High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow

Abstract

Cytokine-mediated phosphorylation of Erk (pErk), ribosomal S6 (pS6), and Stat5 (pStat5) in CD34(+)/CD117(+) blast cells in normal bone marrow from 9 healthy adult donors were analyzed over 60 minutes. Treatment with stem cell factor (SCF), Flt3-ligand (FL), IL-3, and GM-CSF and measurement by multiparametric flow cytometry yielded distinctive, highly uniform phosphoprotein kinetic profiles despite a diverse sample population. The correlated responses for SCF- and FL-stimulated pErk and pS6 were similar. Half the population phosphorylated Erk in response to SCF between 0.9 and 1.2 minutes, and S6 phosphorylation followed approximately a minute later (t½(pS6 rise) = 2.2-2.7 minutes). The FL response was equally fast but more variable (t½(pErk rise) = 0.9-1.3 minutes; t½(pS6 rise) = 2.5-3.5 minutes). Stat5 was not activated in 97% of the cells by either cytokine. IL-3 and GM-CSF were similar to each other with half of blast cells phosphorylating Stat5 and 15% to 20% responding through Erk and S6. Limited comparison with leukemic blasts confirmed universal abnormal signaling in AML that is significantly different from normal bone marrow blasts. These differences included sustained signals, a larger fraction of responding cells, and amplification of phosphorylation levels for at least one phosphoprotein. These data support the eventual use of this approach for disease diagnosis and monitoring.

Figure 1

Gating strategy for pErk, pS6, and pStat5 in CD34⁺, CD117⁺ blast cells. (A) A region used to exclude debris. (B) The circle represents the blast region defined by CD45 expression and side scatter. (C) The box shows the CD34⁺/CD117⁺ region. (D,G) pErk histograms at t = 0 (gray) or 2 (green) minutes for SCF and t = 0 (gray) or 8 (green) minutes for IL-3, respectively. (E,H) pS6 histograms at t = 0 (gray) or 4 (blue) minutes for SCF and t = 0 (gray) or 8 (blue) minutes for IL-3, respectively. (F,I) pStat5 histograms at t = 0 (gray) or 2 (red) minutes for SCF and t = 0 (gray) or 8 (red) minutes for IL-3, respectively. The histograms are representative of all the flow cytometry data reported here.

Figure 2

Cytokine-mediated phosphoprotein kinetic profiles: frequency. Frequency indicates percentage of responding blast cells. Error bars represent SEM.

Figure 3

SCF- and FL-mediated Erk and S6 signal shape analysis. Response indicates percentage of responding cells with t = 0 level subtracted. Individual signaling profiles were normalized to t = 0 (equal to 0 response) and the largest value (maximum). t½_{Erk/S6 rise} indicates time to 50% of maximum; and t½_{Erk/S6 decay}, time to 50% loss of maximum. Gray zones represent 95% CI for t½_{Erk rise} (A-B) and t½_{Erk decay} (C). The response shapes for S6 phosphorylation were not significantly different (D). Error bars represent 95% CI.

Figure 4

IL-3– and GM-CSF–mediated Stat5, Erk, and S6 signal shape comparisons. Response indicates percentage of responding cells with t = 0 level subtracted. Error bars represent SEM. Data were processed as described in the legend to Figure 3.

Figure 5

Cytokine-mediated phosphoprotein kinetic profiles: MFI. Error bars represent SEM.

Figure 6

Phosphoprotein kinetic profiles of AML patient samples: frequency analysis. (A-B) The frequency of blast cells that are not positive for any of the 3 downstream markers are plotted with the frequency at t = 0 set to 100%. (C-F) Actual frequencies (in percentage) of cells positive for each marker at time t. Gray zones represent 95% CI for normal blast cell response; and black line, the center (mean) of that distribution. Arrows point to high constitutive activity for AML3. ⟐ represents AML2; □, AML3; ⊠, AML1; ⊡, AML5; and ▒, NBM.

Figure 7

Phosphoprotein kinetic profiles of AML patient samples: MFI analysis. AMLs classified as 3 potential “phenotypes” by signaling patterns: AML3 (rows A and J); AMLs 1 and 2 (rows D and M); AML5 (rows G and P). Data have been normalized to NBM for SCF → Erk, SCF → S6, and IL-3 → Stat5 = 1.0 at max. Colored zones represent the 95% CI for the NBM blast response MFIs.