American Society of Hematology

Figure 6.

$MHCIIlo HSCs exhibit enhanced megakaryocytic differentiation and are preferentially expanded in mutant CALR mice with thrombocythemia/myelofibrosis. (A) Heatmap showing Vwf-expressing HSCs cluster separately from HSCs with high levels of MHCII gene expression. LT-HSCs from Nestorowa scRNA-seq dataset were analyzed; a large proportion of LT-HSCs expressing high levels of Vwf is shown to cluster separately from HSCs with high-level expression of MHCII genes. (B) Expression of Cd74, Vwf genes plotted on the force-directed graph generated from HSPC cells in Nestorowa’s scRNA-seq dataset. (C) Representative flow cytometry plots showing Vwf+ HSCs were within the MHCIIlo fraction. (D) Bar graphs showing the negative correlation between Vwf and MHCII cell surface expression within ESLAM HSCs. (E) Experimental scheme showing single-cell in vitro assays of ESLAM HSC differentiation. Single ESLAM HSCs gated with MHCIIhi or MHCIIlo were FACS sorted into 96-well plates and cultured in StemSpan medium with 10% fetal bovine serum (FBS), 250 ng/mL stem cell factor (SCF), 10 ng/mL IL-3, and 10 ng/mL IL-6, and at day 7, each individual cell-derived clone was scored and categorized using criteria as described in Prins et al.54 (F) Bar graphs showing a reduced number of clones derived from MHCIIhi ESLAM HSCs with presence of large cells at day 7. MHCIIhi, n = 187 wells; MHCIIlo, n = 193; Chi-squared test; P = .0035. (G) Representative flow cytometry plots showing increased frequency of MHCIIlo ESLAM HSCs in knock-in mice expressing homozygous mutant CALR (CALRdel/del). (H) Bar graphs showing preferential expansion of MHCIIlo ESLAM HSCs in mutant CALR mice. Data are shown as mean ± standard error; asterisks indicate significant differences by Student t test (∗P < .05; ∗∗∗∗P < .0001). iA/iE, MHCII antibody; Large, colonies of any cell number (usually 1-30 cells), containing only very large flattened cells; Mixed, colonies of any cell number, containing small round cells and very large flattened cells; ns, not significant; Small, colonies of any cell number, containing cells that are uniformly round and small; STHSC, short term HSCs.$

MHCII^lo HSCs exhibit enhanced megakaryocytic differentiation and are preferentially expanded in mutant CALR mice with thrombocythemia/myelofibrosis. (A) Heatmap showing Vwf-expressing HSCs cluster separately from HSCs with high levels of MHCII gene expression. LT-HSCs from Nestorowa scRNA-seq dataset were analyzed; a large proportion of LT-HSCs expressing high levels of Vwf is shown to cluster separately from HSCs with high-level expression of MHCII genes. (B) Expression of Cd74, Vwf genes plotted on the force-directed graph generated from HSPC cells in Nestorowa’s scRNA-seq dataset. (C) Representative flow cytometry plots showing Vwf⁺ HSCs were within the MHCII^lo fraction. (D) Bar graphs showing the negative correlation between Vwf and MHCII cell surface expression within ESLAM HSCs. (E) Experimental scheme showing single-cell in vitro assays of ESLAM HSC differentiation. Single ESLAM HSCs gated with MHCII^hi or MHCII^lo were FACS sorted into 96-well plates and cultured in StemSpan medium with 10% fetal bovine serum (FBS), 250 ng/mL stem cell factor (SCF), 10 ng/mL IL-3, and 10 ng/mL IL-6, and at day 7, each individual cell-derived clone was scored and categorized using criteria as described in Prins et al.⁵⁴ (F) Bar graphs showing a reduced number of clones derived from MHCII^hi ESLAM HSCs with presence of large cells at day 7. MHCII^hi, n = 187 wells; MHCII^lo, n = 193; Chi-squared test; P = .0035. (G) Representative flow cytometry plots showing increased frequency of MHCII^lo ESLAM HSCs in knock-in mice expressing homozygous mutant CALR (CALR^del/del). (H) Bar graphs showing preferential expansion of MHCII^lo ESLAM HSCs in mutant CALR mice. Data are shown as mean ± standard error; asterisks indicate significant differences by Student t test (∗P < .05; ∗∗∗∗P < .0001). iA/iE, MHCII antibody; Large, colonies of any cell number (usually 1-30 cells), containing only very large flattened cells; Mixed, colonies of any cell number, containing small round cells and very large flattened cells; ns, not significant; Small, colonies of any cell number, containing cells that are uniformly round and small; STHSC, short term HSCs.

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