Fig 2: Co-expression with CoV2 Mpro produces intracellular cleavage of the RFP biosensor. A) Graphic diagram of the RFP biosensor depicting two domains of RFP that had previously been mutated to weaken the interaction such that the two domains must be tethered as a single polypeptide (54 kDa) to allow domain dimerization to fluoresce (top). Upon Mpro cleavage of the AVLQS amino acid sequence, the two RFP domains (~27 kDa) dissociate, and fluorescence is lost (lower). B) Immunoblotting: of untransfected AD293 cells (lane 1) and cells transfected with either: the parental pRFPA1B1-DEVD vector (lane 2), or the wt pRFP-AVLQS vector (lane 3), with the Q of AVLQS mutated to A (pRFP-AVLAS, lane 4), pGST-Mpro (wt) (lane 5), pGST-Mpro with Q to A mutation in the N-terminal cleavage site (lane 6), pGST-Mpro with the catalytic C145 mutated to A (lane 7). Dual transfections were carried out with the following combinations: pRFP-AVLQS (wt) with pGST-Mpro (wt) (lane 8), pRFP-AVLAS with pGST-Mpro (wt) (lane 9), pRFP-AVLQS (wt) with pGST-Mpro (Q > A) (lane 10), pRFP-AVLQS (wt) with pGST-Mpro (C > A) (lane 11), and pRFP-AVLAS with pGST-Mpro (C > A) (lane 12). All were immunoblotted for RFP (upper panel) and for ß-ACT (lower panel) as a loading control. (The same extracts immunoblotted for GST to analyze Mpro expression and cleavage are shown in Supplemental Fig. 2.) C) Microscopy: The same transfections as in B were visualized using phase contrast and fluorescence microscopy, as indicated (same numbering as Fig. 3B). Bar = 156 µm. D) Quantification of the RFP fluorescence of the panels in Fig. 3C using the BioTek Cytation 1 as described in the Methods. E) Determination of effective pGST-Mpro:pRFP-AVLQS ratios: The Fiji software suite was used to quantify the signals of RFP immunoblots in HEK293T cells transfected with 1 µg pRFP-AVLQS and the indicated levels of pGST-Mpro. The intensity of the 54 kDa RFP band, normalized to LT, provided a measure of RFP levels, compared to the RFP 54 kDa level in cells transfected with pRFP-AVLQS alone set as 100%. Relative RFP levels in the cells co-transfected with varying amounts (1–0.005 µg) of pGST-Mpro established that ratios of ~1:4 pGST-Mpro to pRFP-AVLQS resulted in near linear changes in RFP intensity with changing Mpro levels, while still retaining sufficient RFP levels to readily detect fluorescence (also see Supplemental Fig. 3). A representative immunoblot used to generate this graph is shown in Supplemental Fig. 3A. Microscopy verifying these plasmid levels produce readily detectable levels of fluorescence is shown in Supplemental Fig. 3B. F) Time course of fluorescence: AD293 cells were either untransfected (squares), or transfected with pRFP-AVLQS (circles) or co-transfected with pGST-Mpro and pRFP-AVLQS (triangles) in the ratio of 1:4 for 21 h. The fluorescence output of live cells was monitored for Hoechst-DNA (blue) and biosensor expression (red), every hour for 20 h following the media exchange as described in Methods. (Micrographs at the 36th hour are shown in Supplemental Fig. 4).

Fig 3: Induction of PD-L1 expression on the target cells. (A) PD-L1 expression induced by supernatant from activated CAR T or CAR–NK-92 cells on cancer cells was assessed by flow cytometry. The control (only medium) and conditioned supernatants from the 24 hours coincubation cultures of control (unmodified) or PD-L1–CAR T/NK-92 cells with the target MDA-MB-231 cells were transferred onto the culture of MCF-7 and incubated for 48 hours. Next, PD-L1 surface presence was assessed using anti-PD-L1 antibody (clone MIH1) by flow cytometry. The experiment was repeated three times. (B) Representative images of different subpopulations of macrophages (M0, M0+IFN?, M2) stained for PD-L1 assessed by immunocytochemistry assay using Cytation 1 Cell Imaging Multi-Mode Reader (BioTek, Agilent). Macrophages were incubated with 10 ng/mL IFN? or IL-4 and IL-10 (for M2) for 5 days before staining (every 2-day fresh portion of cytokines was added). The control (only medium) and conditioned supernatants from the 24 hours coincubation cultures of control (unmodified) or PD-L1–CAR T cells with the target MDA-MB-231 cells were transferred onto different subpopulations of macrophages and incubated for 48 hours. Next, PD-L1 surface presence was assessed using anti-PD-L1 antibody (clone MIH1, cat. no. 14-5983-82, eBioscience, diluted 1:100). The signal was developed using AF488-conjugated secondary antibody (green), and nuclei were counterstained with DAPI (blue), scale bar: 100 µm. The background fluorescence was removed, and the low threshold for green fluorescence was set to create a mask of the area covered by macrophages. Bar graphs represent the mean fluorescent intensity of PD-L1 within the thresholded area. Data aggregated from three experiments performed in duplicates with two to four donors in each experiment (n=8–10). Bars represent the mean value±SD. Normality was checked using the Shapiro-Wilk test. The p values derived from Student’s t-test or Wilcoxon test (comparing to control), depending on data distribution: **p<0.01. Two-sample Kolmogorov-Smirnov test was used to calculate statistics between T cells and CAR T groups: #p<0.01. (C) The relative levels of human cytokines in supernatants collected from control (unmodified T cells and NK-92 cells) or PD-L1–CAR T cells and NK-92 cells cocultured with MDA-MB-231 breast cancer cells for 24 hours at 1:1 E:T ratio were determined by cytokine array assay. The changes in the expression profile of 36 cytokines are presented on the heat map. The experiment was performed with two donors in duplicates. (D) Representative PD-L1 expression in MCF-7 (left-hand panel) or SKBR-3 (right-hand panel) cells after a 24 hours incubation in the presence of 20 ng/mL IFN?, supernatants from coculture of unmodified T cells or PD-L1–CAR T cells with MDA-MB-231 and directly with HER2–CAR T cells at 1:1 E:T ratio. PD-L1 surface presence was assessed by flow cytometry using anti-PD-L1 antibody (clone MIH1). The experiment was performed three times in duplicates. CAR, chimeric antigen receptor, PD-L1, programmed death-ligand 1, IFN?, interferon ?, NK- natural killer, HER2, human epidermal growth factor receptor 2, IL-4, interleukin 4

Fig 4: Effects of drop delay on the yield and purity of sorted cells and beads. The effect of drop delay on the yield of beads was tested. Each of the 10 puddles was collected using a drop delay setting with 0.1 intervals. Precisely 50 droplets were deposited into each puddle at different drop delay settings. (A) The effect of drop delay on the yield was tested with automatic and manually adjusted drop delays. The red line is the automatic one, and the blue line is the other one. (B) Map of the correlation between drop delay and yield. (C) Effect of long-term sorting-related drop delay deviation on yield. (D, E) The R-max method was used to assess the effect of drop delay on yield and purity. CS&T beads consist of 3 µm bright or mid and 2 µm dim beads. According to the R-max method reported, two beads of 3 µm bright or mid and central sheath catch (CSC) were sorted at the same time. After flow cytometry detection, the R-max value and purity of two kinds of beads were analyzed. (D) Dot plots of flow cytometry detection. (a) Dot plots of CS&T beads. (b–g) Dot plots of CSC, mid and bright beads were sorted with delay value 31.26 (b–d, red frame) and 30.96 (e–g, blue frame). 31.26 is the optimal value. 30.96 is the value of deviation. (F) The yield and purity of cells were assessed under manually adjusted conditions. (G) The estimated drop delay for different experiments of testing drop delay changes. Jurkat cells stained with CFSE and Hoechst 33342 were mixed at 1 : 1 with unstained cells to obtain a suspension (1 × 107 cells·mL-1). The double-positive cells were sorted into 1536-well plates. Then, each well of cells in the 1536-well plate was scanned with a BioTek Cytation 1 Cell Imaging Multi-Mode Reader and imaged individually. The data were analyzed by gen5 3.04 software (BioTek, Winooski, VT, USA). Data are presented as the mean ± SEM. n = 3. Each experiment was repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance, by one-way ANOVA.

Fig 5: PMA-stimulated OCR measures NADPH oxidase activity. (A-B) OCR was measured by Seahorse in primary human monocytes treated with media alone or IFN? for 24 hours prior to the start of the assay (n = 5 technical replicates). (A) Monocytes from 4 healthy controls (black and green lines) and 4 CGD patients (red lines) were compared. (B) Monocytes were treated with (red lines) or without (black and green lines) DPI (100 uM) in addition to IFN? or media alone for 24 hours. All OCR values were normalized by staining cells with 2 µg/mL Hoechst 33342 (ThermoFisher Scientific) for 30 minutes. Cells were then imaged and counted using the Biotek Cytation1. Cell counts were calculated by cell imaging software (Agilent) and imported into Wave (Agilent) using the normalization function. Raw OCR values were normalized by cell counts per well. (C) Bar graphs displaying PMA-stimulated OCR values normalized to the basal OCR levels in healthy control plus medium samples run in the same experiment (data not shown). Data are representative of at least 3 independent of experiments and were analyzed by 1-way ANOVA with Tukey’s multiple comparisons test. Error bars are mean ± standard error of the mean. ****P < .0001.