Phenotypic Comparison of Regulatory T Cells: Mobilized Peripheral Blood Versus Umbilical Cord Blood
Abstract
Regulatory T cells (Tregs) play a central role in maintaining immune tolerance and modulating inflammatory responses. Different cellular sources may give rise to Tregs with distinct phenotypic properties, which could influence their clinical applicability in immune-mediated diseases.
In this exploratory comparative study, we performed a comparative flow cytometric analysis of Treg populations derived from mobilized peripheral blood (mPB) and umbilical cord blood (UCB). Tregs were identified based on a standard CD4+CD25+CD127low/− gating strategy, followed by phenotypic assessment using markers associated with differentiation (CD45RA/RO, CD27), activation (CD69), and immune checkpoint–related regulation (PD-1, TIM-3).
Our results demonstrate source-dependent differences in the distribution of naive and memory-like Treg subsets, as well as in the expression of activation and checkpoint-associated markers.
These preliminary findings suggest phenotypic heterogeneity between mPB- and UCB-derived Tregs and warrant validation in larger cohorts and functional studies.
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3. Cheru N, Hafler DA, Sumida TS. Regulatory T cells in peripheral tissue tolerance and diseases. Front Immunol. 2023;14:1154575.
4. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of Treg in immune regulation of allergic diseases. Europ J Immunol. 2010;40(5):1232-40.
5. Gershon RK, Kondo K. Cell interactions in the induction of tolerance: the role of thymic lymphocytes. Immunology. 1970;18(5):723.
6. Dikiy S, Rudensky AY. Principles of regulatory T cell function. Immunity. 2023;56(2):240-55.
7. Magenau JM, Qin X, Tawara I, Rogers CE, Kitko C, Schlough M, et al. Frequency of CD4+ CD25hiFOXP3+ regulatory T cells has diagnostic and prognostic value as a biomarker for acute graft-versus-host-disease. Biolo Blood Marrow Transplant. 2010;16(7):907-14.
8. Botturi K, Lacoeuille Y, Cavaillès A, Vervloet D, Magnan A. Differences in allergen-induced T cell activation between allergic asthma and rhinitis: Role of CD28, ICOS and CTLA-4. Resp Res. 2011;12(1):25.
9. Georgiev P, Benamar M, Han S, Haigis MC, Sharpe AH, Chatila TA. Regulatory T cells in dominant immunologic tolerance. Journal of Allergy and Clinical Immunology. 2024;153(1):28-41.
10. Reddy RL. Mobilization and collection of peripheral blood progenitor cells for transplantation. Trans Apheresis Sci. 2005;32(1):63-72.
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12. Roura S, Pujal J-M, Gálvez-Montón C, Bayes-Genis A. The role and potential of umbilical cord blood in an era of new therapies: a review. Stem Cell Res Therapy. 2015;6(1):123.
13. Seay HR, Putnam AL, Cserny J, Posgai AL, Rosenau EH, Wingard JR, et al. Expansion of human Tregs from cryopreserved umbilical cord blood for GMP-compliant autologous adoptive cell transfer therapy. Mol Therapy-Methods Clin Develop. 2017;4:178-91.
14. Booth NJ, McQuaid AJ, Sobande T, Kissane S, Agius E,
Jackson SE, et al. Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO. J Immunol. 2010;184(8):4317-26.
15. Muth S, Klaric A, Radsak M, Schild H, Probst HC. CD27 expression on Treg cells limits immune responses against tumors. J Mol Med. 2022;100(3):439-49.
16. Cibrián D, Sánchez‐Madrid F. CD69: from activation marker to metabolic gatekeeper. European journal of immunology. 2017;47(6):946-53.
17. Francisco LM, Sage PT, Sharpe AH. The PD‐1 pathway in tolerance and autoimmunity. Immunol Rev. 2010;236(1):219-42.
18. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature. 2002;415(6871):536-41.
19. Jin H-T, Anderson AC, Tan WG, West EE, Ha S-J, Araki K, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proceedings of the National Academy of Sciences. 2010;107(33):14733-8.
20. Sage PT, Francisco LM, Carman CV, Sharpe AH. The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood. Nat Immunol. 2013;14(2):152-61.
21. Gautron AS, Dominguez‐Villar M, de Marcken M, Hafler DA. Enhanced suppressor function of TIM‐3+ FoxP3+ regulatory T cells. Europ J Immunol. 2014;44(9):2703-11.
22. Sakaguchi S, Kawakami R, Mikami N. Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective. Immunotherapy Advances. 2023;3(1):ltad007.
23. Romano M, Tung SL, Smyth LA, Lombardi G. Treg therapy in transplantation: a general overview. Transplant Int. 2017;30(8):745-53.
24. Roncarolo M-G, Battaglia M. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol. 2007;7(8):585-98.
25. Santosh Nirmala S, Hu Y, Floegel FD, Cruz H, Morgenstern J, Platz A, et al. Delineating phenotypic heterogeneity in human regulatory T cells across developmental stages and therapeutic sources. Front Immunol. 2026;17:1697723.
26. To LB, Levesque J-P, Herbert KE. How I treat patients who mobilize hematopoietic stem cells poorly. Blood, The Journal of the American Society of Hematology. 2011;118(17):4530-40.
27. Gluckman E. History of cord blood transplantation. Bone Marrow Transplant. 2009;44(10):621-6.
28. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood. 2013;122(4):491-8.
29. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009;30(6):899-911.
30. Zhao C, Davies JD. A peripheral CD4+ T cell precursor for naive, memory, and regulatory T cells. J Exp Med. 2010;207(13):2883-94.
31. Hendriks J, Gravestein LA, Tesselaar K, Van Lier RA, Schumacher TN, Borst J. CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol. 2000;1(5):433-40.
32. Condomines M, Quittet P, Lu Z-Y, Nadal L, Latry P, Lopez E, et al. Functional regulatory T cells are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and granulocyte colony-stimulating factor. J Immunol. 2006;176(11):6631-9.
33. Brunstein CG, Miller JS, McKenna DH, Hippen KL, DeFor TE, Sumstad D, et al. Umbilical cord blood–derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect. Blood. 2016;127(8):1044-51.
34. Rutella S, Pierelli L, Bonanno G, Sica S, Ameglio F, Capoluongo E, et al. Role for granulocyte colony–stimulating factor in the generation of human T regulatory type 1 cells. Blood. 2002;100(7):2562-71.
35. Arpinati M, Green CL, Heimfeld S, Heuser JE, Anasetti C. Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells. Blood. 2000;95(8):2484-90.
36. Takahashi S, Minnie SA, Ensbey KS, Schmidt CR, Sekiguchi T, Legg SR, et al. Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation. Blood. 2024;143(16):1656-69.
37. Banerjee H, Nieves-Rosado H, Kulkarni A, Murter B, McGrath KV, Chandran UR, et al. Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment. Cell Reports. 2021;36(11).
38. Lowther DE, Goods BA, Lucca LE, Lerner BA, Raddassi K, van Dijk D, et al. PD-1 marks dysfunctional regulatory T cells in malignant gliomas. JCI insight. 2016;1(5):e85935.
39. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015;15(8):486-99.
40. Gootjes C, Zwaginga JJ, Roep BO, Nikolic T. Defining human regulatory T cells beyond FOXP3: the need to combine phenotype with function. Cells. 2024;13(11):941.
41. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 2006;203(7):1701-11.
42. Hossen MM, Ma Y, Yin Z, Xia Y, Du J, Huang JY, et al. Current understanding of CTLA-4: from mechanism to autoimmune diseases. Front Immunol. 2023;14:1198365.
43. Elkord E, Abd Al Samid M, Chaudhary B. Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP. Oncotarget. 2015;6(24):20026.
2. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133(5):775-87.
3. Cheru N, Hafler DA, Sumida TS. Regulatory T cells in peripheral tissue tolerance and diseases. Front Immunol. 2023;14:1154575.
4. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of Treg in immune regulation of allergic diseases. Europ J Immunol. 2010;40(5):1232-40.
5. Gershon RK, Kondo K. Cell interactions in the induction of tolerance: the role of thymic lymphocytes. Immunology. 1970;18(5):723.
6. Dikiy S, Rudensky AY. Principles of regulatory T cell function. Immunity. 2023;56(2):240-55.
7. Magenau JM, Qin X, Tawara I, Rogers CE, Kitko C, Schlough M, et al. Frequency of CD4+ CD25hiFOXP3+ regulatory T cells has diagnostic and prognostic value as a biomarker for acute graft-versus-host-disease. Biolo Blood Marrow Transplant. 2010;16(7):907-14.
8. Botturi K, Lacoeuille Y, Cavaillès A, Vervloet D, Magnan A. Differences in allergen-induced T cell activation between allergic asthma and rhinitis: Role of CD28, ICOS and CTLA-4. Resp Res. 2011;12(1):25.
9. Georgiev P, Benamar M, Han S, Haigis MC, Sharpe AH, Chatila TA. Regulatory T cells in dominant immunologic tolerance. Journal of Allergy and Clinical Immunology. 2024;153(1):28-41.
10. Reddy RL. Mobilization and collection of peripheral blood progenitor cells for transplantation. Trans Apheresis Sci. 2005;32(1):63-72.
11. Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH. Identification and functional characterization of human CD4+ CD25+ T cells with regulatory properties isolated from peripheral blood. J Exp Med. 2001;193(11):1285-94.
12. Roura S, Pujal J-M, Gálvez-Montón C, Bayes-Genis A. The role and potential of umbilical cord blood in an era of new therapies: a review. Stem Cell Res Therapy. 2015;6(1):123.
13. Seay HR, Putnam AL, Cserny J, Posgai AL, Rosenau EH, Wingard JR, et al. Expansion of human Tregs from cryopreserved umbilical cord blood for GMP-compliant autologous adoptive cell transfer therapy. Mol Therapy-Methods Clin Develop. 2017;4:178-91.
14. Booth NJ, McQuaid AJ, Sobande T, Kissane S, Agius E,
Jackson SE, et al. Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO. J Immunol. 2010;184(8):4317-26.
15. Muth S, Klaric A, Radsak M, Schild H, Probst HC. CD27 expression on Treg cells limits immune responses against tumors. J Mol Med. 2022;100(3):439-49.
16. Cibrián D, Sánchez‐Madrid F. CD69: from activation marker to metabolic gatekeeper. European journal of immunology. 2017;47(6):946-53.
17. Francisco LM, Sage PT, Sharpe AH. The PD‐1 pathway in tolerance and autoimmunity. Immunol Rev. 2010;236(1):219-42.
18. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature. 2002;415(6871):536-41.
19. Jin H-T, Anderson AC, Tan WG, West EE, Ha S-J, Araki K, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proceedings of the National Academy of Sciences. 2010;107(33):14733-8.
20. Sage PT, Francisco LM, Carman CV, Sharpe AH. The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood. Nat Immunol. 2013;14(2):152-61.
21. Gautron AS, Dominguez‐Villar M, de Marcken M, Hafler DA. Enhanced suppressor function of TIM‐3+ FoxP3+ regulatory T cells. Europ J Immunol. 2014;44(9):2703-11.
22. Sakaguchi S, Kawakami R, Mikami N. Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective. Immunotherapy Advances. 2023;3(1):ltad007.
23. Romano M, Tung SL, Smyth LA, Lombardi G. Treg therapy in transplantation: a general overview. Transplant Int. 2017;30(8):745-53.
24. Roncarolo M-G, Battaglia M. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol. 2007;7(8):585-98.
25. Santosh Nirmala S, Hu Y, Floegel FD, Cruz H, Morgenstern J, Platz A, et al. Delineating phenotypic heterogeneity in human regulatory T cells across developmental stages and therapeutic sources. Front Immunol. 2026;17:1697723.
26. To LB, Levesque J-P, Herbert KE. How I treat patients who mobilize hematopoietic stem cells poorly. Blood, The Journal of the American Society of Hematology. 2011;118(17):4530-40.
27. Gluckman E. History of cord blood transplantation. Bone Marrow Transplant. 2009;44(10):621-6.
28. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood. 2013;122(4):491-8.
29. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009;30(6):899-911.
30. Zhao C, Davies JD. A peripheral CD4+ T cell precursor for naive, memory, and regulatory T cells. J Exp Med. 2010;207(13):2883-94.
31. Hendriks J, Gravestein LA, Tesselaar K, Van Lier RA, Schumacher TN, Borst J. CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol. 2000;1(5):433-40.
32. Condomines M, Quittet P, Lu Z-Y, Nadal L, Latry P, Lopez E, et al. Functional regulatory T cells are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and granulocyte colony-stimulating factor. J Immunol. 2006;176(11):6631-9.
33. Brunstein CG, Miller JS, McKenna DH, Hippen KL, DeFor TE, Sumstad D, et al. Umbilical cord blood–derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect. Blood. 2016;127(8):1044-51.
34. Rutella S, Pierelli L, Bonanno G, Sica S, Ameglio F, Capoluongo E, et al. Role for granulocyte colony–stimulating factor in the generation of human T regulatory type 1 cells. Blood. 2002;100(7):2562-71.
35. Arpinati M, Green CL, Heimfeld S, Heuser JE, Anasetti C. Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells. Blood. 2000;95(8):2484-90.
36. Takahashi S, Minnie SA, Ensbey KS, Schmidt CR, Sekiguchi T, Legg SR, et al. Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation. Blood. 2024;143(16):1656-69.
37. Banerjee H, Nieves-Rosado H, Kulkarni A, Murter B, McGrath KV, Chandran UR, et al. Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment. Cell Reports. 2021;36(11).
38. Lowther DE, Goods BA, Lucca LE, Lerner BA, Raddassi K, van Dijk D, et al. PD-1 marks dysfunctional regulatory T cells in malignant gliomas. JCI insight. 2016;1(5):e85935.
39. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015;15(8):486-99.
40. Gootjes C, Zwaginga JJ, Roep BO, Nikolic T. Defining human regulatory T cells beyond FOXP3: the need to combine phenotype with function. Cells. 2024;13(11):941.
41. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 2006;203(7):1701-11.
42. Hossen MM, Ma Y, Yin Z, Xia Y, Du J, Huang JY, et al. Current understanding of CTLA-4: from mechanism to autoimmune diseases. Front Immunol. 2023;14:1198365.
43. Elkord E, Abd Al Samid M, Chaudhary B. Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP. Oncotarget. 2015;6(24):20026.
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| Keywords | ||
| Flow cytometry Immune checkpoint markers Immunophenotyping Mobilized peripheral blood Regulatory T cells Umbilical cord blood | ||
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How to Cite
1.
Tajrishi M, Mohammadi S, Vaezi M, Ahmadvand M, Abroun S. Phenotypic Comparison of Regulatory T Cells: Mobilized Peripheral Blood Versus Umbilical Cord Blood. Iran J Allergy Asthma Immunol. 2026;:1-13.
