Generation and Characterization of Mouse Regulatory Macrophages.

International audienceIn the last years, cell therapy has become a promising approach to therapeutically manipulate immune responses in autoimmunity, cancer, and transplantation. Several types of lymphoid and myeloid cells origin have been generated in vitro and tested in animal models. Their efficacy to decrease pharmacological treatment has successfully been established. Macrophages play an important role in physiological and pathological processes. They represent an interesting cell population due to their high plasticity in vivo and in vitro. Here, we describe a protocol to differentiate murine regulatory macrophages in vitro from bone marrow precursors. We also describe several methods to assess macrophage classical functions, as their bacterial killing capacity and antigen endocytosis and degradation. Importantly, regulatory macrophages also display suppressive characteristics, which are addressed by the study of their hypostimulatory T lymphocyte capacity and polyclonal T lymphocyte activation suppression

Comparative Study of the Immunoregulatory Capacity of In Vitro Generated Tolerogenic Dendritic Cells, Suppressor Macrophages, and Myeloid-Derived Suppressor Cells

International audienceBackground. Regulatory myeloid cell (RMC) therapy is a promising strategy for the treatment of immunological disorders such as autoimmune disease and allograft transplant rejection. Various RMC subsets can be derived from total bone marrow using different protocols, but their phenotypes often overlap, raising questions about whether they are truly distinct.Methods. In this study, we directly compared the phenotype and function of 3 types of RMCs, tolerogenic dendritic cells, suppressor macrophages, and myeloid-derived suppressor cells, generated in vitro from the same mouse strain in a single laboratory.Results. We show that the 3 RMC subsets tested in this study share some phenotypic markers, suppress T cell proliferation in vitro and were all able to prolong allograft survival in a model of skin transplantation. However, our results highlight distinct mechanisms of action that are specific to each cell population.Conclusions. This study shows for the first time a side-by-side comparison of 3 types of RMCs using the same phenotypic and functional assays, thus providing a robust analysis of their similarities and differences

Phenotype of BM-derived MDSC.

<p>(A) BM cells from naive mice were cultured in the presence of GM-CSF and IL-6 for 4 days. Surface expression of CD11b and Gr1 was measured by flow cytometry. (B) Quantification of the relative proportions of CD11b⁺ Gr1^hi and CD11b⁺ Gr1^low populations in independent preparations. (C) Expression of CD11c and MHC II on total CD11b⁺ cells or in Gr1^hi and Gr1^low populations. Gray areas represent fluorescence minus one (FMO) controls. Data show representative results from at least four independent experiments.</p

Adoptive transfer of BM-derived MDSC is associated with increased numbers of CD25⁺ and CD69⁺ cells, mainly in the spleen.

<p>Male skin grafts were transplanted onto females recipients treated or not at days −1, 6 and 13 post-transplantation with four million autologous (female) MDSC generated in vitro with GM-CSF and IL-6. Draining lymph nodes and spleen were harvested from skin-grafted mice 14 days after transplantation or from naive mice for flow cytometry analysis. (A) Representative staining and quantification of donor-specific Pentamer⁺ CD8⁺ T cells in naive or skin-grafted mice. (B) Quantification of FoxP3⁺ cells among CD3⁺ CD4⁺ T cells. (C, D) Representative stainings and quantifications of CD25⁺ (C) and CD69⁺ (D) among CD3⁺ CD4⁺ or CD3⁺ CD8⁺ T cells. Data show results from two independent experiments with 4 to 9 mice per group. *p<0.05, **p<0.01, ***p<0.001.</p

BM-derived MDSC efficiently inhibit T cell proliferation <i>in vitro.</i>

<p>CD8⁺ T cells were purified from OT-1 transgenic mice and labeled with CFSE before anti-CD3/CD28 bead stimulation. MDSC generated in vitro with GM-CSF and IL-6 were added to T cells at different ratios. After three days of culture, the percentage of proliferating cells (CFSE^low) in CD8⁺ cells was assessed by flow cytometry. Representative histograms of CFSE dilution (A) and quantification of triplicates for each condition are shown (B). Data are representative of three independent experiments.</p

Adoptive transfer of BM-derived MDSC does not prevent lymphocyte infiltration in skin allografts.

<p>Male skin grafts were transplanted onto females recipients treated or not at days −1, 6 and 13 post-transplantation with four million autologous (female) MDSC generated in vitro with GM-CSF and IL-6. Skin grafts were harvested 14 days after transplantation and infiltrated leukocytes were analyzed by flow cytometry. Results are expressed in percentages of CD3⁺ CD4⁺ T cells (A), CD3⁺ CD8⁺ T cells (B) and donor-specific Pentamer⁺ cells among CD8⁺ T cells (C). Data show results from two independent experiments with 4 to 9 mice per group. **p<0.01.</p

Adoptive transfer of BM-derived MDSC does not alter antigen-specific CD8⁺ T cell responses.

<p>(A–B) In vivo proliferation assay: COS cells transfected with a plasmid coding for GFP fused to the OVA_257–264 peptide (COS OVA) or GFP alone (COS) were injected i.v. into mice with or without MDSC generated in vitro with GM-CSF and IL-6. Responder CD8⁺ T cells purified from OT-1 TCR-transgenic mice were labeled with CFSE and injected i.v. the following day. After 3 days, spleens of recipient mice were harvested to assess CFSE dilution by flow cytometry. Data are representative of two experiments. (C–D) In vivo cytotoxicity assay: CD8⁺ T cells purified from OT-1 TCR-transgenic mice (non labeled with CFSE) were injected in COS GFP/OVA-immunized mice as described above. After 8 days, CFSE-labeled CD45.1⁺ target cells either loaded with OVA_257–264 (CFSE^hi) or control (CFSE^low) peptides were injected. Specific lysis was determined the next day by flow cytometry by measuring the relative proportion of each population in the spleen of MDSC-treated or untreated mice compared to non-immunized mice. Data show results from four independent experiments with 9 to 11 mice per group.</p