15417786mcr200622-sup-247403_2_supp_6749810_qlphfh.pdf (4.2 MB)

Supplementary Figures 1- 7 from ILT3 (LILRB4) Promotes the Immunosuppressive Function of Tumor-Educated Human Monocytic Myeloid-Derived Suppressor Cells

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journal contribution
posted on 2023-04-03, 19:50 authored by Latika Singh, Eric S. Muise, Anannya Bhattacharya, Jeff Grein, Sarah Javaid, Peter Stivers, Jun Zhang, Yujie Qu, Barbara Joyce-Shaikh, Andrey Loboda, Chunsheng Zhang, Michael Meehl, Derek Y. Chiang, Sheila H. Ranganath, Michael Rosenzweig, Philip E. Brandish

Figure S1A-C - Dot plots and histograms from flow cytometry analysis of SK-MEL-5-educated M-MDSCs and PBMCs cultured in medium alone.

Figure S1D - FACS result of cell surface staining of cytokine-induced M-MDSCs and PBMCs cultured in medium alone.

Figure S1E - Cell surface staining of monocytes cultured in the presence of M-CSF and IL4 and SK-MEL-5-educated M-MDSCs by staining with HLA-DR.

Figure S2 A-B - ILT3 is highly expressed on CD45+CD33+CD14+ myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.

Figure S3 - Single-cell RNAseq (scRNAseq) of human immune cells in non-small cell lung cancer (NSCLC) public dataset.

Figure S4 - Association of LILRB4 expression with deconvoluted cell fractions for human tumor types in the TCGA database.

Figure S5 - Suppression of autologous CD8+ T cell proliferation by cytokine-induced M-MDSCs.

Figure S6 - CD33+ cells from PBMCs cultured in medium alone are not suppressive.

Figure S7 - Differentially expressed genes (DEG) between SK-MEL-5 educated M-MDSCs and monocytes.



Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

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