Sig15 promotes an immunosuppressive tumor microenvironment in B-ALL. A–M, Unirradiated WT C57BL6 mice were injected via tail vein with 5 × 105 Cas9-only (n = 8 from two independent experiments) or Sig15 KO (n = 8) leukemia. Bone marrow was harvested 7 days later for highly dimensional flow cytometry of non-leukemia bone marrow populations. Healthy, leukemia-naïve mice (Naive) were included as controls (n = 6). CD3+ T cells (A), NK cells (B), classical dendritic cells (cDC; C), and neutrophils (D) were variably present at higher totals in the bone marrow of Sig15 KO recipient mice compared with control leukemia. E, Representative contour plots of T-cell populations. Proportions of CD8+ (F) but not CD4+ (G) T cells were higher in Sig15 KO recipients than control leukemia, increasing the CD8+/CD4+ T-cell ratio (H) to levels comparable with Naive mice. I, Degranulated CD8+ T cells were higher in Sig15 KO recipient mice, as were SLEC (J) and MPEC (K) CD8+ T-cell populations. Early memory-like populations consistent with central (L) and effector memory (M) CD8+ T-cell populations were also higher in Sig15 KO recipients. N–P, From the same experiments, bone marrow supernatant from control and Sig15 KO leukemia recipient mice was analyzed via 44-plex cytokine/chemokine assay. Levels of IL6 (N), LIF (O), and IL5 (P) were all significantly lower in the bone marrow of Sig15 KO recipients as compared with control leukemia (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Sig15 expression on B-ALL cells suppresses CD8+ T-cell expansion and activation and contributes overall to formation of a more proleukemia bone marrow microenvironment.

Siglec-15 (Sig15) has been implicated as an immune checkpoint expressed in solid tumor-infiltrating macrophages and is being targeted in clinical trials with mAbs to normalize the tumor immune microenvironment and stimulate antitumor immunity. However, the role of Sig15 in hematologic malignancies remains undefined. Sig15 mRNA and protein expression levels in hematologic malignancies were determined from publicly available databases, cell lines, and primary patient samples. Human B-cell acute lymphoblastic leukemia (B-ALL) cell lines were used to identify signaling pathways involved in the regulation of Sig15 expression. Secreted/soluble Sig15 and cytokine levels were measured from the plasma of children with leukemia and healthy controls. Knockdown and knockout of Siglec15 in a murine model of B-ALL was used to evaluate the effect of leukemia-derived Sig15 on the immune response to leukemia. We observed pathologic overexpression of Sig15 in a variety of hematologic malignancies, including primary B-ALL samples. This overexpression was driven by NFκB activation, which also increased the surface localization of Sig15. Secreted/soluble Sig15 was found to circulate at elevated levels in the plasma of children with B-ALL and correlated with an immune-suppressive cytokine milieu. Genetic inhibition of Sig15 in murine B-ALL promoted clearance of the leukemia by the immune system and a marked reversal of the immune-privileged leukemia bone marrow niche, including expanded early effector CD8+ T cells and reduction of immunosuppressive cytokines. Thus, Sig15 is a novel, potent immunosuppressive molecule active in leukemia that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Significance: We demonstrate that Sig15 is overexpressed in hematologic malignancies driven by NFκB, is required for immune evasion in a mouse model of leukemia, and, for the first time, that it circulates at high levels in the plasma of children with leukemia.

B-ALL–expressed Sig15 has dynamic subcellular localization. A, Flow cytometry of human B-ALL cell lines probed for SIG15 in both permeabilized and unpermeabilized (Surface) cells. Dotted line marks mean fluorescence intensity (MFI) of healthy PBMC signal (data representative of three independent experiments). Leukemia and PBMCs had modest surface expression of SIG15, while leukemia cells expressed higher intracellular SIG15 compared with PBMCs. SIG15 was probed using the NP159 mAb (NextCure). B and C, Immunofluorescence of REH cells staining for DAPI, Sig15, and Golgin-97. B, Imaging depicts limited surface localization of Sig15 and strong colocalization with the Golgi apparatus and TGN in unstimulated REH cells. C, Representative images depict REH cells treated for 24 hours with DMSO (vehicle control) or 81 nmol/L PMA and 1.3 μmol/L ionomycin. Stimulated REH cells show decreased colocalization of Sig15 signal with Golgin-97. SIG15 was probed using the Invitrogen polyclonal antibody. D, Flow cytometry of unpermeabilized REH cells treated with PMA with or without BOT64 for 6 hours, demonstrating that NFkB activation stimulates Sig15 localization at the cell surface (data representative of three independent experiments; dotted line represents MFI of isotype staining). SIG15 was probed using the NP159 mAb (NextCure) conjugated to Alexa Fluor 647.

Sig15 expression is regulated by NFκB activation in B-ALL. A, Western blot analysis shows upregulated SIG15 expression in REH cells stimulated for 24 hours with a PMA/ionomycin stimulation (81 nmol/L PMA and 1.3 μmol/L ionomycin) but not with 30 ng/mL of recombinant human MCSF and/or IL10. Densitometric analysis of SIG15 is quantified above. SIG15 was probed using the Invitrogen polyclonal antibody. B, qRT-PCR from REH cells treated with PMA (81 nmol/L) with or without pan-PKC inhibitor Gö 6983 (5 μmol/L) for 24 hours, showing that PKC inhibition abrogates SIG15 induction by PMA. (****, P < 0.0001). C, qRT-PCR from REH cells stimulated with PMA (81 nmol/L) and/or treated with calcineurin inhibitor CSA (1 μmol/L) or IKK-2 inhibitor BOT64 (10 μmol/L) for 24 hours, demonstrating that NFκB inhibition but not calcineurin inhibition abrogates PMA-induced SIG15 transcription (****, P < 0.0001). D, Western blot analysis shows SIG15 expression in REH cells following 24 hours of stimulation with 81 nmol/L PMA alone or in combination with 10 μmol/L of the IKK-2 inhibitor BOT64. NFκB inhibition attenuates PMA-induced upregulation of SIG15. SIG15 was probed using the 1F7 mAb (NextCure). E,SIG15 expression from the B-ALL TARGET database correlated with protein kinase C β (PRKCB; r = 0.35; P < 0.0001, Pearson correlation), calcineurin catalytic subunit alpha (PPP3CA; r = 0.42; P < 0.0001), and IL1β (IL1B; r = 0.33; P < 0.0001, Pearson correlation). F, Western blot analysis shows increased SIG15 protein in REH cells stimulated for 24 hours with increasing doses of recombinant human CD40L. Densitometric analysis of SIG15 is quantified above. SIG15 was probed using the 1F7 mAb (NextCure).