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Maria Christofi, Lucia Kuffova, John V Forrester; Preparation of tolerogenic GM-CSF murine bone marrow cells for potential use in autoimmune experimental uveoretinitis model. Invest. Ophthalmol. Vis. Sci. 2017;58(8):529.
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© ARVO (1962-2015); The Authors (2016-present)
Autoimmune uveitis, a potentially sight-threatening disease is induced by immunogenic dendritic cells (DCs) via Th1/Th17 cells. Studies have shown that tolerogenic (tol) DCs prevent development of autoimmunity including autoimmune experimental uveoretinitis (EAU). However, studies have shown that conventionally prepared GM-CSF derived bone marrow (BM) DC population yielded a low percentage of CD11c+CD11b+MHCII+CD135+ DCs, with CD115+ macrophage/monocyte myeloid cells as the predominant population. Therefore, we aimed to generate a tolDC population from bone marrow progenitor cell population by depleting lineage-defined cells, particularly cells expressing MHCII prior to culture.
Bone marrow cells (BMCs) were extracted from C57Bl/6 mice and depleted for MHCII+, CD4+, CD8+ and B220+ cells. These Lin- cells were cultured for 6 days in GM-CSF and then depleted for granulocytes (Gr-1). Further, BMCs were cultured for 24 hours (h) in the presence of tolerogenic agent (Vitamin D3+Dexamethasone) and an immunogenic stimulant (Mycobacterium tuberculosis (Mtb) extract (15μg/ml) (24h) or lipopolysaccharide (LPS) (1μg/ml) (1h)). Using multicolour flow cytometry, these cells were tested for maturation status (MHCII, CD40), and characterised using progenitor (CX3CR1, c-kit) and differentiation (CD11b, CD11c, Ly6C, Zbtb46) markers. Additionally, BMCs were investigated for their functional capacity in T cell proliferation induction using OTII specific assay.
Our data suggests that after 7 days of culture more than 60% of untreated BMCs are committed cDC progenitors expressing Zbtb46, yet have low levels of activation markers (MHCII, CD40). These results are similar to the Vitamin D3+Dexamethasone treated BMCs. DC-enriched BMCs induced T regulatory cell (Tregs) (FoxP3+) as effectively as the Vitamin D3+Dexamethasone only treated BMCs. In contrast LPS stimulated cells failed to promote Tregs but induced Th1 (Tbet+) cells. Our protocol reduced the CD115+ macrophage contamination from 80% to 30% and yielded a maximum of 98.5% of committed progenitor cell population in the DC fraction.
Lin- BMCs generated in GM-CSF were highly enriched for progenitor/tolDC population that expressed low levels of MHCII and CD40. The tolerogenic state of these cells was confirmed by Treg induction in vitro. These cells are currently been investigated in the in vivo model of EAU.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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