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Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

Kurkó J, Vida A, Ocskó T, Tryniszewska B, Rauch TA, Glant TT, Szekanecz Z, Mikecz K - PLoS ONE (2014)

Bottom Line: The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide.Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Medicine, Department of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, United States of America; Department of Rheumatology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.

ABSTRACT

Background: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.

Methods: Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.

Results: BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.

Conclusions: Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

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Suppression of antigen (Ag)-specific and non-specific T-cell responses by BM-MDSCs.(A) T cells, purified from the spleens of mice expressing a PG-specific T cell receptor transgene (PG-TCR-Tg) were cultured for 5 days with dendritic cells (DCs) loaded with recombinant G1 domain of human PG (rhG1) in the absence or presence of the following “suppressors”: BM-MDSCs (red bar), arthritic SF cells (gray bar), or Ly6Chi (monocytic) cell-depleted BM-MDSCs (black bar). The ability of suppressors to inhibit Ag (rhG1)-specific T-cell proliferation (which is also dependent on Ag presentation by DCs) was assessed on the basis of inhibition of [3H]thymidine incorporation by the T cells. Percent suppression was calculated as described in the Methods. All suppressors exhibited robust inhibition of T-cell proliferation. The results shown are from 5 independent experiments. (B) T cells from PG-TCR-Tg mice were cultured for 2 days with rhG1-loaded DCs and BM-MDSCs as described for panel A. The percent of CD4+ T cells containing IFNγ, IL-10, or FoxP3 (CD4+CD25+FoxP3+ T regulatory cells, Tregs) was determined by flow cytometry. The results shown are the individual values (n = 5–6) and the means. On average, the percentages of IFNγ+ cells, IL-10+ cells, and Tregs were lower in the presence of BM-MDSCs (*p<0.001, 0.001, and 0.05, respectively; Mann-Whitney U test) than in their absence (None). (C) T cells from PG-TCR-Tg mice were cultured in anti-CD3/CD28-coated plates for 4 days in the absence or presence of the listed suppressors. Percent suppression was calculated and results expressed as described for panel A. Non-depleted BM-MDSCs and BM-MDSCs depleted in Ly6Chi cells were equally potent in suppressing anti-CD3/CD28-induced T-cell proliferation, while arthritic SF cells exhibited much weaker inhibition (*p<0.01, n = 5; Kruskal-Wallis test followed by Dunn’s multiple comparisons test) in this induction system.
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pone-0111815-g002: Suppression of antigen (Ag)-specific and non-specific T-cell responses by BM-MDSCs.(A) T cells, purified from the spleens of mice expressing a PG-specific T cell receptor transgene (PG-TCR-Tg) were cultured for 5 days with dendritic cells (DCs) loaded with recombinant G1 domain of human PG (rhG1) in the absence or presence of the following “suppressors”: BM-MDSCs (red bar), arthritic SF cells (gray bar), or Ly6Chi (monocytic) cell-depleted BM-MDSCs (black bar). The ability of suppressors to inhibit Ag (rhG1)-specific T-cell proliferation (which is also dependent on Ag presentation by DCs) was assessed on the basis of inhibition of [3H]thymidine incorporation by the T cells. Percent suppression was calculated as described in the Methods. All suppressors exhibited robust inhibition of T-cell proliferation. The results shown are from 5 independent experiments. (B) T cells from PG-TCR-Tg mice were cultured for 2 days with rhG1-loaded DCs and BM-MDSCs as described for panel A. The percent of CD4+ T cells containing IFNγ, IL-10, or FoxP3 (CD4+CD25+FoxP3+ T regulatory cells, Tregs) was determined by flow cytometry. The results shown are the individual values (n = 5–6) and the means. On average, the percentages of IFNγ+ cells, IL-10+ cells, and Tregs were lower in the presence of BM-MDSCs (*p<0.001, 0.001, and 0.05, respectively; Mann-Whitney U test) than in their absence (None). (C) T cells from PG-TCR-Tg mice were cultured in anti-CD3/CD28-coated plates for 4 days in the absence or presence of the listed suppressors. Percent suppression was calculated and results expressed as described for panel A. Non-depleted BM-MDSCs and BM-MDSCs depleted in Ly6Chi cells were equally potent in suppressing anti-CD3/CD28-induced T-cell proliferation, while arthritic SF cells exhibited much weaker inhibition (*p<0.01, n = 5; Kruskal-Wallis test followed by Dunn’s multiple comparisons test) in this induction system.

Mentions: To study the effect of BM-MDSC-like cells on Ag-specific T-cell proliferation, we cultured Ag (rhG1)-loaded DCs with T cells isolated from the spleens of naive PG-TCR-Tg mice in the presence or absence of BM-MDSCs as suppressors. Additional “suppressors” (as comparators) were SF cells, and BM-MDSCs depleted in Ly6Chi cells. Ag-dependent T-cell proliferation was dramatically reduced in the presence of BM-MDSCs, i.e., BM-MDSC-mediated suppression reached nearly 100% (Fig. 2A, red bar). Compared with SF cells (Fig. 2A, gray bar) BM-MDSCs were equally potent in suppressing T-cell proliferation. As also reported for SF cells [21], depletion of the Ly6Chi monocytic subset from the BM-MDSCs (Fig. 2A, black bar) did not reduce their suppressive capacity. BM-MDSC-mediated suppression of Ag-specific T-cell proliferation was accompanied by significant decreases in the percentage of CD4+ T helper (Th) cells containing intracellular cytokines (IFNγ in Th1 and IL-10 in Th2 cells) as well as in the percentage of Tregs (CD4+CD25+ cells containing FoxP3) (Fig. 2B).


Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

Kurkó J, Vida A, Ocskó T, Tryniszewska B, Rauch TA, Glant TT, Szekanecz Z, Mikecz K - PLoS ONE (2014)

Suppression of antigen (Ag)-specific and non-specific T-cell responses by BM-MDSCs.(A) T cells, purified from the spleens of mice expressing a PG-specific T cell receptor transgene (PG-TCR-Tg) were cultured for 5 days with dendritic cells (DCs) loaded with recombinant G1 domain of human PG (rhG1) in the absence or presence of the following “suppressors”: BM-MDSCs (red bar), arthritic SF cells (gray bar), or Ly6Chi (monocytic) cell-depleted BM-MDSCs (black bar). The ability of suppressors to inhibit Ag (rhG1)-specific T-cell proliferation (which is also dependent on Ag presentation by DCs) was assessed on the basis of inhibition of [3H]thymidine incorporation by the T cells. Percent suppression was calculated as described in the Methods. All suppressors exhibited robust inhibition of T-cell proliferation. The results shown are from 5 independent experiments. (B) T cells from PG-TCR-Tg mice were cultured for 2 days with rhG1-loaded DCs and BM-MDSCs as described for panel A. The percent of CD4+ T cells containing IFNγ, IL-10, or FoxP3 (CD4+CD25+FoxP3+ T regulatory cells, Tregs) was determined by flow cytometry. The results shown are the individual values (n = 5–6) and the means. On average, the percentages of IFNγ+ cells, IL-10+ cells, and Tregs were lower in the presence of BM-MDSCs (*p<0.001, 0.001, and 0.05, respectively; Mann-Whitney U test) than in their absence (None). (C) T cells from PG-TCR-Tg mice were cultured in anti-CD3/CD28-coated plates for 4 days in the absence or presence of the listed suppressors. Percent suppression was calculated and results expressed as described for panel A. Non-depleted BM-MDSCs and BM-MDSCs depleted in Ly6Chi cells were equally potent in suppressing anti-CD3/CD28-induced T-cell proliferation, while arthritic SF cells exhibited much weaker inhibition (*p<0.01, n = 5; Kruskal-Wallis test followed by Dunn’s multiple comparisons test) in this induction system.
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Related In: Results  -  Collection

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pone-0111815-g002: Suppression of antigen (Ag)-specific and non-specific T-cell responses by BM-MDSCs.(A) T cells, purified from the spleens of mice expressing a PG-specific T cell receptor transgene (PG-TCR-Tg) were cultured for 5 days with dendritic cells (DCs) loaded with recombinant G1 domain of human PG (rhG1) in the absence or presence of the following “suppressors”: BM-MDSCs (red bar), arthritic SF cells (gray bar), or Ly6Chi (monocytic) cell-depleted BM-MDSCs (black bar). The ability of suppressors to inhibit Ag (rhG1)-specific T-cell proliferation (which is also dependent on Ag presentation by DCs) was assessed on the basis of inhibition of [3H]thymidine incorporation by the T cells. Percent suppression was calculated as described in the Methods. All suppressors exhibited robust inhibition of T-cell proliferation. The results shown are from 5 independent experiments. (B) T cells from PG-TCR-Tg mice were cultured for 2 days with rhG1-loaded DCs and BM-MDSCs as described for panel A. The percent of CD4+ T cells containing IFNγ, IL-10, or FoxP3 (CD4+CD25+FoxP3+ T regulatory cells, Tregs) was determined by flow cytometry. The results shown are the individual values (n = 5–6) and the means. On average, the percentages of IFNγ+ cells, IL-10+ cells, and Tregs were lower in the presence of BM-MDSCs (*p<0.001, 0.001, and 0.05, respectively; Mann-Whitney U test) than in their absence (None). (C) T cells from PG-TCR-Tg mice were cultured in anti-CD3/CD28-coated plates for 4 days in the absence or presence of the listed suppressors. Percent suppression was calculated and results expressed as described for panel A. Non-depleted BM-MDSCs and BM-MDSCs depleted in Ly6Chi cells were equally potent in suppressing anti-CD3/CD28-induced T-cell proliferation, while arthritic SF cells exhibited much weaker inhibition (*p<0.01, n = 5; Kruskal-Wallis test followed by Dunn’s multiple comparisons test) in this induction system.
Mentions: To study the effect of BM-MDSC-like cells on Ag-specific T-cell proliferation, we cultured Ag (rhG1)-loaded DCs with T cells isolated from the spleens of naive PG-TCR-Tg mice in the presence or absence of BM-MDSCs as suppressors. Additional “suppressors” (as comparators) were SF cells, and BM-MDSCs depleted in Ly6Chi cells. Ag-dependent T-cell proliferation was dramatically reduced in the presence of BM-MDSCs, i.e., BM-MDSC-mediated suppression reached nearly 100% (Fig. 2A, red bar). Compared with SF cells (Fig. 2A, gray bar) BM-MDSCs were equally potent in suppressing T-cell proliferation. As also reported for SF cells [21], depletion of the Ly6Chi monocytic subset from the BM-MDSCs (Fig. 2A, black bar) did not reduce their suppressive capacity. BM-MDSC-mediated suppression of Ag-specific T-cell proliferation was accompanied by significant decreases in the percentage of CD4+ T helper (Th) cells containing intracellular cytokines (IFNγ in Th1 and IL-10 in Th2 cells) as well as in the percentage of Tregs (CD4+CD25+ cells containing FoxP3) (Fig. 2B).

Bottom Line: The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide.Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Medicine, Department of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, United States of America; Department of Rheumatology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.

ABSTRACT

Background: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.

Methods: Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.

Results: BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.

Conclusions: Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

Show MeSH
Related in: MedlinePlus