SIRPα expression delineates subsets of intratumoral monocyte/macrophages with different functional and prognostic impact in follicular lymphoma

Abstract

Signal regulatory protein-α (SIRPα) is a key member of the "do-not-eat-me" signaling pathway, but its biological role and clinical relevance in B-cell NHL is relatively unknown. Using biopsy specimens from follicular lymphoma (FL), we identified three subsets (CD14⁺SIRPα^hi, CD14^-SIRPα^low, and CD14^-SIRPα^neg) of monocyte/macrophages (Mo/MΦ) based on CD14 and SIRPα expression. CD14⁺SIRPα^hi cells expressed common Mo/MΦ markers; exhibited characteristic differentiation, migration, and phagocytosis; and suppressed T-cell function. CD14^-SIRPα^low cells expressed fewer typical Mo/MΦ markers; migrated less and phagocytosed tumor cells less efficiently; and stimulated rather than suppressed T-cell function. Interestingly, the CD14^-SIRPα^neg subset expressed distinct Mo/MΦ markers compared to the other two subsets; had limited ability to migrate and phagocytose; but stimulated T-cell function. When using SIRPα-Fc to block the interaction between SIRPα and CD47, alone or in combination with rituximab, phagocytosis of tumor cells was differentially increased in the three Mo/MΦ subsets. Clinically, increased numbers of CD14⁺SIRPα^hi cells were associated with an inferior survival in FL. In contrast, increased numbers of the CD14^-SIRPα^low subset appeared to correlate with a better survival. Taken together, our results show that SIRPα expression delineates unique subsets of intratumoral Mo/MΦs with differing prognostic importance.

Fig. 1. Expression of CD14 on intratumoral Mo/MΦs does not correlate with CD68 expression in FL.

a Representative images from immunohistochemistry showing staining of CD14 and CD68 with different scores of +++, ++, or +/− in FL patients. b Graph showing percentage of patient samples with different scores of CD14 and CD68 staining. N = 159. c Images of immunohistochemistry from a representative patient biopsy specimen showing staining of CD68, CD14, and CD163. d Graph showing cell recovery after CD14-positive selection from PBMCs and NHL tissue. Cell recovery was measured by counting cells before and after CD14-positive selection

Fig. 2. SIRPα expression delineates intratumoral Mo/MΦs.

a Dot plots showing SIRPα expression on Lin⁻ cells before and after enrichment. b The viSNE plots showing expression of SIRPα and CD14 on CD45⁺ cells from a representative patient biopsy specimen of FL. Graph (right) showing percentage of SIRPα⁺ and CD14⁺ cells in CD45⁺ cell population. c Histogram plots showing expression of SIRPα and CD14 on CD45⁺CD19⁻CD3⁻CD56⁻ cells from a representative FL biopsy specimen. Graph (right) showing percentage of SIRPα⁺ and CD14⁺ cells in CD45⁺CD19⁻CD3⁻CD56⁻ cells. d Graphs showing percent expression of various markers on SIRPα⁺ and SIRPα⁺ cells. e Dot plots showing coexpression of SIRPα and CD14 on CD45⁺CD19⁻CD3⁻CD56⁻ cells from a representative patient biopsy specimen of FL. Graph (right) showing percentage of CD14⁺SIRPα^hi, CD14^-SIRPα^low, and CD14⁻SIRPα^neg cells within the CD45⁺CD19⁻CD3⁻CD56⁻ cell population. f Dot plots showing coexpression of CD14 and SIRPα after Mo/MΦs enrichment. Graph (right) showing percentages of CD14⁺SIRPα^hi, CD14⁻SIRPα^low, and CD14⁻SIRPα^neg cells in peripheral blood (PBMC), benign lymph nodes and lymphoma tissue (NHL) after enrichment. P values indicate the comparison between PBMC and NHL. g Representative images of immunofluorescent staining showing localization of CD14⁺ and SIRPα⁺ cells in FL lymphoma tissue. h The viSNE plots showing expression of surface markers from CD14⁺SIRPα^hi, CD14⁻SIRPα^low, and CD14⁻SIRPα^neg cells in FL. Graphs (below) showing the summarization of percentage of the three subsets (CD14⁺SIRPα^hi, CD14⁻SIRPα^low, and CD14⁻SIRPα^neg) expressing each marker (n = 13)

Fig. 3. SIRPα-defined Mo/MΦs display distinct polarization and migration properties.

a Microscopic images showing morphological changes of Mo/MΦs treated with or without GM-CSF or M-CSF for 3 days. b Dot plots showing expression of CD14 and SIRPα by Mo/MΦs treated with or without GM-CSF or M-CSF for 3 days. c Microscopic images showing morphological changes of CD14⁺SIRPα^hi, CD14⁻SIRPα^low and CD14⁻SIRPα^neg cells treated with GM-CSF, M-CSF, or IL-34 for 3 days. d Dot plots of SSC and FSC showing the numbers of migrated Mo/MΦs in respond to 10% FBS or escalated doses of MCP-1. e Dot plots showing expression of CD14 and SIRPα by Mo/MΦs with (Lower chamber) or without (Upper chamber) migration in response to 10% FBS. f Graphs showing percentages of CD14⁺SIRPα^hi, CD14⁻SIRPα^low, and CD14⁻SIRPα^neg cells in upper (Before) or lower (After) chamber

Fig. 4. SIRPα-defined Mo/MΦs possess different abilities to regulate of T-cell function and different phagocytosis properties.

a Overlapped histograms showing CFSE staining of CD3⁺, CD4⁺, or CD8⁺ T cells cocultured with or without CD14⁺SIRPα^hi, CD14⁻SIRPα^low or CD14⁻SIRPα^neg cells for 5 days. b Graphs showing cytokine/chemokine production by CD14⁺SIRPα^hi, CD14⁻SIRPα^low, or CD14⁻SIRPα^neg cells determined by the SCBC assay from IsoPlexis system. Results were expressed as the percentage of cells producing cytokines/chemokines. *p < 0.05; **p < 0.01; n.s. no significant difference. N = 3. c Graph showing multiple cytokine/chemokine production (polyfunctionality) by CD14⁺SIRPα^hi, CD14⁻SIRPα^low or CD14⁻SIRP^αneg subset. The numbers of cytokines (2, 3, 4, or 5 plus) produced by single cell from these three subsets of Mo/MΦs were measured by SCBC assay from IsoPlexis system. *p < 0.05; **p < 0.01; n.s. no significant difference. N = 3. d Histograms showing phagocytosis of Lin⁺ or Lin⁻ cells to latex beads indicated by percentages of cells stained with FITC positive. e Fluorescent images showing phagocytosis of latex beads by enriched cells that stained with SIRPα. f Histograms showing phagocytosis of CD14⁺SIRPα^hi, CD14⁻SIRPα^low, or CD14⁻SIRPα^neg cells to latex beads indicated by percentages of cells stained with FITC positive. g Fluorescent images showing phagocytosis of latex beads by CD14⁺SIRPα^high, CD14⁻SIRPα^low, or CD14⁻SIRPα^neg cells

Fig. 5. Blockade SIRPα/CD47 signaling enhances phagocytosis of tumor cells.

a Histograms showing CD47 expression on cell lines Raji (Burkitt lymphoma), MWCL (Waldenstrome macroglubinemia), and Toledo (Diffuse large B-cell lymphoma) cells. b Representative fluorescent images showing phagocytosis of Toledo cells (green) by Mo/MΦs (red). Arrows indicate cells engulfing at least one tumor cell. c Mo/MΦs were cocultured with Toledo cells in the presence or absence of control (Ctrl) or SIRPα Fc or mIgG and Rituximab (RTX) for 2–4 h and phagocytosis was measured by confocal microscopy. Arrows indicate cells engulfing at least one tumor cells. d Graph showing the average number of Mo/MΦs that phagocytosed Toledo cells from five fields of each experiment of multiple samples by confocal microscopy. e Histogram showing CD47 expression on Toledo cells treated with different doses of SIRPα-Fc. f Representative fluorescent images showing phagocytosis of Toledo cells (green) by Mo/MΦs (red). SIRPα (blue) was stained after phagocytosis assay was completed. Arrows indicated Mo/MΦs engulfing tumor cells without SIRPα (blue) on cell surface. d Fluorescent images showing phagocytosis of CD14⁺SIRPα^high, CD14⁻SIRPα^low, or CD14⁻SIRPα^neg cells treated with SIRPα-Fc

Fig. 6. SIRPα-defined Mo/MΦ subsets differentially correlate patient outcomes in FL.

a Graphs showing percentage of total SIRPα⁺, total CD14⁺, CD14⁺SIRPα^hi, CD14⁻SIRPα^low, or CD14⁻SIRPα^neg Mo/MΦs in two patient groups. FL patients were grouped using histology (grade 1/2 vs. 3a/3b), hemoglobin (HGB) level (<12 g/dL vs. ≥12 g/dL), lactate dehydrogenase (LDH) level (abnormal vs. not) and absolute lymphocyte counts (ALC, <0.89 × 10⁹/l vs. ≥0.89 × 10⁹/l). b Kaplan–Meier curves for overall survival of FL patients (n = 83) by the total number of SIRPα⁺, total CD14⁺, CD14⁺SIRPα^hi, CD14⁻SIRPα^low, or CD14⁻SIRP^αneg Mo/MΦs