Blocking Siglec-6–sialyltransferase axis activates pDCs and CD3+ T cells. MM BM-MNCs were treated with the sialyltransferase inhibitor 3Fax-Neu5Ac for 24 hours, followed by the flow analysis of activation markers CD83 (A) and CD86 (B) on MM-pDCs (mean ± SD; FC, treated vs untreated; P < .05). (C) MM BM-MNCs were treated with the sialyltransferase inhibitor 3Fax-Neu5Ac for 48 hours, followed by the flow analysis of the activation marker CD69 on CD3+ T cells. Bar plots show the upregulation of CD3+/CD69+ T-cell populations (left) and change in MFI of CD69 on CD3+ T cells (right; mean ± SD; FC, treated vs untreated; P < .05). (D) MM BM-MNCs were treated with the sialyltransferase inhibitor 3Fax-Neu5Ac for 48 hours, followed by the flow analysis of autologous CD138+ tumor cells. After treatment, the cells were washed and stained with anti-CD138 conjugated to Fluorescein isothiocyanate (FITC), followed by staining with 7-AAD. Viable cells were gated out as 7-AAD–, and the viability of autologous CD138+ tumor cells was analyzed in the presence and absence of the inhibitor (mean ± SD; FC, treated vs untreated; P < .05). (E) FC in the MFI of biotinylated lectins SNA and MAL-II bound to pDC cell surface in the presence and absence of 3Fax-Neu5Ac. Fluorescence signal was detected by flow using secondary streptavidin conjugated to PE. The cells were cultured in the presence of increasing inhibitor concentrations (1-125 μM) for 48 hours. After this, the cells were washed and first treated with biotinylated lectins (5 μg/mL; Vector Laboratories) for 45 to 60 minutes. The cells were then washed, followed by staining with streptavidin-PE secondary (0.8-1.0 μg/mL) for 30 minutes. All samples were preblocked with Tru Fc blocker (BioLegend). 7-AAD dye was used to exclude dead cells, and signal was measured only on live cells. The data are presented as FC in biotinylated lectin–streptavidin-PE signal in the presence vs absence of the inhibitor (mean ± SD; P = .0109 for MAL-II). (F) MM BM-MNCs were treated with Siglec-6–neutralizing Ab (5 μg/mL) or appropriate isotype Ab control for 48 hours, followed by the flow analysis of activation markers CD83, CD86, and HLA-DR on MM-pDCs in the presence or absence of Siglec-6–neutralizing Ab (mean ± SD; FC, treated vs untreated; P <.05). (G) Total BM-MNCs from patients with MM were treated with Siglec-6–neutralizing Ab or isotype control (10 μg/mL) for 48 hours, then washed and resuspended, followed by the addition of CellTrace Violet–stained K562 cells. Cells were incubated for 1 day before multicolor flow analysis to assess K562 cell lysis. The bar plot shows the quantification of flow cytometry data (mean ± SD; treated vs untreated). (H) Siglec–sialic acid immune checkpoint axis-mediated immune dysfunction in myeloma (upper); and the therapeutic potential of Siglec–sialic acid immune checkpoint axis as an immunometabolic target (lower).
Figure 2.

Blocking Siglec-6–sialyltransferase axis activates pDCs and CD3+ T cells. MM BM-MNCs were treated with the sialyltransferase inhibitor 3Fax-Neu5Ac for 24 hours, followed by the flow analysis of activation markers CD83 (A) and CD86 (B) on MM-pDCs (mean ± SD; FC, treated vs untreated; P < .05). (C) MM BM-MNCs were treated with the sialyltransferase inhibitor 3Fax-Neu5Ac for 48 hours, followed by the flow analysis of the activation marker CD69 on CD3+ T cells. Bar plots show the upregulation of CD3+/CD69+ T-cell populations (left) and change in MFI of CD69 on CD3+ T cells (right; mean ± SD; FC, treated vs untreated; P < .05). (D) MM BM-MNCs were treated with the sialyltransferase inhibitor 3Fax-Neu5Ac for 48 hours, followed by the flow analysis of autologous CD138+ tumor cells. After treatment, the cells were washed and stained with anti-CD138 conjugated to Fluorescein isothiocyanate (FITC), followed by staining with 7-AAD. Viable cells were gated out as 7-AAD–, and the viability of autologous CD138+ tumor cells was analyzed in the presence and absence of the inhibitor (mean ± SD; FC, treated vs untreated; P < .05). (E) FC in the MFI of biotinylated lectins SNA and MAL-II bound to pDC cell surface in the presence and absence of 3Fax-Neu5Ac. Fluorescence signal was detected by flow using secondary streptavidin conjugated to PE. The cells were cultured in the presence of increasing inhibitor concentrations (1-125 μM) for 48 hours. After this, the cells were washed and first treated with biotinylated lectins (5 μg/mL; Vector Laboratories) for 45 to 60 minutes. The cells were then washed, followed by staining with streptavidin-PE secondary (0.8-1.0 μg/mL) for 30 minutes. All samples were preblocked with Tru Fc blocker (BioLegend). 7-AAD dye was used to exclude dead cells, and signal was measured only on live cells. The data are presented as FC in biotinylated lectin–streptavidin-PE signal in the presence vs absence of the inhibitor (mean ± SD; P = .0109 for MAL-II). (F) MM BM-MNCs were treated with Siglec-6–neutralizing Ab (5 μg/mL) or appropriate isotype Ab control for 48 hours, followed by the flow analysis of activation markers CD83, CD86, and HLA-DR on MM-pDCs in the presence or absence of Siglec-6–neutralizing Ab (mean ± SD; FC, treated vs untreated; P <.05). (G) Total BM-MNCs from patients with MM were treated with Siglec-6–neutralizing Ab or isotype control (10 μg/mL) for 48 hours, then washed and resuspended, followed by the addition of CellTrace Violet–stained K562 cells. Cells were incubated for 1 day before multicolor flow analysis to assess K562 cell lysis. The bar plot shows the quantification of flow cytometry data (mean ± SD; treated vs untreated). (H) Siglec–sialic acid immune checkpoint axis-mediated immune dysfunction in myeloma (upper); and the therapeutic potential of Siglec–sialic acid immune checkpoint axis as an immunometabolic target (lower).

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