Fig 2: Exclusion of CD5hi and TCRαβhi cells from analysis enhances purity of sorted DEsIn this experiment, a PBMC sample from T1D patients was stained using our four-antibody cocktail, acquired and two gating strategies used to identify and sort DEs as shown below. Purity was determined by rerunning sorted DEs. Top panel. The plots show how inclusion of all CD5+ cells (left plot) and TCRαβ+ cells (middle plot) significantly lowers purity of sorted DEs (right plot). The number in the right plot shows percentage of bona fide DEs (19%) among sorted cells. Bottom panel. The plots show how restricting gating to CD5lowCD19+ (left plot) and TCRαβlow cells (middle plot) cells significantly increases purity of sorted DEs (right plot). The number in the right plot shows percentage of bona fide DEs (82%) among sorted cells.

Fig 3: Consequential gating strategy for acquiring samples and detecting DEs, Bcon, and Tcon by using FACS and FlowJo(A) Dot plots show how to sequentially gate live single lymphocytes and exclude doublets and dead cells in your PBMC sample.(B) Left dot plot shows how to use a 2-parameter plot of CD19 versus CD5 to divide gated lymphocytes into Bcon (CD5−CD19+); Tcon (CD5+CD19−), and CD5+CD19+ populations. Right dot plots show how to analyze each gated subpopulation for DEs as identified by the coexpression of TCRαβ and BCR. Note: most DEs are located within the CD5+CD19+ compartments, rare among Bcon, and virtually absent among Tcon. Consequently, analysis and sorting of DEs, is limited to DEs localized within the CD5+ CD19+ population. Numbers indicate percentages in each quadrant. This figure is adopted from the manuscript by Ahmed and colleagues (Ahmed et al., 2019).

Fig 4: Expression of HK2 in B cell leukemiaPeripheral blood mononuclear cell samples from patients with chronic lymphocytic leukemia or healthy controls were assessed for HK2 expression by flow cytometry.(A) HK2 MFI within gated CD19+ cells. Each dot represents an individual patient or healthy control.(B) Spearman’s correlation of HK2 MFI with percent ZAP70 expression among CLL cells (determined by clinical lab testing).(C) Proliferative versus quiescent CLL cell populations were gated based on CXCR4 and CD5 expression (left). HK2 expression in each individual patient’s proliferative and quiescent cell fractions are connected by lines (right). Significance was determined by Wilcoxon test.(D) Pearson’s correlation of HK2 MFI with CXCR4 expression among CLL cells. See Table S2 for CLL patient demographics and clinical parameters and Figure S5 for the association of HK2 expression with other CLL clinical parameters.(E) CLL cells were cultured overnight with CD40L+IL-4+anti-IgM (Stim) alone or with pan-PI3K inhibitor (1 μM pictilisib) prior to assessment of HK2 expression. Significance was determined by Wilcoxon test.

Fig 5: In vivo SpiD3 treatment yields an immunostimulatory response. (A) Schematic of experiment design: Eµ-TCL1 mice with comparable leukemia burden were treated intravenously with SpiD3 prodrug (SpiD3_AP, 10 mg/kg; n = 6) or equivalent vehicle (Veh; 50% PEG400, 10% DMSO, 40% water; n = 5) once daily for 3 days, as previously reported [20]. Following treatment, spleen cells were collected for flow cytometry analysis and plasma was isolated from murine blood; (B) leukemic (CD19+/CD5+) cells from murine spleens were analyzed by flow cytometry for changes in surface CALR expression (ecto-CALR) and compared to the percentage of leukemic cells detected in spleens of the same mice (as reported in Eiken, et al. [20]). The concentrations of plasma inflammatory cytokines and chemokines were assessed using Mouse Anti-Virus Response (C,E); and Mouse Pro-Inflammatory Chemokine (D,F) LEGENDplex™ flow cytometry-based multiplex immunoassays. (C,D) Heatmaps display fold change in the plasma analyte concentration compared to the average of vehicle-treated mice. Columns represent individual mice per treatment group. (E,F) Raw plasma analyte concentration and correlation with the percentage of CD19+/CD5+ spleen-derived cells are shown for select analytes. Individual data points (Veh = black circles; SpiD3_AP = blue triangles) in addition to summary statistics (mean ± SEM) are shown. Comparisons between treatment groups were analyzed by unpaired t-test. Asterisks denote magnitude of significance: * p < 0.05, ** p < 0.01.