Fig 2: (A) Binding traces for APC anti-CD3 monoclonal antibody (mAb) binding to Jurkat cells (red, blue, and green) (n = 3, reference subtracted with K562 cells) and the result from globally fitting all curves to a “one-to-one” model (black). (B) Binding traces for fluorescein isothiocyanate anti-CD32 mAb binding to HEK293 cells transfected with FcγIIa receptor (red and blue) (n = 2, reference subtracted with non-transfected HEK293 cells). As addition of the second concentration varies slightly in time, the results from fitting a “one-to-one” model to the individual curves are displayed (black). For all binding traces, fluorescent signals were normalized to 0% at baseline level and 100% at the end of the second ligand incubation to facilitate visual comparison.

Fig 3: LSD1 deletion reverses the inhibitory effect of GC cell-derived exosomes in T-cell response by influencing the expression of PD-L1 in target cells. a Confocal image of 20 μg/ml B-EXO or B KO-EXO that were co-incubated with MGC-803 cells for 24 h. Exosomes were stained with PKH26, followed by membrane dye DIO and nucleus dye DAPI staining. Representative confocal images are shown on the left, whereas quantification is shown on the right. Scale bar = 20 µm. b-d Membrane (b) and total expression (c and d) of PD-L1 in BGC-823 cell lines when incubated with B-EXO or B KO-EXO. e Confocal images of recombinant PD-1-Fc binding to BGC-823 cell-PD-L1 when cells were incubated with B-EXO or B KO-EXO. Cells were incubated with anti-rabbit Alexa Fluor 488 dye conjugated antibody. Scale bar = 50 μm. f Mean fluorescence intensity (MFI) of PD-1-Fc binding when cells were incubated with B-EXO or B KO-EXO. g Expression of CD69 in CD8+ T-cell when cells were incubated with B-EXO or B KO-EXO. h Cell survival of BGC-823 cells in anti-CD3/CD28-stimulated PBMC when incubated with B-EXO or B KO-EXO. The data are presented as the mean ± SD; n = 3; n.s, no significance, * P < 0.05 *** P < 0.001, two-tailed unpaired Student’s t-test

Fig 4: Generation and in vitro characterization of Tat-fusion proteins. A. Graphic representation of Tat-MYC and Tat-Bcl-2 fusion proteins. We cloned the cDNAs for either human MYC or human Bcl-2 in frame with the protein transduction domain of HIV-1 Tat as well as a V5 epitope tag and 6xHis tags. B. The fusion proteins localize to the appropriate intracellular compartment. A lawn of confluent 3T3 cells was exposed to either purified recombinant Tat-MYC or Tat-Bcl-2 or left untreated (NT). The cells were washed 2 hours later, fixed and stained with a monoclonal antibody to V5 (Green) and with a Hoechst 9934 nuclear stain (Blue). The Tat-MYC protein largely localized to the nuclear region in this timeframe, whereas the Tat-Bcl-2 remained in the cytoplasmic and perinuclear space. C. Tat-MYC rapidly localizes to the nucleus in primary human HSPCs. Human cord blood derived cells were pulsed with a single exposure of Tat-MYC. The cells were washed 1 hour later and the cells were collected at the indicated time points for analysis. The cells were lysed in a NP-40 buffer in order to separate the plasma membrane and cytoplasmic fraction from the nuclear fraction. All samples were analyzed by SDS-PAGE and western blot using monoclonal antibody to V5 and ß-actin. D. Tat-MYC was present in the nuclear fraction for a transient period of time. Human cord blood cells were treated as described for panel C. The nuclear fractions were analyzed by SDS-PAGE and western blotting using a monoclonal antibody to the V5 tag. The bulk of the protein was lost between 24 and 48 hours. There was no detectable protein left at any point after 48 hours. E. and F. To test the function of Tat-Myc and Tat-Bcl-2, primary murine CD4+ T-cells were activated with monoclonal antibody to CD3 for 48 hours. The frequency of live cells was measured in the starting population of cells (black bars). The cells were then replated in media alone (NT), or media supplemented with Tat-Cre (TC) or increasing concentrations of either Tat-MYC (TM) (E), or Tat-Bcl-2 (TB) (F). Activated T-cells cultured with Tat-MYC, but not Tat-Cre, retain a blasting phenotype and continue to proliferate after the antigenic stimulation G. To further test the biological function of recombinant Tat-MYC, primary murine CD4+ T-cells were CFSE labeled and activated in vitro with monoclonal antibody to CD3 for 48 hours. After activation, the cells were washed and the live cells were isolated by ficoll-hypaque centrifugation. The cells were then replated in media supplemented with 5 µg/ml Tat-Cre (first panel) or 5 µg/ml Tat-MYC (second panel). The frequency of live cells was measured by flow cytometry. H. The activated T-cells were further assessed by CFSE signal in order to determine whether the addition of Tat-MYC can promote proliferation in addition to survival.

Fig 5: CD151 is enriched in memory and Ki67+ T cells. Box and whisker plots indicate frequencies of CD151+ cells among total CD3+ cells, CD4, CD8, naïve, memory and Ki67+ T cells in GG (n = 6; blue circles) and CC (n = 6; red circles) genotype groups, without (open circles) and with (closed circles) Ct stimulation for seven days. The boundaries of the box indicate the 25th and 75th percentiles; the line within the box indicates the median; whiskers indicate the 10th and 90th percentiles. Each dot represents one subject. The relative frequencies of CD151+ lymphocytes were significantly increased in the CC group with and without Ct stimulation compared to GG group among total CD3+ cells, CD8, naïve and Ki67+ T cells by Wilcoxon Rank Sum Test. *:P < 0.05; **:P < 0.01; ***:P < 0.005. No statistical differences were detected between media and Ct-stimulated groups for any cell type.