Session Title: Innate Immunity and Rheumatic Disease
Session Type: Abstract Submissions (ACR)
Background/Purpose: Myeloid-derived suppressor cells (MDSCs) are innate immune cells that expand under pathological conditions (such as cancer and autoimmune diseases) in response to local growth factors or cytokines. MDSCs are a heterogeneous population of immature myeloid lineage (monocyte-like and granulocyte-like) cells with immunosuppressive ability. These cells have the potential to down-regulate autoreactive T cell responses in autoimmune diseases such as rheumatoid arthritis (RA). Using proteoglycan (PG)-induced arthritis (PGIA), a mouse model of RA, we previously reported that MDSCs are present in synovial fluid (SF) of the arthritic joints of mice and suppress antigen-specific T cell proliferation. As the number of cells that can be collected from murine SF is limited and SF MDSCs do not expand in culture, we sought an alternative source for generating greater quantities of MDSCs for potential therapeutic intervention (via cell transfer) in PGIA.
Methods: Bone marrow (BM) cells were isolated from naïve BALB/c mice and cultured in the presence of recombinant murine granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) for up to 7 days. After harvest, the phenotype of cells was evaluated by flow cytometry. Their suppressive function towards PG-specific T cells was tested by co-culture with PG-loaded BM-derived dendritic cells (DCs) and T cells from naïve PG-specific T cell receptor transgenic (PG-TCR-Tg) mice. The mechanisms of MDSC-mediated suppression were investigated using inhibitors of MDSC-produced effector molecules including arginase-1, inducible nitric oxide (NO) synthase (iNOS), and reactive oxygen species. Expression of MDSC effector molecules was analyzed by RT-PCR and Western blot.
Results: Similar to SF MDSCs, BM-derived MDSCs expressed the common myeloid marker CD11b. However, unlike SF MDSCs, BM MDSCs contained a smaller population of Ly6G positive (granulocyte-like) cells, and the majority of them expressed both Ly6G and the monocytoid cell surface marker Ly6C. Upon co-culture with PG-TCR-Tg T cells in the presence of PG-loaded DCs, BM MDSCs profoundly inhibited the proliferation of T cells, thereby confirming their suppressor activity. BM cells grown with GM-CSF, G-CSF, and IL-6 for only 3 days already showed potent suppressive effect on T cell proliferation. Despite expression of Ly6C by most BM MDSCs, these cells retained their suppressor activity after depletion of the Ly6C positive population. Experiments with inhibitors of MDSC effector molecules revealed that the primary mechanism of suppression of T cell proliferation was via NO release. Indeed, iNOS expression in BM MDSCs was found elevated at both mRNA and protein levels.
Conclusion: We developed an in vitro culture method of generating large quantities of immunosupressive murine MDSCs. Characterization of the phenotype, gene expression, suppressor activity of BM-derived MDSCs revealed that these cells are similar to SF MDSCs, but are dominated by a less mature (double Ly6C/Ly6G positive) population. BM-derived MDSCs appear to be suitable for in vivo cell transfer experiments.
T. A. Rauch,
T. T. Glant,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/generation-of-myeloid-derived-suppressor-cells-in-vitro-from-murine-bone-marrow-precursors/