Cd4 bb700

A 1: 1 ratio was used in co-culturing NT or CAR-T cells pretreated with Dec (0.05, 0.1μM) or Aza (0.5, 1μM) with MV4-11 cells for 16 h. Meanwhile CD107a-BV421 (BD Biosciences, Cat#562623) and protein transport inhibitor (BD Biosciences, Cat#554724) were added to the culture medium. After cells collection, Antibodies CD3-APC/Cy7 (Biolegend, Cat#300318), CD4-BB700 (BD Biosciences, Cat# 566392), and CD8-BB515 (BD Biosciences, Cat# 564526) were applied to stain the cells.

Cryopreserved PBMC from patients hospitalized because of COVID-19 severe interstitial pneumonia (severe) and OTD subjects were thawed, washed and rested for 30 min in complete RPMI-1640 medium supplemented with 10% FCS, 2mM L-glutamine and antibiotic antimycotic solution (100 U/ml penicillin, 0.1 μg/ml streptomycin, 0.25 μg/ml amphotericin B (Sigma- Aldrich, St. Louis, MO, USA) (Complete Medium). Subsequently, PBMC were washed and stained for flow cytometric analysis using the following fluorochrome conjugated antibodies: CD8 BV421, CCR7 BV510, PD1 PE-CF594, CD45RA BV650, CD56 BV786, TIM-3 BB515, CD69 PE, CD4 BB700, CD3 APC-H7 (BD Biosciences, San Diego, CA, USA). After fixation and permeabilization (Fixation/Permeabilization Solution Kit; BD Biosciences), cells were stained with anti-Granzyme B Alexa 647 (BD Biosciences). To detect circulating T follicular helper (TFH) cells 1×10⁶ PBMC were stained with the following antibodies: CD3 BV510, CD4 BB700, CD8 BV786, PD1 PE and CXCR5 BV421. The following antibodies were used to identify plasma cells: CD19 BV605, CD27 BB515, CD38 BV421and CD138 BV480. Flow cytometry was performed with a 12-color Celesta (BD Biosciences) instrument and data analyzed with FlowJo 10 software.

The CAR-expressing T cells were identified by incubating with Biotinylated Protein L (GeneScript, Cat# M00097) at 4°C for 45 min and then washing them off before incubating with streptavidin-FITC (BD Biosciences, Cat# 554060) for 15 min in the dark. To analyze apoptosis, Annexin V-FITC/PI kit (BD Biosciences, Cat# 556547) was used to detect Dec (Selleck, NSC 127716) or Aza (Sigma, A2385) treated CAR-T cells or tumor cells. Assays involving cells surface antigens were performed in the dark at 4°C for 30 min with the corresponding antibodies. Antibodies (BD Biosciences) TIM3-PE (Cat# 563422) and CD69-BV421 (Cat# 562884) were applied to test the exhaustion and activation of CD44v6 CAR-T. CD4-BB700 (Cat# 566392), CD8-PE-cy7 (Cat# 557746), CCR7-Alexa 647 (Cat#557734), CD45RA-PE (Cat#555489) or CD45RA-BV421 (Cat# 562885) were utilized for detecting the phenotype of non-transduced T (NT) or CAR-T cells. CD44v6-PE (Cat# 566803) was applied to test the CD44v6 expression in AML cells. The samples were all detected by BD LSRFortessa X-20 flow cytometry, and these data were analyzed with FlowJo_V10.4 software.