Reduced Percentage of CD14dimCD16+SLAN+ Monocytes Producing TNF and IL-12 as an Immunological Sign of CLL Progression

Abstract

Monocytes are one of the least studied immune cells with a potentially important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Nevertheless, data regarding the role of subpopulations of monocytes in the CLL microenvironment are still limited. For the very first time, this study presents an assessment of monocyte subsets divided according to SLAN and CD16 expression in CLL patients. The study involved 70 freshly diagnosed CLL patients and 35 healthy donors. Using flow cytometry, monocyte subpopulations were assessed among PBMCs. CD14⁺ monocytes can be divided into: "classical" (CD14⁺CD16^-SLAN^-), "intermediate" (CD14⁺CD16⁺SLAN^-) and "non-classical" (CD14^dimCD16⁺SLAN⁺). In our study, we noted an increased percentage of non-classical monocytes with intracellular expression of TNF and IL-12. On the other hand, among the intermediate monocytes, a significantly higher percentage of cells synthesizing anti-inflammatory IL-10 was detected. The percentage of CD14^dimCD16⁺SLAN⁺ monocytes producing TNF and IL-12 decreased with the stage of CLL and inversely correlated with the expression of the prognostic factors ZAP-70 and CD38. Moreover, the percentage of CD14^dimCD16⁺SLAN⁺ monocytes producing TNF and IL-12 was lower in CLL patients requiring treatment. This may indicate the beneficial effect of non-classical monocytes on the anti-tumor response.

Keywords: SLAN; chronic lymphocytic leukemia; monocytes.

Figure 1

An example of the cytometric evaluation of SLAN-positive and SLAN-negative monocytes’ subpopulations from a CLL patient. (A) FSC-H vs. FSC-A dot plot of the doublets’ elimination (setting up gate covering the singlets population); (B) dot plots of SSC-A vs. FSC-A gating of PBMC cells; (C) dot-plot of the FVS510 vs. SSC exclusion of dead cells from further analysis. Live cells collected in the LIVE CELLS gate were used for further evaluation. (D) From the living cells, CD14⁺ cells (dot plot: SSC vs. CD14 V450; gating of Monocytes) were identified. The presence of the CD14 marker and scatter properties were the basis for the identification of monocytes. (E) SLAN APC vs. CD16 FITC dot plot—the differentiated expression of CD16 and SLAN molecules allowed the identification of classical (CD14⁺CD16⁻SLAN⁻), intermediate (CD14⁺CD16⁺SLAN⁻) and non-classical (CD14^dim CD16⁺SLAN⁺) monocytes.

Figure 2

Comparison of the percentage of (A) classical CD14⁺CD16⁻SLAN⁻, (B) CD14⁺CD16⁺SLAN⁻ intermediate monocytes and (C) non-classical CD14^dimCD16⁺SLAN⁺ monocytes in CLL patients and healthy volunteers. The solid line marks the median value, while the whiskers depict the interquartile range (IQR).

Figure 3

Comparison of the percentage of (A) classical, (B) intermediate and (C) non-classical monocytes in CLL patients at different stages of disease advancement. The solid line marks the median value. Ɪ represents the interquartile range (IQR).

Figure 4

Comparison of the percentage of (A,D) classical, (B,E) intermediate and (C,F) non-classical monocytes in CLL patients ZAP-70-positive and ZAP-70-negative and CD38-positive and CD38-negative group. The solid line marks the median value. Ɪ represents the interquartile range (IQR).

Figure 5

The percentage of monocytes (A) CD14⁺CD16⁻SLAN⁻, (B) CD14⁺CD16⁺SLAN⁻ and (C) CD14^dimCD16⁺SLAN⁺ in the group of CLL patients with del (11q22.3) and/or del (17p13.1), and in the group of patients without these cytogenetic aberrations. The solid line marks the median value. Ɪ represents the interquartile range (IQR).

Figure 6

The percentage (A) classical, (B) intermediate and (C) non-classical monocytes in the group of CLL patients requiring treatment and in the group of patients not requiring antitumor treatment. The solid line marks the median value. Ɪ represents the interquartile range (IQR).

Figure 7

Low CD14^dimCD16⁺SLAN⁺ percentage as a negative prognostic marker for the time-to-treatment (A) Graph of the ROC curve for the percentage of non-classical monocytes (CD14^dimCD16⁺SLAN⁺) in ZAP-70-positive patients versus ZAP-70-negative CLL patients. ROC, receiver operating characteristic; AUC, area under the curve. ROC and AUC were used to calculate the most significant cut-off value of the non-classical monocytes (CD14^dimCD16⁺SLAN⁺) percentage that best distinguished ZAP-70-positive and ZAP-70-negative CLL cases. (B) Kaplan–Meier curve comparing time-to-treatment-initiation (TTT) in groups of CLL patients with the percentage of non-classical CD14^dimCD16⁺SLAN⁺ monocytes (≤6.37% and >6.37%). The division of CLL patients into two groups was made with reference to the cut-off point determined using the ROC curve analysis. HR, hazard ratio; CI, confidence interval.

Figure 8

Percentage of classical (CD14⁺CD16⁻SLAN⁻), intermediate (CD14⁺CD16⁺SLAN⁻) and non-classical monocytes (CD14^dimCD16⁺SLAN⁺) assessed for intracellular expression IL-10, TNF and IL-12 in “ex vivo” conditions. The solid line marks the median value. Ɪ represents the interquartile range (IQR).

Figure 9

Expression of IL-10, TNF and IL-12 at the mRNA level in classical (CD14⁺CD16⁻SLAN⁻), intermediate (CD14⁺CD16⁺SLAN⁻) and non-classical monocytes (CD14^dimCD16⁺SLAN⁺) in ex vivo conditions in 20 randomly selected CLL patients.