Fig 2: Gating Strategy. Overlapping events were ruled out from the FSC-H/FSC-A analysis. Subsequently, the populations were selected by size and complexity. From the lymphocyte region, T lymphocytes were identified as CD3 + and CD4 + cells, and CD19+ for B lymphocytes. Monocytes were identified as CD14+ cells. Once the three populations had been selected, the positive cells for CD74, CD44, CXCR2, CXCR4 and CXCR7 receptors were identified from the pre-established FMOs (CD44 are represented in CD4+ T lymphocytes, CD74 in B lymphocytes, CD44 in monocytes and CXCR2 in granulocytes). FSC-H/FSC-A; FSC-Height (FSC-H) by FSC-Area (FSC-A). FMO; Fluoresence minus one.

Fig 3: Validation of RG7769 receptor occupancy (RO) assay for whole-blood T cell RO analysis. (A) Gating strategy for RO calculation. I. In all conditions, single lymphocytes were identified by excluding cell doublets via FSC-H versus FSC-W followed by SSC-H versus SSC-W. II. Total lymphocytes were gated on via CD45 staining versus SSC-A. III. Within the CD45+ compartment, T cells were identified via CD3 expression. A non-T cell gate (NOT-CD3+) was set. IV. Within total CD3+ T cells, CD4+ (Q3) and CD8+ single-positive (Q1) T cells were identified. V. In the non-T cell population, NK cells were identified via CD56 versus CD16 (QI-III). Total NK cells were defined as CD56+CD16+/-. Lower right: Comparing the anti-PGLALA-PE staining intensity of CD3+ T cells, CD4+ and CD8+ T cells pulsed with RG7769. Orange: 1 µg/ml, red: Saturation (100 µg/ml); gray: Negative control (1000 ng/ml + mIgG2b-isotype-PE). (B) Dose–response of RG7769 binding to healthy donor lymphocytes. Using the gating strategy in A, CD45+ lymphocyte single cells from blood pulsed ex vivo with RG7769 (0 ng/ml?100 ng/ml), were identified. The control group (0 ng/ml) was stained with mIgG2b-PE isotype, cells with RG7769 with anti-PGLALA-PE. Five thousand concatenated CD45+ events were clustered according to CD3, CD4, CD8, CD56 and CD16 by UMAP. Left: Cell lineages identified using FlowSOM include CD4+ T cells, CD8+ T cells, CD4-CD8- unconventional T cells as well as NK cell subsets. Right: PE+ (RG7769+) T cells (blue) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: CD147 is an alternative receptor for SARS-CoV-2 infection in ACE2-deficient cell types. a No interaction of CD147 and ACE2 was detected by Co-IP assay. The mIgG and rIgG were served as negative controls. b No co-localization was found between CD147 and ACE2 by FRET. The color bar denotes FRET ratio. Scale bars: 10 µm. c No co-localization of CD147-ACE2 and the co-localizations of spike-ACE2 and spike-CD147 were observed by immuno-electron microscope (scale bars: 200 nm) and multicolor immunofluorescence (magnification: ×200) in lung tissues from COVID-19 patient. Spike protein, 10 nm-gold colloid, purple arrows; CD147, 20 nm-gold colloid, blue arrows; and ACE2, 40 nm-gold colloid, green arrows. d Virions (red arrows) were observed in lymphocytes of lung tissues from COVID-19 patient. Scale bars: 500 nm. The localization of spike protein and CD3 was analyzed by multicolor immunofluorescence staining. Magnification: ×200. e The gene expressions of CD147 and ACE2 in CD4+ and CD8+ T cells were detected by real-time PCR (***p < 0.001, two-tailed t-test, mean ± SEM). f The expressions of CD147 and ACE2 in unactivated or activated T cells were detected by real-time PCR (*p < 0.05, Mann–Whitney test, mean ± SEM). g The infection efficiency of SARS-CoV-2 pseudovirus was detected by luciferase reporter assay in unactivated and activated T cells (*p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t-test, mean ± SEM). h SARS-CoV-2 pseudovirus infection of CD4+ and CD8+ T cells was inhibited by Meplazumab (***p < 0.001, two-tailed t-test, mean ± SEM). i The gene expressions of CD147 and ACE2 in Vero E6 and BEAS-2B cells were detected by real-time PCR (***p < 0.001, two-tailed t-test, mean ± SEM)

Fig 5: Assessing RG7769 binding to T cells in anti-PD-1 experienced patients. (A) Comparison of the frequency of NP40435 patients with prior aPD-1 experience with aPD-1 naïve patients. (B) Comparison of RG7769 CD3+ T cell receptor occupancy (RO) pre- and post-infusion in NP40435 patients. (C) Control in vitro experiment assessing the effect of PD-1 competition on RG7769 binding to CD3+ T cells. Activated peripheral blood mononuclear cells (PBMCs) were incubated with IgG4 isotype, nivolumab or pembrolizumab (100 pg/ml?100 µg/ml), then pulsed with RG7769 (10 µg/ml), and analyzed for T cell anti-PGLALA-PE binding. The pooled MFI for PE is shown (data from two independent experiments). (D) Linear regression of max. RG7769 (MFI) to CD3+ T cells in NP40435 anti-PD-1+ patients prior to RG7769 infusion (C1D1_pre) as a function of the last reported prior anti-PD-1 administration. R 2 = .8338, p = 0.0015. (E) anti-PGLALA-PE staining on CD3+ T cells of representative NP40435 patients. Maximum anti-PGLALA-PE assessed post ex vivo blood saturation with RG7769, in recently (up to 14 weeks) or historically experienced (>14 weeks) patients versus naïve patients prior to RG7769 (C1D1_pre). Anti-PGLALA–PE intensities are shown for ex-vivo saturation and neg. control. (F) CD3+ T cell RG7769 mean ABC in recently (<14 weeks) or historically (>14 weeks) aPD-1+ patients versus naïve. Data are pooled from (E). ABCs were compared to aPD-1 naïve (Welch ANOVA test). (G) Schematic of RG7769 binding to T cells in aPD-1+ patients. Left: RG7769 engages T cells via high-affinity anti-PD-1 engagement if PD-1 is freely available. Right: In situations where prior aPD-1 is engaging PD-1, RG7769 has to displace prior bound aPD-1 antibodies. Steric hindrance may occur if competing mAbs target an epitope in the PD-L1 interaction side of PD-1 [Color figure can be viewed at wileyonlinelibrary.com]