Identification Of Bone Marrow Naive Myeloid-Derived Suppressor Cells Subpopulations With Distinct Phenotype And T Cell Suppressive Activity

Objectives:MDSCs are a group of heterogeneous cells, usually increased in tumor, infection, and inflammation and transplantation disease. It has a broad inhibitory function to immune system in pathological conditions and plays a regulator of the immune system; it is a debate about the inhibitory function of na(i|¨)ve MDSCs in normal condittions. Currently, mixed-MDSCs or different subsets of MDSCs have been researched in different pathological conditions such as cancer and infection. This study used the bone marrow of Gfi1: GFP knockin mouse model, which isolated na(i|¨)ve MDSCs, and sorted four subsets cells of naive MDSCs by FACS Aria, and using of modern immunological methods to understand the na(i|¨)ve MDSCs and its four subsets of normal mice may have different phenotypes and T cell immune suppression; focus on different subsets of na(i|¨)ve MDSCs inhibited T cells peroliferation and its mechanism.Methods:The bone marrow cells of C57BL/6J Gfi1: GFP knockin mouse model were separated and purificated, na(i|¨)ve MDSCs of bone marrow were isolated by immunomagnetic beads (CD11b microbeads kit), the purity of na(i|¨)ve MDSCs was detected by FCM; the morphology na(i|¨)ve MDSCs was analyzed of by smear of centrifugation and staining of Wright-Giemsa; Identification na(i|¨)ve MDSCs with CD4⁺T cells or CD8⁺T cells pre-stimulated by Dynabeads CD3/28 T-Activator and were cultured to detect CD4⁺T and CD8⁺T cells function by FCM. CD11b^lowGr1^low GFP⁺ (G1), CD11b^lowGr1^highGFP⁺(G2), CD11b^highGr1^highGFP⁺ (G3), CD11b⁺Gr1^lowGFP^- (M1) four subsets dependened on the differences of CD11b,Gr1 and Gfi1-GFP expression. Identification and isolation of purified four subsets function and identified the phenotype and analysed morphology by smear of centrifugation and staining of Wright-Giemsa; respectively purified G1, G2, G3 and M1 subsets cells with CD4⁺T cells or CD8⁺T cells pre-stimulated by Dynabeads CD3/28 T-Activator and were cultured to detect CD4⁺T and CD8⁺T cells function by FCM. G1, G2,G3 and M1 subsets cells were isolated from the model mice bone marrow and detected mRNA expression in a vary of cytokines of na(i|¨)ve MDSC subsets, which play a detail molecular mechanisms of immunosuppression. Furthermore,to investigate the status of ArgI and NOS2 in the mechanism of immunosuppression, blocked ArgI ,NOS, and NOS2 respectly by L-NMMA, L-NIL and nor-NOHA and detected by FCM..Results:Na(i|¨)ve MDSCs are important components of normal mice immune system, the morphology of total MDSCs in the bone marrow is large and small, the cytoplasm is blue, and the shape of nucleus is sub-leaf, ring and oval. CD4⁺T or CD8⁺T cells proliferation were inhibited by na(i|¨)ve MDSCs (CD11b⁺ cells) of normal mice bone marrow, and the mortality of acute hepatic failure (AHF) mice models were delayed and decreased by na(i|¨)ve MDSCs. It could be devided into CD11b^lowGr1^low GFP⁺ (G1), CD11b^lowGr1^highGFP⁺(G2), CD11b^highGr1^highGFP⁺ (G3), CD11b⁺Gr1^lowGFP^- (M1) four subsets dependened on the differences of CD11b,Gr1 and Gfi1-GFP expression. Furthermore, G1 subsets of na(i|¨)ve MDSCs are about 1.19±0.38% of bone marrow total cells, and about 4.04±0.9% of na(i|¨)ve MDSCs. G2 subsets of na(i|¨)ve MDSCs are about 8.49±2.69% of bone marrow total cells, and about 28.9±5.25% of na(i|¨)ve MDSCs. G3 subsets of na(i|¨)ve MDSCs are about 6.04±1.55% of bone marrow total cells, and about 20.95±4.98% of na(i|¨)ve MDSCs. M1 subsets of na(i|¨)ve MDSCs are about 3.86±1.06% of bone marrow total cells, and about 13.25±2.65% of na(i|¨)ve MDSCs. The volume of G1 subset are huge and the nucleus of large group cells are ring shape , the nucleus of G2 subset cells are ring and sub-leaf shape, the nucleus of G3 subset cells are sub-leaf and polymorphonuclear shape, the nucleus of G3 subset cells are sub-leaf and polymorphonuclear shape, while the volume of M1 subsets are smaller, and most of cells are immature monocytes. The subsets of G1 and G2 high expression of CD62L and Ly6C , M1 and G3 high expression of Ly6C, and high expression of CD62L partially. CD204, CD206, TLR2 and CD80, CD86 (costimulatory molecules) and CD124 (receptor of IL-4) are low expression on four subsets of na(i|¨)ve MDSCs, and not expression of TLR4 and CD210 (IL-10). G2 and G3 subsets of na(i|¨)ve MDSCs inhibit CD4⁺T or CD8⁺T cell proliferation respectly at different levels; however, G1 and M1 subsets of na(i|¨)ve MDSCs had no significant inhibitory effect to CD4⁺T cells or CD8⁺T cells. G2 and G3 subsets of na(i|¨)ve MDSCs IL-4, IL-10, IL-13, IFN-γand other cytokines were increased expression of G2 and G3 subsets of na(i|¨)ve MDSCs. In addition, Argâ… a nd NOS2 is maybe no significant relationship with the immunosuppressive effects.Conclutions:1. CD4⁺T or CD8⁺T cells proliferation were inhibited by na(i|¨)ve MDSCs (CD11b+ cells) of normal mice bone marrow, and the mortality of acute hepatic failure (AHF) mice models were delayed and decreased by na(i|¨)ve MDSCs.2. CD11blowGr1lowGFP+(G1),CD11blowGr1highGFP+(G2),CD11bhighGr1highGFP+(G3), CD11b+Gr1lowGFP-(M1) subsets of na(i|¨)ve MDSCs were isolated from bone marrow of Gfi1: GFP knockin normal mice by the difference of Gfi1-GFP , CD11b and Gr1 expression.3. In four subsets of na(i|¨)ve MDSCs, G2 and G3 subsets have different levels of inhibition of CD4 ⁺T or CD8⁺T cell proliferation respective. However, G1 and M1 subsets of naive MDSCs had no significant inhibition to CD4⁺T or CD8⁺T cells. G2 subset of na(i|¨)ve MDSCs maybe play an immunosuppressive effect by increasing the levels of anti-inflammatory cytokines, such as IL-4 and IL-10. G3 subset of na(i|¨)ve MDSCs maybe play an immunosuppressive effect by increasing the levels of anti-inflammatory cytokines, such as IL-10 and IFN-γ.4. The T cells inhibitory mechanism of G2 and G3 subsets may be not related to NOS2 and ArgI.