Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines
The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-10-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2021.686530/full |
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| author | Arimelek Cortés-Hernández Evelyn Katy Alvarez-Salazar Saúl Arteaga-Cruz Katya Rosas-Cortina Nadyeli Linares Josefina M. Alberú Gómez Gloria Soldevila |
| author_facet | Arimelek Cortés-Hernández Evelyn Katy Alvarez-Salazar Saúl Arteaga-Cruz Katya Rosas-Cortina Nadyeli Linares Josefina M. Alberú Gómez Gloria Soldevila |
| author_sort | Arimelek Cortés-Hernández |
| collection | DOAJ |
| description | The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients. |
| first_indexed | 2024-12-19T00:12:23Z |
| format | Article |
| id | doaj.art-7dc500fb02914354947a76b87e8a70d8 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-19T00:12:23Z |
| publishDate | 2021-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-7dc500fb02914354947a76b87e8a70d82022-12-21T20:45:58ZengFrontiers Media S.A.Frontiers in Immunology1664-32242021-10-011210.3389/fimmu.2021.686530686530Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory CytokinesArimelek Cortés-Hernández0Evelyn Katy Alvarez-Salazar1Saúl Arteaga-Cruz2Katya Rosas-Cortina3Nadyeli Linares4Josefina M. Alberú Gómez5Gloria Soldevila6Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MexicoDepartment of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MexicoDepartment of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MexicoDepartment of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MexicoDepartment of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MexicoNational Laboratory of Flow Cytometry, Instituto de Investigaciones Biomedicas, Universidad Nacional Autónoma de México, Mexico City, MexicoDepartment of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MexicoThe adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of Foxp3. Interestingly, alloTregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.https://www.frontiersin.org/articles/10.3389/fimmu.2021.686530/fulltransplantationregulatory T cellsallospecificexpansionsuppression |
| spellingShingle | Arimelek Cortés-Hernández Evelyn Katy Alvarez-Salazar Saúl Arteaga-Cruz Katya Rosas-Cortina Nadyeli Linares Josefina M. Alberú Gómez Gloria Soldevila Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines Frontiers in Immunology transplantation regulatory T cells allospecific expansion suppression |
| title | Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines |
| title_full | Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines |
| title_fullStr | Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines |
| title_full_unstemmed | Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines |
| title_short | Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines |
| title_sort | highly purified alloantigen specific tregs from healthy and chronic kidney disease patients can be long term expanded maintaining a suppressive phenotype and function in the presence of inflammatory cytokines |
| topic | transplantation regulatory T cells allospecific expansion suppression |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2021.686530/full |
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