CD45-positive (CD45^pos) and CD45^ko/CD19CAR-T cells three days after the CD45-KO electroporation. The cells were labeled using CD45 (TIL) MicroBeads (Miltenyi) and sorted through MACS columns into CD45^pos and CD45^ko fractions according to the manufacturer’s protocol. Successful sorting was confirmed via FC 24 h later.

Patients provided consent for the use of biospecimens for research as approved by the UT Southwestern Institutional Review Board. Freshly resected human tissues (squamous cell carcinoma from the base/lateral of tongue, cervical tumour tissues and a sentinel lymph node) were rinsed and divided into several sections (1–5 mm³) using a scalpel, followed by injection at multiple sites using 5% glucose control, free cGAMP (80 ng), PC7A polymer (50 μg) or cGAMP–PC7A NPs (80 ng cGAMP in 50 μg PC7A NPs) in 5% glucose solution within 30 min of resection. Each section was cultured in 0.5 ml RPMI 1640 medium (supplemented with 10% heat-inactivated human serum, 1% insulin-transferrin-selenium, 1% GlutaMAX, and 1% penicillin–streptomycin) in a 24-well plate for 24 h. RNA was isolated and RT–qPCR was performed as previously described. For CD45 selection, tumour tissues were first digested by 1 mg ml⁻¹ collagenase IV and 0.2 mg ml⁻¹ DNase I (Sigma-Aldrich) for 45 min at 37 °C, then passed through a 70 μm nylon cell strainer to obtain single cells. CD45⁺ leukocytes and CD45⁻ cell populations were collected using magnetic separation using CD45 TIL microbeads and MS columns (Miltenyi Biotec) according to the manufacturer’s instructions before RT–qPCR analysis.