CXCR2⁺CD10⁺ neutrophils are inflammatory and immunosuppressive. (A) The percentage of CXCR2⁺CD10⁺ neutrophils in BM and FLs from patients with MM (MM BM and MM FLs, respectively) and HBM. CD138^–CD3^–CD56^–CSF1R^–CD11b⁺CD10⁺CXCR2⁺C5AR1⁺ were characterized as mature neutrophils. CXCR2⁺CD10⁺ neutrophils accumulated in MM BM and MM FL samples. (B) Confocal microscopy and Wright Giemsa staining of CXCR2⁺ and CXCR2^– neutrophils from MM microenvironment; CXCR2⁺ neutrophils were mature with increased nuclear segmentation (lobulation), and CXCR2^– neutrophils were immature cells with nuclear hypolobulation. CXCR2 was marked by fluorescein isothiocyanate anti-CXCR2 (green), while the nucleus was stained with NucSpot 750/780 (red). These imaging results further emphasize the high purity of the CXCR2⁺ neutrophil sorting, underscoring the precision in identifying distinct neutrophil populations within the MM microenvironment. (C) CD11b⁺C5AR1⁺CXCR2^– (immature) and CD11b⁺C5AR1⁺CXCR2⁺ (mature) neutrophils were isolated from MM BM and MM FLs. Sorted neutrophils were cultured in complete media for 24, 48, and 72 hours. Cells were harvested and stained for Annexin V at each time point. CXCR2⁺ cells had significantly shorter survival compared with CXCR2^– cells. (D) CD138^–CD3^–CD56^–CSF1R^–CD11b⁺CD10⁺CXCR2⁺C5AR1⁺ neutrophils isolated from HBM (n = 3), MM BM (n = 3), and MM FLs (n = 3). Sorted neutrophils were cultured in complete media for 24 hours, and supernatants were collected (see “Materials and methods” for details). Multiplex enzyme-linked immunosorbent assay protein measurement was performed on collected supernatants. Several markers of inflammation were elevated in MM FLs compared with MM BM and HBM. CXCL10 was elevated in HBM. (E-F) HBM, MM BM, and MM FL neutrophils were cocultured (1:4 ratio of neutrophils to T cells) with healthy carboxyfluorescein succinimidyl ester–stained T cells activated with CD28/CD3 antibodies in the presence of IL-2. On day 4, T-cell activation was analyzed using CFSE dilution by flow cytometry. Proliferation index, with a value of 1.0 representing baseline (no proliferation), serves as the reference point. (E) Representative flow cytometry histograms of T-cell proliferation. (F) FL neutrophils showed significantly higher immunosuppression of T-cell proliferation compared with BM neutrophils from the same (paired) patient with MM. (G) Kaplan-Meier curves of OS (left) and PFS (right) for patients with MM from the MMRF CoMMpass data set stratified by the “immature neutrophil” signature. (H) OS (left) and PFS (right) curves for patients with MM from the same data set stratified by the “mature neutrophil” signature. (I) T-cell activation and exhaustion signatures were calculated from the MMRF CoMMpass data set, and compared based on the ratio of “high mature neutrophil” and “low mature neutrophil” signatures. An exhausted T-cell signature was significantly associated with the “high mature neutrophil” signature. One-way analysis of variance with Tukey multiple comparison tests was used to test significant differences between groups in panels A,C. One-way analysis of variance with Kruskal-Wallis multiple comparisons test was used to compare means between groups in panel F; statistics were derived from BM and FLs from 3 HBM and 3 patients with MM. Experiments were performed in triplicate in panels D,F. ∗P < .05; ∗∗P < .01 (in all experiments). Data are presented as mean ± standard deviation.