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Immunoregulatory properties of erythroid nucleated cells induced from CD34+ progenitors from bone marrow - PubMed

️Sun Jan 01 2023

Immunoregulatory properties of erythroid nucleated cells induced from CD34+ progenitors from bone marrow

Julia A Shevchenko et al. PLoS One. 2023.

Abstract

CD 71+ erythroid nucleated cells have pronounced immunoregulatory properties in normal and pathological conditions. Many populations of cells with immunoregulatory properties are considered candidates for cellular immunotherapy for various pathologies. This study characterized the immunoregulatory properties of CD71+ erythroid cells derived from CD34-positive bone marrow cells under the influence of growth factors that stimulate differentiation into erythroid cells. CD34-negative bone marrow cells were used to isolate CD71+ erythroid nuclear cells. The resulting cells were used to assess the phenotype, determine the mRNA spectrum of the genes responsible for the main pathways and processes of the immune response, and obtain culture supernatants for the analysis of immunoregulatory factors. It was found that CD71+ erythroid cells derived from CD34+ cells carry the main markers of erythroid cells, but differ markedly from natural bone marrow CD71+ erythroid cells. The main differences are in the presence of the CD45+ subpopulation, distribution of terminal differentiation stages, transcriptional profile, secretion of certain cytokines, and immunosuppressive activity. The properties of induced CD71+ erythroid cells are closer to the cells of extramedullary erythropoiesis foci than to natural bone marrow CD71+ erythroid cells. Thus, when cultivating CD71+ erythroid cells for clinical experimental studies, it is necessary to take into account their pronounced immunoregulatory activity.

Copyright: © 2023 Shevchenko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

**Fig 1. Staging of natural bone marrow CD71+ erythroid cells.**
A–Dot-plot for natural bone marrow CD71+ erythroid cells. B–Histogram of the distribution of bone marrow CD71+ erythroid cells by marker CD 45. C–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 71 and CD 235a from CD 45-negative natural bone marrow CD71+ erythroid cells, ProEB–proerythroblasts (CD235a^lowCD71^high cells), BasoEB+PolyEB+OrthoEB+Ret–basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts/reticulocytes (CD235^highCD 71^pos cells), Erythrocytes–CD235a^highCD71^neg. D–Histogram of the distribution of stages of development of erythroid cells with a phenotype CD235^highCD 71^pos depending on the FSC. Ret–reticulocytes (CD 235a^highCD71^pos FSC^low), Ortho–orthochromatophilic erythroblasts (CD235a^highCD71^posFSC^low cells), Poly–polychromatophilic erythroblasts (CD235a^highCD 71^posFSC^middle cells), Baso–basophilic erythroblasts (CD 235a^high CD71^pos FSC^high cells).

**Fig 2. Staging of induced CD71+ erythroid cells derived from CD34+ bone marrow cells.**
A–Dot-plot for induced CD71+ erythroid cells derived from CD34+ bone marrow cells. B–Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 45. C–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 71 and CD 235a from CD 45-positive induced CD71+ erythroid cells. D–Histogram of the distribution of stages of development of CD71+ erythroid cells with a phenotype CD45^posCD235a^highCD 71^pos depending on the FSC. E–Dot-plot of the distribution of bone marrow CD71+ erythroid by marker CD 71 and CD 235a from CD 45-negative induced CD71+ erythroid cells. F–Histogram of the distribution of stages of development of CD71+ erythroid cells with a phenotype CD45^negCD235a^highCD 71^pos depending on the FSC, ProEB–proerythroblasts (CD235a^lowCD71^high cells), BasoEB+PolyEB+OrthoEB+Ret–basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts/reticulocytes (CD235a^highCD 71^pos cells), Erythrocytes–CD235a^highCD71^neg, Ret–reticulocytes (CD 235a^highCD71^pos FSC^low), Ortho–orthochromatophilic erythroblasts (CD235a^highCD71^posFSC^low cells), Poly–polychromatophilic erythroblasts (CD235a^highCD 71^posFSC^middle cells), Baso–basophilic erythroblasts (CD 235^high CD71^pos FSC^high cells).

**Fig 3. Comparison of the distribution of stages of development of bone marrow CD71+ erythroid cells and CD71+ erythroid cells induced from CD34+ bone marrow cells.**
Kruskal-Wallis test ANOVA with Dunn’s correction for multiple comparisons was carried out to evaluate statistical significance of the differences between the cell groups. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range.

**Fig 4. Expression of lymphoid cell markers on the surface of induced CD71+ erythroid cells.**
A–Dot-plot for induced CD71+ erythroid cells derived from CD34+ bone marrow cells. B–Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 45. С –Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 3 from CD 45-negative induced CD71+ erythroid cells. D–Histogram of the distribution of induced CD71+ erythroid cells derived from CD34+ bone marrow cells by marker CD 3 from CD 45-positive induced CD71+ erythroid cells. E–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 8 and CD 4 from CD 45-positive induced erythroblasts. F–Dot-plot of the distribution of bone marrow CD71+ erythroid cells by marker CD 16 and CD 19 from CD 45-positive induced erythroblasts.

**Fig 5. Heat map of studied cells’ transcriptome.**

**Fig 6. Volcano-plots of differentially expressed genes.**
The purple dots correspond to the samples of induced CD71+ erythroid cells, the orange dots correspond to the samples of natural CD71+ erythroid cells of bone marrow.

**Fig 7. MA-plots of differentially expressed genes.**
The purple dots correspond to the samples of induced CD71+ erythroid cells, the orange dots correspond to the samples of natural CD71+ erythroid cells of bone marrow.

**Fig 8. Plot of Gene Set Enrichment Analysis (GSEA) results showing down-regulated differentially expressed genes.**

Fig 9. The PLS-DA plot of cytokine production by different types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34⁺ bone marrow cells).
An example distribution of data points for chemokines, interleukins, and growth factors is shown. (x)–ABM–bone marrow erythroblasts. (·)–CD 34 induced–induced erythroblasts from CD34⁺ bone marrow cells.

Fig 10. The PLS-DA plot of cytokine production by different types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34⁺ bone marrow cells).
An example distribution of data points for colony-stimulating factors, interferons, and TNF family cytokines is shown. (x)–ABM–bone marrow erythroblasts. (·)–CD 34 induced–induced erythroblasts from CD34⁺ bone marrow cells.

Fig 11. Cytokines that manifested significant differences in secretion of interleukins between the two types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34⁺ bone marrow cells).
The Mann–Whitney test was performed to compare the production of cytokines between the different types of CD71+ erythroid cells. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range.

Fig 12. Cytokines that manifested significant differences in secretion of chemokines and growth factors between the two types of CD71+ erythroid cells (n = 6 for bone marrow CD71+ erythroid cells and induced CD71+ erythroid cells from CD34⁺ bone marrow cells).
The Mann–Whitney test was performed to compare the production of cytokines between the different types of CD71+ erythroid cells. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range.

**Fig 13. Effects of the conditioned media from either the natural or induced bone marrow CD71+ erythroid cells on the mixed lymphocyte reaction (n = 4).**
The results are presented as optical density of allogeneic cultures. Statistical analysis of the data was conducted in the GraphPad Prism 8.4 software. Two-way ANOVA with Tukey’s correction for multiple comparisons was carried out to evaluate statistical significance of the differences between the cell groups. Results were assumed to be statistically significant at p-value < 0.05, and the data are presented as a median with an interquartile range. “Allostimulator”: culture of mononuclear cells with the addition of allogeneic donor cells treated with mitomycin C; “Allostimulator + BM EBs”: culture of mononuclear cells with the addition of mitomycin C–treated allogeneic donor cells and supplementation with the conditioned medium from natural bone marrow CD71+ erythroid cells; “Allostimulator + induced EBs”: culture of mononuclear cells with the addition of mitomycin C–treated allogeneic donor cells and supplementation with the conditioned medium from the induced CD71+ erythroid cells derived from CD34+ bone marrow cells of healthy donors.

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References

1. Zivot A, Lipton JM, Narla A, Blanc L. Erythropoiesis: insights into pathophysiology and treatments in 2017. Mol Med. 2018, 24(1), 11. doi: 10.1186/s10020-018-0011-z - DOI - PMC - PubMed
1. Koury S, Yarlagadda S, Moskalik-Liermo K, Popli N, Kim N, Apolito C, et al.. Differential gene expression during terminal erythroid differentiation Genomics. 2007, 90(5), 574–82 doi: 10.1016/j.ygeno.2007.06.010 - DOI - PMC - PubMed
1. Huang P, Zhao Y, Zhong J, Zhang X, Liu Q, Qiu X, et al.. Putative regulators for the continuum of erythroid differentiation revealed by single-cell transcriptome of human BM and UCB cells. Proc Natl Acad Sci U S A. 2020,117(23), 12868–12876. doi: 10.1073/pnas.1915085117 - DOI - PMC - PubMed
1. Morera D, MacKenzie SA. Is there a direct role for erythrocytes in the immune response? Vet Res. 2011, 42(1), 89. doi: 10.1186/1297-9716-42-89 - DOI - PMC - PubMed
1. Mohseni YR, Tung SL, Dudreuilh C, Lechler RI, Fruhwirth GO, Lombardi G. The Future of Regulatory T Cell Therapy: Promises and Challenges of Implementing CAR Technology. Front Immunol. 2020, 11, 1608. doi: 10.3389/fimmu.2020.01608 - DOI - PMC - PubMed

Immunoregulatory properties of erythroid nucleated cells induced from CD34+ progenitors from bone marrow - PubMed