A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus
Thymus-committed regulatory T cells (Tregs) are essential for immune homeostasis. Recent findings stress their heterogeneity, suggesting possible alternate routes for thymic Treg development with unique features in humans, namely the clear evidence of Treg commitment at the double-positive (DP) stag...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-03-01
|
| Series: | Frontiers in Immunology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1553535/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850240691456704512 |
|---|---|
| author | Beatriz Moleirinho Beatriz Moleirinho Margarida Paulo-Pedro Margarida Paulo-Pedro Nicole C. Martins Nicole C. Martins Emily Jelagat Emily Jelagat Eller Conti Eller Conti Tiago R. Velho Tiago R. Velho Miguel Abecasis Rui Anjos Afonso R. M. Almeida Afonso R. M. Almeida Ana E. Sousa Ana E. Sousa |
| author_facet | Beatriz Moleirinho Beatriz Moleirinho Margarida Paulo-Pedro Margarida Paulo-Pedro Nicole C. Martins Nicole C. Martins Emily Jelagat Emily Jelagat Eller Conti Eller Conti Tiago R. Velho Tiago R. Velho Miguel Abecasis Rui Anjos Afonso R. M. Almeida Afonso R. M. Almeida Ana E. Sousa Ana E. Sousa |
| author_sort | Beatriz Moleirinho |
| collection | DOAJ |
| description | Thymus-committed regulatory T cells (Tregs) are essential for immune homeostasis. Recent findings stress their heterogeneity, suggesting possible alternate routes for thymic Treg development with unique features in humans, namely the clear evidence of Treg commitment at the double-positive (DP) stage and the presence of a significant population of CD8 single-positive (SP) FOXP3pos Tregs. Here, we present a dedicated analysis strategy to a spectral flow cytometry-based study of thymus from children and aged adults (≥ 74-years-old), to further elucidate Treg development and heterogeneity in the human thymus. We applied an unsupervised analysis pipeline to data generated from 6 high-dimensional panels, taking advantage of a common backbone of 11 markers, and we were able to map thymocytes along T cell maturation stages. Generating UMAP and FlowSOM cluster coordinates from the backbone, we projected all other markers onto these, characterizing clusters with the information of all markers. Focusing this analysis on events inside a putative total Treg gate, we could portray rarer subsets of human thymic Tregs and investigate their trajectories using pseudotime analysis. We uncover clusters within human DP thymocytes uniquely expressing FOXP3 or CD25, a DP-branching trajectory towards a CD103posCD8SP Tregs endpoint, and define trajectories towards CD4SP Tregs, including towards a cluster of CXCR3posCD4SP Tregs, that may consist of thymic resident or recirculating Tregs, and do not expand in the elderly. Our flow cytometry approach separates Treg populations with likely distinct functions and facilitates the design of future studies to unravel the complexity of human regulatory T cells. |
| format | Article |
| id | doaj-art-c6bf933b08bd45bda273732bcce2977d |
| institution | OA Journals |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj-art-c6bf933b08bd45bda273732bcce2977d2025-08-20T02:00:47ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-03-011610.3389/fimmu.2025.15535351553535A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymusBeatriz Moleirinho0Beatriz Moleirinho1Margarida Paulo-Pedro2Margarida Paulo-Pedro3Nicole C. Martins4Nicole C. Martins5Emily Jelagat6Emily Jelagat7Eller Conti8Eller Conti9Tiago R. Velho10Tiago R. Velho11Miguel Abecasis12Rui Anjos13Afonso R. M. Almeida14Afonso R. M. Almeida15Ana E. Sousa16Ana E. Sousa17GIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalGIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalGIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalGIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalGIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalCardiothoracic Surgery Research Unit, Centro Cardiovascular da Universidade de Lisboa, Faculdade de Medicina da Universidade de Lisboa, Lisbon, PortugalDepartment of Cardiothoracic Surgery, Hospital de Santa Maria, Unidade Local de Saúde de Santa Maria, Lisbon, PortugalHospital de Santa Cruz, Unidade Local de Saúde de Lisboa Ocidental, Carnaxide, PortugalHospital de Santa Cruz, Unidade Local de Saúde de Lisboa Ocidental, Carnaxide, PortugalGIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalGIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, PortugalFaculdade de Medicina, Universidade de Lisboa, Lisbon, PortugalThymus-committed regulatory T cells (Tregs) are essential for immune homeostasis. Recent findings stress their heterogeneity, suggesting possible alternate routes for thymic Treg development with unique features in humans, namely the clear evidence of Treg commitment at the double-positive (DP) stage and the presence of a significant population of CD8 single-positive (SP) FOXP3pos Tregs. Here, we present a dedicated analysis strategy to a spectral flow cytometry-based study of thymus from children and aged adults (≥ 74-years-old), to further elucidate Treg development and heterogeneity in the human thymus. We applied an unsupervised analysis pipeline to data generated from 6 high-dimensional panels, taking advantage of a common backbone of 11 markers, and we were able to map thymocytes along T cell maturation stages. Generating UMAP and FlowSOM cluster coordinates from the backbone, we projected all other markers onto these, characterizing clusters with the information of all markers. Focusing this analysis on events inside a putative total Treg gate, we could portray rarer subsets of human thymic Tregs and investigate their trajectories using pseudotime analysis. We uncover clusters within human DP thymocytes uniquely expressing FOXP3 or CD25, a DP-branching trajectory towards a CD103posCD8SP Tregs endpoint, and define trajectories towards CD4SP Tregs, including towards a cluster of CXCR3posCD4SP Tregs, that may consist of thymic resident or recirculating Tregs, and do not expand in the elderly. Our flow cytometry approach separates Treg populations with likely distinct functions and facilitates the design of future studies to unravel the complexity of human regulatory T cells.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1553535/fullthymushuman immunologyregulatory T cellsT cell developmentspectral flow cytometry |
| spellingShingle | Beatriz Moleirinho Beatriz Moleirinho Margarida Paulo-Pedro Margarida Paulo-Pedro Nicole C. Martins Nicole C. Martins Emily Jelagat Emily Jelagat Eller Conti Eller Conti Tiago R. Velho Tiago R. Velho Miguel Abecasis Rui Anjos Afonso R. M. Almeida Afonso R. M. Almeida Ana E. Sousa Ana E. Sousa A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus Frontiers in Immunology thymus human immunology regulatory T cells T cell development spectral flow cytometry |
| title | A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus |
| title_full | A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus |
| title_fullStr | A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus |
| title_full_unstemmed | A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus |
| title_short | A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus |
| title_sort | backbone based flow cytometry approach to decipher regulatory t cell trajectories in the human thymus |
| topic | thymus human immunology regulatory T cells T cell development spectral flow cytometry |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1553535/full |
| work_keys_str_mv | AT beatrizmoleirinho abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT beatrizmoleirinho abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT margaridapaulopedro abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT margaridapaulopedro abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT nicolecmartins abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT nicolecmartins abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT emilyjelagat abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT emilyjelagat abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT ellerconti abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT ellerconti abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT tiagorvelho abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT tiagorvelho abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT miguelabecasis abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT ruianjos abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT afonsormalmeida abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT afonsormalmeida abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT anaesousa abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT anaesousa abackbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT beatrizmoleirinho backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT beatrizmoleirinho backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT margaridapaulopedro backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT margaridapaulopedro backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT nicolecmartins backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT nicolecmartins backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT emilyjelagat backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT emilyjelagat backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT ellerconti backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT ellerconti backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT tiagorvelho backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT tiagorvelho backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT miguelabecasis backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT ruianjos backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT afonsormalmeida backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT afonsormalmeida backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT anaesousa backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus AT anaesousa backbonebasedflowcytometryapproachtodecipherregulatorytcelltrajectoriesinthehumanthymus |