Cd4 bv421

The following Abs were purchased from BD bioscience: human CD3-APC-H7 (Cat#: 560176, dilution: 1:100), CD4-BV421 (Cat#: 562424, dilution: 1:100), CD8-APC (Cat#: 340584, dilution: 1:100), CD45-BV510 (Cat#: 563204, dilution: 1:30), CD45RA-PE (Cat#: 555489, dilution: 1:30), CCR7-FITC (Cat#: 561271, dilution: 1:50) and B7-H3-BV421 (Cat#: 565829, dilution: 1:100); human CCR2-BV421 (Cat#: 357210, dilution: 1:100) Ab was purchased from Biolegend. Expression of human B7-H3 in tumor cell lines and organoid cells was assessed with the 376.96 mAb followed by APC-goat anti-mouse IgG Ab (BD Biosciences, Cat# 550826, RRID: AB_398465, dilution 1:100), and confirmed with another B7-H3 mAb (Clone 7–517; BD Bioscience, Cat#: 565829, RRID: AB_2739369, dilution 1:100)^{32 (link)}. Expression of the B7-H3.CARs was detected using the fusion protein 2Ig-B7-H3-Fc (R&D Cat# 1027-B3) and followed by AF647-goat anti-human IgG (H+L) Ab (Jackson ImmunoResearch Laboratories Inc., Cat# 109-606-088, dilution 1:100); CD19.CAR was detected with the previously described anti-CD19.CAR mAb^{47 (link)}. Samples were acquired with BD FACS Canto II or BD FACS Fortessa using the BD Diva software (BD Biosciences). For each sample, a minimum of 10,000 events were acquired and data were analyzed using Flowjo 10.

To determine the percentage of different T cell subsets, at least 1 × 10⁶ PBMCs were suspended in isoflow sheath fluid (Beckman Coulter) and stained in a DuraClone IM T cell tube (Beckman Coulter, Miami, FL) according to the manufacturer's protocol. The T cell tube contained anti-CD45, CD3, CD8, CD4, CD45RA, and CCR7. Samples were measured by use of the Navios flow-cytometer (Beckman Coulter).
To determine the Tfh, at least 1 × 10⁶ PBMC were washed by Fascflow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark.

Patient's PBMC were stimulated for 3 days at 37°C, 5% CO₂ and 95% humidity with CSFE-labeled, irradiated (40 Gy) donor PBMCs, depleted for CD3⁺ T cells, in culture medium. At the end of day 2 monensin and brefeldin A (GolgiStop and GolgiPlug, BD Biosciences) were added for 16 h in a concentration of 1:1,500 and 1:1,000, respectively, to allow the measurement of intracellularly accumulated cytokines in PBMCs. Thereafter, intracellular IL-21 was measured, and surface marker staining was used to investigate which subsets produced these cytokines. Monoclonal antibodies used for surface marker staining and intracellular cytokine staining were CD3 BV510 (BioLegend), CD4 BV421 (BioLegend), CD8 PerCP (BD Biosciences), CXCR5 APC (BD Biosciences), PD-1 APC-Cy7 (BioLegend), and IL-21 PE (Biolegend).

For flow cytometry, single-cell suspensions of mouse cLNs and lungs were prepared according to published protocols.^{53 (link),62 (link)} Red blood cells were removed by lysis buffer (00-4300-54, eBioscience). Single-cell suspensions were incubated with flow cytometry antibodies targeting surface markers. For intracellular cytokine staining, the suspensions were stimulated using Leukocyte Activation Cocktail with BD GolgiPlug (423303, Biolegend) for 5 h. Antibodies against CD45-BV510 (103137, Biolegend), CD3-FITC (553061, BD Pharmingen), CD4-BV421 (100543, Biolegend), CD8-PE-Cy7 (100721, Biolegend), IL-17A-PE (506903, Biolegend) and IFN-γ-APC (505809, Biolegend) were used at a 1:200 dilution ratio. Dead cells were excluded from analysis using the Zombie NIR™ Fixable Viability Kit (423106, Biolegend). Samples were analyzed using a BD LSRFortessa X-20 (BD Biosciences) or CytoFLEX (Beckman Coulter). Results were analyzed using FlowJo 10.5.3 software (BD Biosciences).

Flow-cytometry antibodies included CD3e-PE, PD1-BV605, CD45-BV786 (BD-Biosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-APC, CD45.1-PE (eBioscience), CD45.2-Alexa700, CD-8α-Percp-Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas-red (Life Technologies, Carlsbad, CA, USA). Blocking PD-1 antibody (clone RMP1-14, BioXCell, Branford, CT, USA) was administered systemically by intraperitoneal injections of 250 μg [41 (link)]. Blocking anti-CD40L (clone MR1, BioXCell) was administered systemically by intraperitoneal injections of 250 μg [19 (link)].