Identification of Novel Human Monocyte Subsets and Evidence for Phenotypic Groups Defined by Interindividual Variations of Expression of Adhesion Molecules

Abstract

Monocytes contribute to immune responses as a source for subsets of dendritic cells and macrophages. Human blood monocytes are classified as classical, non-classical and intermediate cells. However, the particular functions of these subsets have been hard to define, with conflicting results and significant overlaps. One likely reason for these ambiguities is in the heterogeneity of these monocyte subsets regrouping cells with divergent functions. To better define monocyte populations, we have analysed expression of 17 markers by multicolour flow cytometry in samples obtained from 28 control donors. Data acquisition was tailored to detect populations present at low frequencies. Our results reveal the existence of novel monocyte subsets detected as larger CD14⁺ cells that were CD16⁺ or CD16^neg. These large monocytes differed from regular, smaller monocytes with respect to expression of various cell surface molecules, such as FcR, chemokine receptors, and adhesion molecules. Unsupervised multidimensional analysis confirmed the existence of large monocytes and revealed interindividual variations that were grouped according to unique patterns of expression of adhesion molecules CD62L, CD49d, and CD43. Distinct inflammatory responses to TLR agonists were found in small and large monocytes. Overall, refining the definition of monocyte subsets should lead to the identification of populations with specific functions.

Figure 1

Gating strategy and identification of monocyte subpopulations. PBMC were separated and stained with antibodies directed at various cell surface molecules (Supplementary Table S2). Lineage markers were used to exclude lymphocyte subsets (a) and CD14+ cells were selected (b). Forward and side scatter identified small and large clusters of monocytes (c). After exclusion of doublets from each cluster (d,e), the expressions of CD14 and CD16 were analysed in gated cells (f,g). The higher expression of CD14 in large monocytes is shown in panel h where the profiles of CD14 and CD16 expressions in large (blue contours) and small monocytes (red contours) were overlaid. A substantial part of la14⁺16^neg monocytes had a higher CD14 expression than sm14⁺16^neg cells, and almost all la14⁺16⁺ monocytes expressed more CD14 than sm14⁺16⁺ cells. Data presented were obtained from one representative donor.

Figure 2

Imaging flow cytometry analysis of monocyte subpopulations. PBMC (n = 5) were stained and analysed by imaging flow cytometry (ImageStream, Amnis). After exclusion of lymphocytes, selection of CD14-positive cells, and doublet exclusion, small and large monocytes were visualized. (a) Representative images of small and large monocytes. (b) Representative images of cells in the CD14⁺ doublet gate. (c) Shape analysis of all events in small and large monocyte gates and in doublet gate using the aspect ratio feature (IDEAS software) in a representative donor. The ratio between the minor and major axis of each event in monocyte gates was calculated and compared to the corresponding ratio of cells in the doublet gate. A vertical bar drawn at the nadir between singlet and doublet curves (Aspect Ratio Intensity around 0.7) served as threshold to quantify singlets and doublets in each population. (d) Quantification of doublets present in gates used to define small and large monocyte subpopulations, calculated as events located left of the threshold bar drawn in panel c (mean ± s.d.) and expressed as percent of cells in the gate; doublets represented less than 5% of the cells in large monocyte gates, a percentage similar to that of small monocyte gates. (e) Cell size was determined using Area Feature (IDEAS software) with a mask delimited by CD14 expression in small and large monocytes, (f) in small monocyte subpopulations, and (g) in large monocytes subpopulations from a representative donor. (h) quantification of cell sizes for each subpopulation in 5 donors; sm14⁺16^neg, sm14⁺16⁺, and sm14^dim16⁺ monocytes had a similar size distribution, and large monocyte subpopulations la14⁺16^neg and la14⁺16⁺ had also very similar sizes (mean ± SEM, **p ≤ 0.01; ****p ≤ 0.0001, one way ANOVA).

Figure 3

Phenotypes of monocyte subpopulations as analysed with our typing platform. PBMC from 28 donors were separated and stained with antibodies directed at various cell surface receptors such as Ig FcR (CD64 and CD32 in addition to CD16), chemokine receptors (CCR2, CCR5, and CX3CR1), antigen presentation and co-stimulatory molecules (HLA-DR, CD86, and CD80), adhesion molecules (CD62L, CD162, CD43, CD49d, and CD56). The expressions of scavenger receptor CD163^, and immunoglobulin superfamily molecule CD7 were also determined (y-axis and Supplementary Table S2). Cells were analysed as described in Fig. 1. Fluorochrome-matched isotype controls were used to determine specific MFI and percentage of positive cells. Expression levels are presented as dots of colour and size reflecting MFI and percentage of positive cells, respectively, according to colour and size scales shown in legend. Monocytes subsets: (a) sm14⁺16^neg, (b) la14⁺16^neg, (c) sm14⁺16⁺, (d) la14⁺16⁺, (e) sm14^dim16⁺ and (f) sm14^dim16^neg. Variations in the number of donors analysed in each monocyte population were due to the inability to assess the expression of markers when cell numbers were too low. For each sub-population, donors were grouped according to similar expression of markers as noted at the top of the panels and recapitulated with the OP nomenclature at the bottom of the panels.

Figure 4

Connectivity map between donors and defined phenotypic profiles. To determine which phenotypic profiles identified in subpopulations of monocytes were more likely to be associated in a given donor, a network analysis was performed using Gephi. Green dots represent phenotypic profiles (OP, Fig. 3) and pink dots represent donors. The size of the dots is proportional to the number of links. (a) Complete network with ovals around clustered donors as listed in Table 1; (b–e) one donor representative of each cluster with links to corresponding phenotypic profiles; (f) one non-clustered donor. Thus, monocyte phenotype I was composed of profiles OP-01, -10, -22 or -20, -30, -40 or -42, and -50 and was present in 5 donors (see Table 1). In monocyte phenotype II, no profile was strongly associated with la14⁺16^neg monocytes, with donors 3 and 14 having profile OP-11, donor 20 having profile OP-12, and donors 13, 18, and 19 having no characteristic la14⁺16^neg profile. However, in other monocyte sub-populations, these donors shared profiles OP-04 or -05, -20, -32, -41, and -51. Monocyte phenotype III included profiles OP-01 or -02, -11, -21, -31, and -43 or -44 with however some divergence in donors 22 (with OP-33), 23 and 25 (with OP-10). Donors 17 and 25 had profile OP-51 in sub-population sm14^dim16^neg whereas no other donor had distinctive profiles in this sub-population. Monocyte phenotype IV was much less defined and included profiles OP-03 and -12 found in only two donors (5 and 8). Conspicuous in these two donors was the paucity of CD16 positive monocytes, a feature also found in donor 9. Monocyte phenotypes II and III were also linked by donors 7, 15, and 24 that shared parts of their profiles.

Figure 5

viSNE profile of sm14⁺16⁺ monocytes. Small and large monocyte populations identified with SPADE (Supplementary Fig. S5) were analysed with viSNE and specific expression profiles of adhesion molecules were identified in donors. The expression of CD43, CD49d and CD62L (x, y, z) in sm14⁺16⁺ monocytes was represented here in dot plot profiles, with CD62L expression shown according to a colour scale (right hand side axis). Profiles shown in  (a–e) correspond to monocyte sub-populations  a, b, c, d, e identified in Table 2. In panel a, a schematic representation of monocyte populations identified in profile a and that are found in various combinations in profiles  b, c, d, e is shown. Plots shown are from representative donors expressing a to e profiles.

Figure 6

Differential expression of selected transcription factors in monocyte subpopulations. PBMC (n = 3) were isolated, stained, and analysed as described in Fig. 1, and sub-populations were purified by cell sorting. Purified cells were lysed, and RNA was purified and used as a template for cDNA synthesis. Samples were probed for the quantitative expression of indicated genes in a Taqman expression system. Data were expressed using the 2^−ΔΔCt method. (*p < 0.05; **p < 0.01 Mann-Whitney U test).

Figure 7

TNF and IL-1β production induced by TLR agonists in monocyte subpopulations isolated from 3 donors. Subpopulations of monocytes from donors 10, 9, and 7 (upper to lower row, respectively) were sorted and incubated overnight with increasing concentrations of LPS and Pam3CSK4, as indicated. TNF and IL-1β were assayed in supernatants by ELISA and concentrations were normalized to 1 × 10⁵ monocytes. For each condition, monocyte subpopulations with related phenotypes were grouped in a same graph: sm14⁺16^neg (light blue) and la14⁺16^neg (orange); sm14⁺16⁺ (dark blue) and la14⁺16⁺ (red). sm14^dim16^neg monocytes were inconsistently detected in donors and were not included in the study; therefore, their CD16⁺ counterpart, sm14^dim16⁺ monocytes, were not shown. Results are presented as mean and standard deviation of duplicate determinations. When error bars are not seen, they fall within the symbol. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s unpaired t test.