Fig 1: Expansion and characterization of γδ[T2] cells(A) Healthy donor PBMCs were activated with zoledronic acid (ZOL, n = 27) or immobilized anti-γδ TCR antibody (n = 15) and then cultured for 14–17 days in SFM containing IL-2 alone (γδ[2] cells) or IL-2 + TGF-β (γδ[T2] cells). The percentage of γδ T cells was measured in PBMCs (day 1) and after expansion in the indicated cytokines.(B) Absolute number of γδ T cells on day 1 and post-expansion. Median fold expansion is indicated for γδ[2] and γδ[T2] cells.(C–I) Post-γδ T cell expansion, the following markers were assessed in γδ[2] and γδ[T2] cells by flow cytometry: (C) % CLA; (D) % CXCR4; (E) binding to E-selectin-Fc fusion protein; (F) state of differentiation: naive (CD45RA+ CD27+), central memory (CM; CD45RA− CD27+), effector memory (CD45RA− CD27−), and terminally differentiated with CD45RA re-expression (TEMRA; CD45RA+ CD27−; (G) viability, apoptosis, and necrosis; (H) % CD103; and (I) % KLRG1.(J) γδ[T2] T cells and γδ T cells expanded from the same donor in human serum + IL-2 (γδ[2S]) were engineered to co-express firefly luciferase (ffLuc) and red fluorescent protein (RFP) and then analyzed by flow cytometry. GFP-expressing Jurkat cells were injected i.v. in NSG mice. After 4 days, 6 mice each were treated with 10 million RFP/ffLuc-expressing γδ[2S] or γδ[T2] T cells. Untrans., untransduced.(K) Mice were analyzed by BLI after 24 and 48 h to determine persistence of γδ T cells in the whole body and in a region of interest drawn around the femora (means ± SDs; n = 6 at 24 h; n = 3 at 48 h; 2-way ANOVA).(L) After each imaging session, 3 mice per group were culled. The % RFP+ (γδ) T cells present in spleen and bone marrow were determined (means ± SDs).(M) The % GFP+ (leukemic) cells present in bone marrow was also determined (means ± SDs). NS, not significant.(C)–(E) and (G)–(I) show means ± SDs and (A), (B), and (F) show medians ± interquartile ranges, in which data were or were not normally distributed, respectively. Accordingly, statistical analysis was performed using a Student’s t test or Wilcoxon signed-rank test, respectively.
Fig 2: IELLQAR inhibits selectin binding to monocytes. PBMCs and THP-1 cells were incubated with increasing concentrations of IELLQAR (1, 5, and 10 μM) in the presence of 10 μg/mL of P-, E-, or L-selectin for 30 min, analyzed by flow cytometry, and plotted as the percent inhibition vs. bovine serum albumin (BSA) control. IELLQAR exhibited dose-dependent inhibitory effects against selectin family binding to monocytes, with the highest inhibition of P-selectin binding to PBMCs and THP-1 cells (a), a moderate inhibition of E-selectin (b), and the lowest inhibition of L-selectin (c). Data are presented as the mean ± SEM (n = 3).
Fig 3: E-selectin binding is enhanced in CD44high populations.A ST3GAL1 expression level per cell in each cluster from scRNAseq analysis. Data are shown as points denoting values for each cell. B The percentage of HECA-452-positive cells in human CD45+/CD7+ T-ALL cells from the femur (orange), thorax (blue) and tail vertebrae (yellow) assessed via flow cytometry. PDX of five huT-ALL samples (17 mice). Flow cytometry analysis of M106-PDX, huT-ALL cells unstained (black line), femur (orange line), thorax (blue line) and tail (yellow line) (inset). C The frequency of huT-ALL cells from the femur (orange), thorax (blue), tail (yellow) of sham-treated mice and the femur of VADA-treated mice (purple). D Representative E-selectin binding/CD44 staining of huT-ALL (M106) cells from the femur, thorax and tail of sham-treated mice and the femur of VADA-treated mice as analyzed via flow cytometry. E MFI of CD44 in huT-ALL cells from the femur (orange), thorax (blue) and tail (yellow) of sham-treated mice and the femur of VADA-treated mice (purple) with in vitro E-selectin binding and non-binding controls. PDX of four huT-ALL samples (27 mice) F The proportion of E-selectin-bound huT-ALL cells according to treatment response (“NO” vs “YES”). G MFI of CD44 expression in huT-ALL cells according to E-selectin binding status (“NEG” vs “POS”). E–G The data are shown as box-and-whisker plots. The boxes indicate the 25th and 75th percentiles, whiskers indicate the range, and horizontal lines in each row represent the median. B–D Statistical significance was assessed via Kruskal–Wallis test followed by Dunn’s multiple comparisons test (**p < 0.01; ****p < 0.001). F, G Statistical significance was assessed via Mann & Whitney test (**p < 0.01). The huT-ALL samples are described in Supplementary Table 1.
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