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Tissue-specific clonal selection and differentiation of CD4⁺ T cells during infection

Nature Immunology volume 27, pages 725–737 (2026)Cite this article

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Abstract

Pathogen-specific CD4⁺ T cells expand and contract during infection, generating memory clones that shape subsequent immune responses. How distinct tissue environments influence differentiation and clonal selection of polyclonal T cells remains unclear. Here we develop Tracking Recently Activated Cell Kinetics (TRACK) mice, a dual-recombinase fate-mapping system enabling the spatial and temporal labeling of recently activated CD4⁺ T cells. Using TRACK mice during influenza infection, we observed organ-specific transcriptional differentiation and clonal selection in lung, mediastinal lymph nodes (medLNs) and spleen. During the effector phase, spleen-derived CD4⁺ T cells adopted a stem-like migratory phenotype, whereas medLN-activated cells differentiated into T follicular helper cells. T cell receptor sequencing showed low clonal overlap between tissues during the effector response, consistent with distinct antigenic landscapes. During memory formation, overlap increased between lung- and medLN-derived cells, while splenic clones retained a distinct repertoire. These findings define tissue-dependent mechanisms that shape CD4⁺ T cell fate and clonal architecture.

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Fig. 1: Tracking of pathogen-specific CD4+ T cells during intestinal and respiratory infection.
Fig. 2: Enrichment of perturbation-specific T cells.
Fig. 3: Distinct transcriptional profiles among pathogen-specific CD4+ T cells across lymphoid and nonlymphoid organs.
Fig. 4: Divergent CD4+ T cell functional fates in secondary lymphoid organs during influenza infection.
Fig. 5: Distinct clonal expansion and distribution across organs.
Fig. 6: Clonal selection is shaped by organ-specific antigenic landscapes.
Fig. 7: Distinct contributions of TCR avidity and specificity to clonal dynamics.

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Data availability

scRNA-seq data generated in this study have been deposited in the Gene Expression Omnibus under accession code GSE317035. Source data are provided with this paper.

Code availability

Custom scripts supporting the data analysis are available via GitHub at https://github.com/tiagobrc/parsa_2024.

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Acknowledgements

We thank A. Rogoz and J. Bortolatto for embryo bioengineering, animal care, mouse colony management and genotyping; RU Genomics core for assistance with sequencing; K. Gordon and J. P. Truman for sorting; and Y. Khan and additional Rockefeller University employees for continuous assistance. We thank the NIH Tetramer Facility for providing the LLO and NP tetramers. We thank all the members of the Mucida, Victora (Rockefeller) and Lafaille (NYU) laboratories for fruitful discussions. This work was supported by The Swedish-American Foundation and The Swedish Research Council (to R.P.; grant no. 2023/02507-7), the São Paulo Research Foundation (H.CA.) and a Life Sciences Research Foundation postdoctoral fellowship (to T.B.R.d.C.). G.L.d.R. was supported by a scholarship from Fundação Maria Emília, FME (Brazil); NIH grants R01DK093674 and R01DK113375; NIAID grant P01AI179273; the CZI Science, Food Allergy FARE/FASI Consortium; Stavros Niarchos Foundation Institute for Global Infectious Diseases; and The Howard Hughes Medical Institute (to D.M.).

Author information

Author notes

  1. Helder C. Assis

    Present address: Department of Genetics and Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas, Campinas, Brazil

Authors and Affiliations

  1. Immune Cell Dynamics and Function, Biohub New York, New York, NY, USA

    Roham Parsa & Arpita Sushil

  2. Laboratory of Mucosal Immunology, The Rockefeller University, New York, NY, USA

    Roham Parsa, Helder C. Assis, Gabriella Lima dos Reis, Angelina M. Bilate & Daniel Mucida

  3. Laboratory of Lymphocyte Dynamics, The Rockefeller University, New York, NY, USA

    Tiago B. R. de Castro

  4. Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA

    Harald Hartweger

  5. Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA

    Daniel Mucida

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Contributions

Conceptualization by R.P. and D.M. Investigation by R.P., H.C.A., T.B.R.d.C., G.L.d.R., A.S. and A.M.B. Writing—original draft by R.P. and D.M. Editing by all authors. Funding acquisition by R.P. and D.M. Resources by T.B.R.d.C. and H.H. Supervision by D.M.

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Correspondence to Roham Parsa or Daniel Mucida.

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Nature Immunology thanks Adrian Liston and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: S. Houston in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Tracking of pathogen-specific CD4 + T cells during intestinal and respiratory infection.

a, Overview of genetic strategy regarding Cd69-CreER and Sell-DreER mice. b, Gating strategy for CD4+CD44hiTomato+ T cells. c, TRACK mice were infected with L. monocytogenes and CD8+CD44hiTomato+ T cells were analyzed at day 9 post infection. Representative plot. Minimum 3 independent experiments, For infected, n = 4 for IE, LP and spleen, n = 3 for medLN. For control, n = 3. Statistical significance was tested between infected organ to uninfected organ by two-tailed Student’s T-test. d, TRACK mice were treated with tamoxifen as described and medLN, spleen and lung were analyzed for tdTomato labeling of CD4+CD44hi T cells 9 days post-influenza PR8 infection. n = 3/group, data from one experiment. e, Stated mice were infected with L. monocytogenes and CD4+CD44hiTomato+ were analyzed in the LP 9-day post infection. Representative plot from 2 independent experiments, n = 3 per group. One-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. f, T cells from the spleen of TRACK mice were isolated and stimulated with anti-CD3/anti-CD28 with or without 4-Hydroxytamoxifen (4-OHT). CD69, CD62L and Tomato expression on CD4+ T cells were measured over time. Pooled data from two experiments, n = 6. Data are presented as mean ± SEM. g, Similar to e, but T cells were stimulated with IFN-γ, IFN-β or IL-2 without CD3/CD28 stimulation. CD69, CD62L, CD44 and Tomato expression were measured on CD4+ T cells 24 hours post stimulation. Data from 4 mice. h, i, Analysis of CD4+CD44lowTomato+ within the spleen at 4, 8 or 13 weeks of age. Representative plot of minimum 3 independent experiments. For h, n = 3 in uninfected group and n = 4 in infected group. For I, n = 3 in 4 weeks and 13 weeks group. In the 8 weeks group, n = 3 in uninfected group and n = 4 in infected group. Two-tailed Student’s T-test. j, Analysis of CD69 and CD62L expression within the thymus in TRACK mice at 8 weeks of age. Pooled data from 2 independent experiments, n = 6. One-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test.

Source data

Extended Data Fig. 2 Analysis of RNAseq data from influenza infected TRACK mice at day 9 and day 56 post infection.

a and b, CD45.1 isogenic B6 mice were infected with influenza PR8 and treated with anti-CD62L at day 1 and 3. NP-specific naive 500.000 CD4 T cells were transferred to the recipients at day 2 post infection and the transferred T cells in the medLN and spleen were analyzed for activation markers at day 4 post infection. Statistical differences were determined with two-tailed Student’s t-test c, Analysis of CD4+CD44hiTomato+ T cells across timepoints and organs in TRACK mice post-influenza PR8 virus infection. Pooled data from 5 independent experiments n = 17. d, Cell counts across clusters divided by either organs (left) or mice (right) at day 9 post infection, n = 3. e, Cell counts across clusters divided by either organs (left) or mice (right) at day 56 post infection, n = 4. f, Gene signatures used for classifications in UMAP plots. g, All expanded clonotypes (clonal size >10) at day 9 post infection were analyzed to determine the Gini index per clonotype across medLN, spleen and lung. The top 25th and 75th quantile of the Gini index were determined and those selected clonotypes, that is low Gini index clonotypes ( < 0.26) or high Gini index clonotypes ( > 0.57) where pooled and analyzed for cluster distribution. Outer numbers: Cluster, Outer ring: organ origin, Inner ring: Cluster size, Inner number: Clone counts across organs. Pooled data from 3 mice.

Source data

Extended Data Fig. 3 Distinct clonal expansion and distribution across organs.

a, Clonotype size ranking from scRNAseq data across organs and timepoints. Dashed x-axis line indicates top 10 expanded clones. Pool ranked clonotypes from 3 mice for d9 p.i. and from 4 mice for d56 p.i. b, Cluster and organ distribution of top 5 expanded clones located in the lungs at day 9 post infection. Each dot represents one unique mouse. n = 3. c, Cluster and organ distribution of top 5 expanded clones located in the lungs at day 56 post infection. Each dot represents one unique mouse. n = 4.

Source data

Extended Data Fig. 4 Analysis of TCR specificity and functional avidity in fate-mapped T cells.

a, Experimental overview of testing TCR specificity with murine T cell hybridomas. The 15 most expanded TCRs across lungs, medLN and spleen from day 9 (b) and day 56 (d) post infection stimulated with anti-CD3 to determine functionality. TCRs are isolated from the scRNA-seq data in Fig. 3. c, TCR hybridomas from day 9 (c) or day 56 (e)were tested for viral specificity by co-culturing them with splenic DCs that had been pulsed with 105 plaque forming units (pfu) of PR8 particles for 24 hours. In day 9 group n = 14 and in day 56 group n = 15 unique T cell hybridomas. Representative data from 2 independent experiments. f, TRACK mice were infected with influenza PR8 and CD4+ T cells were enriched from the lungs, medLN and spleen, 9 or 56 days post infection. The isolated CD4 T cells were stained with CFSE and co-cultured with splenic DCs that were pulsed with various amounts of PR8 viral particles for 48 hours. Pooled data from 2 independent experiments, n = 5-7/group. One-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test, per dilution point. Statistics determined at 104 PFU. g, Clonotype bias at day 9 (g) or day 56 (h) post infection (p.i.), analysis only considered expanded clonotypes with 5 or more cells and corrected for clonotype size by generating expected background distributions by random permutation (dotted line). Red circles indicate Z score > 1.97 and two-sided p < 0.05. Pooled RNAseq data from 3 individual (d9) or 4 individual mice (d56) mice. i, NP311-325-specific TCR hybridomas was co-cultured with splenic CD11c+ DCs and stimulated with various concentrations of NP311-325 peptide (10 µM, 5 µM, 2.5 µM, 1.25 µM, 0.63 µM and 0 µM) for 24 hours and NFAT-GFP expression was measured.

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TCRs used for hybridomas, isolated from most expanded clones across organs.

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Parsa, R., Assis, H.C., de Castro, T.B.R. et al. Tissue-specific clonal selection and differentiation of CD4⁺ T cells during infection. Nat Immunol 27, 725–737 (2026). https://doi.org/10.1038/s41590-026-02451-4

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