Fig 2: Surface expression of CD62L and CD11b on neutrophils (A) and serum levels of selected analytes (B) from GCA patients who responded to therapy (n = 25), at baseline visit (To) and 1, 4, 12, 24, and 48 weeks of follow-up. After pre-treatment sampling (time point 0), all patients received GC and therapy tapering started after week 4 (indicated with vertical dotted line). After week 12 some of the patients (15/25) received leflunomide, in addition to GC therapy (green). The horizontal lines connect the medians from each follow-up time point. GCA, giant cell arteritis; MFI; median fluorescence intensity. * indicates statistical significance between the corresponding timepoint and T0 in patients receiving GC monotherapy (blue); # indicates statistical significance between the corresponding timepoint and T0 in patients receiving GC in combination with leflunomide (green).

Fig 3: LPS induces a decrease in the neutrophilic expression of CXCR1, CXCR2 and CD62L. Isolated neutrophils were incubated with LPS for 4 h. Levels of (A) CXCR1, (B) CXCR2, (C) CD62L, (D) TLR2, (E) TLR4 and (F) CD11b were measured using flow cytometry. The corresponding mean fluorescence intensity was calculated. Each bar represents the mean ± standard error of the mean of three independent experiments. Statistical significance relative to the CON group was determined. *P<0.05, **P<0.01 and ***P<0.001. LPS, lipopolysaccharide; CXCR1, CXC-chemokine receptor 1; CXCR2, CXC-chemokine receptor 2; CD62L, L-selectin; TLR2, toll-like receptor 2; TLR4, toll-like receptor 4; CON, control.

Fig 4: Multipotent Myelofibrosis Hematopoietic Stem and Progenitor Cells (HSPCs) Are Biased for Megakaryocyte Differentiation(A) Left: model of classically defined CD34+ lin− HSPC subpopulations, in which multi-potent cells (HSCs, hematopoietic stem cells; MPPs, multi-potent progenitor cells; LMPPs, lymphoid-primed multi-potent progenitors) are CD38− and down-stream progenitors (CMPs, common myeloid progenitors; MEPs, megakaryocyte-erythroid progenitors; GMPs, granulocyte-monocyte progenitors) are CD38+. CD45RA+ populations (LMPP/GMP) do not have erythroid or megakaryocyte potential. Middle: % of each classically defined HSPC population in the CD34+ lin− compartment, demonstrating increased MPPs and reduced LMPPs in myelofibrosis (MF) compared to controls. Right: % cells expressing CD41, a surface antigen previously shown to identify cells with increased potential for megakaryocyte differentiation, is increased in both CD38− CD45RA− (HSC/MPP) and CD38+ CD45RA− (CMP/MEP) compartments in myelofibrosis (MF patients, N = 23; controls, N = 14, see also Table S1).(B) Representative FACS plot of a healthy donor control and myelofibrosis patient showing gating strategies.(C) Left: FACS analysis of CD41− HSC (top), CD41− MPP (middle), and CD41+ HSC/MPP (bottom) from healthy donors cultured in megakaryocyte differentiation media (with added recombinant human TPO and stem cell factor [SCF]). CD41+ HSC/MPP demonstrate increased potential for megakaryocyte differentiation, with faster acquisition of the mature megakaryocyte antigen CD42 at an early time point (day 6). Right: images of cultures showing enlarged cell size and proplatelet formation (red star) indicative of accelerated megakaryocyte differentiation from CD41+ HSC/MPP. Representative examples of 3 replicate experiments shown.(D) FACS analysis of CD41− HSC, CD41− MPP and CD41+ HSC/MPP from healthy donors cultured for 12–14 days in megakaryocyte (MK), erythroid (Ery), or myeloid (Mye) differentiation media. CD41+ HSC/MPP showed a higher % of mature CD41+42+ megakaryocytes and glycophorin A+ CD71+ erythroblasts and equivalent CD11b/CD14+ myeloid cells versus CD41− fractions. Representative examples of 3 replicate experiments shown. % of total live (7AAD-), single cells shown.(E) Summary chart (left) and representative FACS plots (right) showing percentage of myelofibrosis and control CD41− HSC/MPP cultured in “bi-potent” erythroid and megakaryocyte differentiation media that give rise to megakaryocyte versus erythroid progeny 6 days after plating (gated on live cells). (controls, n = 7; myelofibrosis [MF], n = 8). Charts show mean + SEM,∗∗∗p < 0.001; ∗∗p ≤ 0.01; ∗p < 0.05). See also Figure S1.

Fig 5: Surface expression of CD62L and CD11b on neutrophils (A) and serum levels of selected analytes (B) from relapsing GCA patients during the 48 weeks of follow-up. Shown are levels/MFI at the last follow-up time point before relapse, at the time point closest to relapse and 12 weeks after relapse. P1 relapsed at week 24, P2 relapsed at week 48 and P3 relapsed at week 12. MFI, median fluorescence intensity, P, patient.