Additional file 1 of Depletion of polyfunctional CD26highCD8+ T cells repertoire in chronic lymphocytic leukemia

Additional file 1: Figure. S1. a Representative flow plot of the purity of isolated CD3+ T cells, (b) CD3+CCR7- cells, and (c) CD19+ B cells. d Representative flow plots of the gating strategy for CD26 staining in CD8+ T cells. e Cumulative data showing the number of CD26low and CD26high CD8+ T cells as normalized in 100,000 CD8+ T cells in HC and CLL patients. f Cumulative data comparing the Mean Fluorescence Intensity (MFI) of CD26 in CD26low and CD26high CD8+ T cells. g Cumulative data comparing the frequency of CD26+CD8+ T cells in female versus male CLL patients. h Correlation between the age of CLL patients and the frequency of CD26+CD8+ T cells. i Cumulative data comparing the frequency of CD26+ and, (j) CD26low, CD26high CD8+ T cells in treated versus non-treated CLL. k Cumulative data are comparing the proportion of CD26+CD8+ T cell in three clinical stages of CLL (Low/Intermediate/high) based on the Rai staging system. l Correlation between CD26+CD8+ T cell frequency and lymphocyte counts (x103/ $$\mu$$ μ l) in CLL. m Representative flow plots, and (n) cumulative data of the frequency of CD26 among CD3- and CD3+ T cells in HC and CLL. (o) Cumulative data showing the frequency of CD26+CD4+ T cells, and (p) CD56+NK cells in HC versus CLL. q Cumulative data showing the MFI of CD26 in B cells from HCs and malignant B cells (B-CLL). r Cumulative data of the concentrations of soluble CD26 (ng/ml) in the plasma of HC and CLL. s Cumulative data of the frequency of CD26+, and (t) CD26low, CD26high CD8+ T cell in the peripheral blood versus bone marrow of CLL. u Cumulative data of CD26 mRNA expression in CD8+ T cells of HCs vs. CLL (n=15). Error bars represent the median with an interquartile range. Each dot represents an individual human sample. Figure. S2. a Representative plots, and (b) cumulative data of the frequency of CD8+ T cell subsets (e.g. naïve, stem cell memory, central memory, transitional memory, effector memory, and effectors). c Representative flow plots, and (d) cumulative data showing the frequency of CD27 expressing cells among CD26neg/low/high effector memory subsets of CD8+ T cells in CLL. e Representative plots, and (f) Cumulative data of the frequency of TV $$\alpha$$ α 7.2+CD161high in CD26high CD8+ T cells in HC. g Cumulative data of the frequency of TV $$\alpha$$ α 7.2+ CD161high in CD26highCD8+ T cells in HC versus CLL. h Cumulative data of the frequency of CD160+, (i) 2B4+, (j) PD-1+, (k) TIGIT+, (l) ICOS+, (m) CD28+ , (n) CD27+, (o) CD39+ , and (p) CD73+ among CD26neg, CD26low, and CD26high CD8+ T cells in HC. q Representative plots of the co-expression of CD26 and CD73 in different CD8+ T cell subsets (naïve, central memory, effector memory, and effector) in CLL. r The pie charts show the pattern of CD26 and CD73 co-expression in different CD8+ T cell subsets in CLL. s Cumulative data of the frequency of CD26+CD73+ versus CD26+CD73- in CD8+ T cells of HC versus CLL. t Cumulative data of the frequency of co-inhibitory/co-stimulatory receptors in CD26highCD8+ T cell subset in HC and CLL. Error bars represent the median with an interquartile range. Each dot represents an individual human sample. Figure. S3. a Cumulative data of the frequency of co-inhibitory/co-stimulatory receptors in CD26neg CD8+ T cell subset in HC and CLL. b Cumulative data of the frequency of GzmB+, (c) Perforin+, and (d) GzmB+Perforin+ among CD26neg, CD26low, and CD26high CD8+CCR7- T cells in CLL. e Cumulative data of the frequency of GzmB+, (f) Perforin+, and (g) GzmB+Perforin+ CD8+ T cells among CD26neg, CD26low, and CD26high subsets in HC. h Representative plots, and (i) cumulative data of the frequency of GzmB and perforin expressing cells among CD26neg, CD26low, and CD26high CD8+ T cells in CLL either unstimulated (black color) or stimulated for following 5 h with anti-CD3/CD28 (3 $$\mu$$ μ g/ml, 1 $$\mu$$ μ g/ml). j Cumulative data of the frequency of TNF- $$\alpha$$ α +, (k) IFN- $$\gamma$$ γ +, and (l) TNF- $$\alpha$$ α +IFN- $$\gamma$$ γ + cells among CD26neg, CD26low, and CD26high CD8+ T cells in CLL. m Cumulative data of the frequency of TNF- $$\alpha$$ α +, (n) IFN- $$\gamma$$ γ +, and (o) TNF- $$\alpha$$ α +IFN- $$\gamma$$ γ + among CD26neg, CD26low, and CD26high CD8+ T cells in HC. p Representative plots, and cumulative data of the frequency of (q) CCR4+CCR6+ cells among CD26neg, CD26low, and CD26high CD8+ T cells in CLL, considered as Tc17 cells. r Representative plots, and (s) cumulative data of the frequency of CXCR3+CCR6+ expressing cells among CD26neg, CD26low, and CD26high CD8+ T cells in CLL, considered as Tc1/Tc17 cells. t Cumulative data of the frequency of CXCR3+CCR6- among CD26neg, CD26low, and CD26high CD8+ T cells in CLL, considered as Tc1 cells. u Cumulative data showing the frequency of CCR4+CCR6- among CD26neg, CD26low, and CD26high CD8+ T cells in CLL, considered as Tc2 cells. Error bars represent the median with an interquartile range. Each dot represents an individual human sample. Florescence minus one (FMO). Figure. S4. a Cumulative data of the expression of T-bet in different CD8+ T cells subsets in CLL. b Cumulative data of the expression of FOXP3 in different CD8+ T cells subsets in CLL. c Representative plots, and (d) cumulative data showing the intensity (MFI) of TOX expression among CD26neg, CD26low, and CD26high CD8+ T cells in CLL. e Representative plots, and (f) cumulative data showing the intensity (MFI) of CCR5 expression among CD26neg, CD26low, and CD26high CD8+ T cells in CLL. g Representative plots, and (h) cumulative data showing the intensity of CCR6 expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. i Representative plots, and (j) cumulative data showing the intensity of Integrin- $$\beta 7$$ β 7 expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. k Representative plots, and (l) cumulative data of the intensity of CCR7 expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. m Representative plots, and (n) cumulative data of the intensity of CLA (Cutaneous Lymphocyte Antigen) expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. o Cumulative data of the frequency, and (p) the intensity of CXCR3+ expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. q Cumulative data of the frequency, and (r) the intensity of CXCR4+ expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. s Cumulative data of the intensity of CCR4 expression in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. Error bars represent the median with an interquartile range. Each dot represents an individual human sample. Figure. S5. a Representative plots, and (b) cumulative data of the frequency of CD69 expressing cells among CD26neg, CD26low, and CD26high CD8+ T cells in CLL. c Cumulative data showing the frequency of TSCM (T Stem Cell Memory: CCR7+ CD45RA+CD95+) among CD26neg, CD26low, and CD26high CD8+ T cells in CLL. d Cumulative data showing the frequency of CD16+ in CD26neg, CD26low, and CD26high CD8+ T cells in CLL. e Representative histogram plots, and (f) cumulative data of CD26 expression in CD8+ T cells upon culture with 10% plasma. (g, h) showing detected plasma concentrations of different cytokines and chemokines in CLL versus HCs. Cumulative data showing the intensity of CD26 in the presence/absence of (i) TNF-α, (j) IL-16, (k) IFN-γ, (l) IL-6, (m) IL-10, and (n) IFN-α. o Detected total plasma TGF-β and (p) free TGF-β in CLL versus HCs. q Cumulative data showing the intensity of CD26 expression in CD8+ T cells in the presence/absence of free TGF-β. r The correlation of plasma free TGF-β with the frequency of CD26+CD8+ T cells in CLL patients. s Cumulative data showing the intensity of Annexin-V expression in CD26neg, CD26low, and CD26high CD8+ T cells from CLL patients following treatment with recombinant human Gal-9 (0.02 $$\mu$$ μ g/ml) in-vitro. t Concentrations of Gal-9 (pg/ml) in supernatants of PBMCs (1× 106 cells/well) from HC and CLL were collected 18 h post culture. u Concentrations of IL-18 (pg/ml) (v) IL-12/IL-23p40 (pg/ml), and (w) IL-15 (pg/ml) in the plasma of HC versus CLL. Error bars represent the median with an interquartile range. Each dot represents an individual human sample. Figure. S6. a Cumulative data showing the intensity of Annexin-V expression in CD26neg, CD26low, and CD26high CD8+ T cells from CLL patients following 8 h of culture in the presence of a cytokine cocktail (IL-18+IL-12+IL-15 (100 ng/ml of each)). b Representative flow cytometry plots, and (c) cumulative data of CD26-expressing cells among CD8+ T cells in the thymus, blood, and spleen of BALB/c mice. d Cumulative data showing the frequency of CD28+CD8+ T cells in HC versus CLL. Error bars represent the median with an interquartile range. Each dot represents an individual human sample or a mouse. Florescence minus one (FMO).

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