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Cited by 324T-cell and B-cell ImmunologyPsoriasis: Treatment and PathogenesisGenetics, Aging, and Longevity in Model Organisms

Aging promotes reorganization of the CD4 T cell landscape toward extreme regulatory and effector phenotypes

Open access
Elyahu, Yehezqel, Hekselman, Idan, Eizenberg-Magar, Inbal, Berner, Omer, Strominger, Itai, Schiller, Maya, Mittal, Kritika, Nemirovsky, Anna +7 more·2019·Science Advances
DOI 10.1126/sciadv.aaw8330PMC PMC6703865
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Abstract

Single-cell analysis of CD4 T cells reveals a population structure associated with inflammation and declined immunity in aging.

AI snapshot

Written from the paper’s full text.

TL;DR

Single-cell RNA-seq of splenic CD4 T cells from young and aged C57BL/6 mice shows that aging reorganizes the CD4 T cell compartment toward regulatory, exhausted, and cytotoxic subsets. These subsets accumulate with age, correlate with inflammatory cytokines, and display extreme suppressive or effector functions.

Key findings

  • Among 24,007 CD4 T cells (13,186 young, 10,821 old), old cells showed mixed inflammatory, regulatory, and exhaustion signatures; top old-cell markers included Aw112010, S100a11, and Izumo1r (AUC > 0.66, power > 0.33).
  • Seven robust clusters were identified; four were enriched in old mice: TEM [log(median ratio) = 0.51, P = 0.03], aTregs [log(median ratio) = 1, P = 0.03], exhausted [log(median ratio) = 1.32, P = 0.03], and cytotoxic [log(median ratio) = 1.46, P = 0.03]. The aTregs, exhausted, and cytotoxic subsets (RECs) accounted for ~30% of old CD4 T cells versus ~1% in young mice.
  • Naïve subsets were enriched in young mice [Naïve: log(median ratio) = −0.27, P = 0.03; Naïve_Isg15: log(median ratio) = −0.23, P = 0.03], while rTregs were equally distributed [log(median ratio) = 0.02, P = 0.89].
  • RECs increased over age: exhausted cells from 6 months (r = 0.94, P = 1.5 × 10−12), aTregs peaking at 16 months (r = 0.64, P = 1.81 × 10−5), and cytotoxic cells from 12 months (r = 0.71, P = 4.0 × 10−8). Functionally, aTregs were more suppressive than rTregs (P < 0.05), and cytotoxic cells showed the highest production of TNF, IFNγ, perforin, and GzmB after activation compared with exhausted and TEM subsets.

Methods

  • Splenic CD4 T cells from young (2–3 months; n = 4) and old (22–24 months; n = 4) C57BL/6 mice were sorted to >99% purity as CD4+TCRβ+CD8−CD19−CD11b−NK1.1− cells and analyzed by 10x Genomics 3′ scRNA-seq; after quality control, 24,007 cells were retained.
  • Seurat was used for dimensionality reduction, clustering, differential expression, and marker analysis; SCENIC was used for regulon activity. Flow cytometry validated subsets and measured their dynamics at 2, 6, 12, 16, and 24 months, along with in vitro suppression and cytokine-production assays and serum cytokine measurements.
  • Statistical analyses included Spearman correlations, t tests, and one-way ANOVA with multiple-comparison correction.

Limitations

  • The paper states that because of the limited number of mice, only strong serum cytokine correlations (absolute Spearman coefficient > 0.6) were considered meaningful; it also states that mechanisms underlying gene induction and REC accumulation/function are “yet unclear” and require further research.
  • Evident from the text: all experiments use C57BL/6 mice, so human relevance is not directly established; cytokine associations are correlational and do not prove causation.
  • Evident from the text: REC frequencies were highly variable among individual old mice, possibly reflecting environmental factors, and the study does not resolve the differentiation pathways or antigen specificity of these subsets.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1126/sciadv.aaw8330

Cites

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Statistics

Nothing to recompute.

Claims the paper makes

0 of 59 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The three top markers common to old cells were Aw112010, S100a11, and Izumo1r (AUC > 0.66 and power > 0.33).

    “The three top markers common to old cells were the genes Aw112010, S100a11, and Izumo1r (AUC > 0.66 and power > 0.33; Fig. 1G)”
    Quote in the paperSubstantial claim
  • Findingcitation

    The aTregs subset overexpressed Foxp3, Cd81, Cd74, and Cst7, together with aTregs-associated genes Tnfrsf4, Tnfrsf9, Tnfrsf18, and Ikzf2.

    “activated regulatory T cells (aTregs) overexpressing Foxp3, Cd81, Cd74, and Cst7 genes, together with aTregs-associated genes such as Tnfrsf4, Tnfrsf9, Tnfrsf18, and Ikzf2 (21, 29, 30)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [21] Zemmour D., Zilionis R., Kiner E., Klein A. M., Mathis D., Benoist C., Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat. Immunol. 19, 291– 301 (2018). 29434354 PMC6069633 source

      Not checked in the free run.

    • cites [29] Zheng C., Zheng L., Yoo J.-K., Guo H., Zhang Y., Guo X., Kang B., Hu R., Huang J. Y., Zhang Q., Liu Z., Dong M., Hu X., Ouyang W., Peng J., Zhang Z., Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169, 1342– 1356.e16 (2017). 28622514 source

      Not checked in the free run.

    • cites [30] Guo X., Zhang Y., Zheng L., Zheng C., Song J., Zhang Q., Kang B., Liu Z., Jin L., Xing R., Gao R., Zhang L., Dong M., Hu X., Ren X., Kirchhoff D., Roider H. G., Yan T., Zhang Z., Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat. Med. 24, 978– 985 (2018). 29942094 source

      Not checked in the free run.

  • Findingown data

    Old versus young splenic CD4 T cells showed an increase in frequency of effector-memory cells (CD4+CD44+CD62L−).

    “we observed a significant reduction in the abundance of naïve cells (CD4+CD62L+CD44−, P = 0.0006) and an increase in the frequency of effector-memory cells (CD4+CD44+CD62L−) in the old versus the young splenic CD4 T cells (Fig. 1C).”
    Quote in the paperSubstantial claim
  • Findingown data

    These subsets gradually accumulate with age.

    “We further show that these subsets gradually accumulate with age and have distinct pro- and anti-inflammatory functional properties.”
    Quote in the paperSubstantial claim
  • Findingown data

    The activated regulatory, exhausted, and cytotoxic CD4 T cell subsets were very rare in young mice.

    “Transcriptomic profiles of 24,007 CD4 T cells from both age groups revealed the emergence of distinct activated regulatory, exhausted, and cytotoxic CD4 T cell subsets in aged mice, which were very rare in young mice, and accounted for about 30% of the CD4 T cell compartment of old mice.”
    Quote in the paperSubstantial claim
  • May combine two claims. 33
  • Classified as probably not a substantial claim. 19

  • May combine two claims.

    Findingown data

    Lef1, Satb1, and Ccr7 were the top three markers common to young cells (AUC > 0.61 and power > 0.23), supporting dominancy of naïve CD4 T cells in young age.

    “Lef1, Satb1, and Ccr7 genes were the top three markers common to young cells [AUC (area under the curve) > 0.61 and power > 0.23], supporting the dominancy of naïve CD4 T cells in young age (Fig. 1F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The cytotoxic subset overexpressed Eomes, Gzmk, and Ctla2a, genes commonly associated with CD8 T cells and previously described in viral infection and cancer as CD4 cytotoxic T cells.

    “cells overexpressing genes such as Eomes, Gzmk, and Ctla2a, which are commonly associated with CD8 T cells (denoted cytotoxic) and were previously described in the context of viral infection and cancer as CD4 cytotoxic T cells (32–34).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [32] Patil V. S., Madrigal A., Schmiedel B. J., Clarke J., O’Rourke P., de Silva A. D., Harris E., Peters B., Seumois G., Weiskopf D., Sette A., Vijayanand P., Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis. Sci. Immunol. 3, eaan8664 (2018). 29352091 PMC5931334 source

      Not checked in the free run.

    • cites [33] Mucida D., Husain M. M., Muroi S., van Wijk F., Shinnakasu R., Naoe Y., Reis B. S., Huang Y., Lambolez F., Docherty M., Attinger A., Shui J. W., Kim G., Lena C. J., Sakaguchi S., Miyamoto C., Wang P., Atarashi K., Park Y., Nakayama T., Honda K., Ellmeier W., Kronenberg M., Taniuchi I., Cheroutre H., Transcriptional reprogramming of mature CD4+ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes. Nat. Immunol. 14, 281– 289 (2013). 23334788 PMC3581083 source

      Not checked in the free run.

    • cites [34] Juno J. A., van Bockel D., Kent S. J., Kelleher A. D., Zaunders J. J., Munier C. M. L., Cytotoxic CD4 T cells—Friend or foe during viral infection? Front. Immunol. 8, 19 (2017). 28167943 PMC5253382 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Old cells grouped together in a distinctive manner in t-SNE, highlighting the impact of aging on the transcriptome of CD4 T cells.

    “Instead, old cells grouped together in a distinctive manner, thus highlighting the impact of aging on the transcriptome of CD4 T cells (Fig. 1D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    rTregs subset expressed higher levels of quiescence genes Ms4a4b and Sell, whereas aTregs subset expressed higher levels of coinhibitory Pdcd1 and Lag3, costimulatory Tnfrsf9 and Tnfrsf4, activation genes S100a11 and Il1r2, and TF Ikzf2.

    “whereas the rTregs subset expressed higher levels of quiescence genes (e.g., Ms4a4b and Sell), aTregs subset expressed higher levels of coinhibitory (e.g., Pdcd1 and Lag3) and costimulatory genes [e.g., Tnfrsf9 and Tnfrsf4; (38)], activation genes [S100a11 and Il1r2 (IL1 decoy receptor); (39)], and the transcription factor (TF) Ikzf2 (30).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [38] Ward-Kavanagh L. K., Lin W. W., Šedý J. R., Ware C. F., The TNF receptor superfamily in co-stimulating and co-inhibitory responses. Immunity 44, 1005– 1019 (2016). 27192566 PMC4882112 source

      Not checked in the free run.

    • cites [39] Wang D., Zhang S., Li L., Liu X., Mei K., Wang X., Structural insights into the assembly and activation of IL-1β with its receptors. Nat. Immunol. 11, 905– 911 (2010). 20802483 source

      Not checked in the free run.

    • cites [30] Guo X., Zhang Y., Zheng L., Zheng C., Song J., Zhang Q., Kang B., Liu Z., Jin L., Xing R., Gao R., Zhang L., Dong M., Hu X., Ren X., Kirchhoff D., Roider H. G., Yan T., Zhang Z., Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat. Med. 24, 978– 985 (2018). 29942094 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Visualizing expression of key TH1 and TH17 markers revealed primarily TH1-rather than TH17-polarization in the cytotoxic subset, validated by high versus low percentage of IFNγ- and IL-17-secreting activated cytotoxic CD4 T cells.

    “Visualizing the expression of key TH1 and TH17 markers on the t-SNE map revealed primarily TH1—rather than TH17—polarization in the cytotoxic subset (fig. S7, B and C), which was validated by the high versus low percentage of IFNγ- and IL-17–secreting activated cytotoxic CD4 T cells, respectively (fig. S7D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Absolute numbers of cytotoxic and aTregs cells significantly increased in old mice (P = 0.016 and P = 0.03, respectively).

    “the absolute numbers of cytotoxic and aTregs cells significantly increased (P = 0.016 and P = 0.03, respectively; Fig. 4E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Flow cytometry identified naïve, TEM, exhausted (based on CD62L, CD44, PD1, LAG3), and two Tregs subsets (all FOXP3+ and differed by CD81, PD1, LAG3).

    “We also identified naïve, TEM, exhausted (based on the expression of CD62L, CD44, PD1, and LAG3), and the two Tregs subsets (all were FOXP3+ and differed by expression levels of CD81, PD1, and LAG3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cytotoxic cells were highly correlated with IL-27 (r = 1) and IFNβ (r = 0.95).

    “Whereas the cytotoxic cells were highly correlated with IL-27 (r = 1) and IFNβ (r = 0.95)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    aTregs were highly abundant in bone marrow; exhausted and cytotoxic cells were enriched also in blood and spleen.

    “Whereas aTregs were highly abundant in BM, exhausted and cytotoxic cells were enriched also in blood and spleen (Fig. 4, F to H, and fig. S5, E and F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    aTregs and exhausted cells were highly correlated with IL-6 (r = 1 and r = 0.95, respectively).

    “aTregs and exhausted cells were highly correlated with IL-6 (r = 1 and r = 0.95, respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Absolute number of exhausted cells was increased in old mice but did not significantly differ from young mice (P = 0.21).

    “The absolute number of exhausted cells was also increased in old mice but did not significantly differ from young mice (P = 0.21).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Flow cytometry validation showed young cells comprised mainly naïve cells and defined clusters of effector-memory and rTregs cells, while old cells included a prominent cytotoxic CD4 T cell subset expressing high levels of EOMES and CCL5.

    “Clustering analysis revealed that whereas the young cells comprised mainly naïve cells and defined clusters of effector-memory and rTregs cells, the old cells included a prominent cytotoxic CD4 T cell subset, expressing high levels of the EOMES (Eomesodermin) TF and the CCL5 chemokine (Fig. 3D and fig. S5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The two naïve subsets were significantly enriched in young mice (Naïve: log(median ratio) = −0.27, P = 0.03; Naïve_Isg15: log(median ratio) = −0.23, P = 0.03).

    “Whereas the two naïve subsets were significantly enriched in young mice [Naïve: log(median ratio) = −0.27, P = 0.03 and Naïve_Isg15: log(median ratio) = −0.23, P = 0.03]”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The RECs accounted for ~30% of the CD4 T cells in old mice and were negligible in young mice (~1%).

    “accounting for ~30% of the CD4 T cells and were negligible in young mice (~1%).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The aTregs, exhausted, and cytotoxic subsets were highly enriched in all aged mice (log(median ratio) = 1, P = 0.03; log(median ratio) = 1.32, P = 0.03; and log(median ratio) = 1.46, P = 0.03; respectively).

    “Notably, the aTregs, exhausted, and cytotoxic subsets (collectively denoted RECs to represent these regulatory, exhausted, and cytotoxic subsets) were highly enriched in all aged mice [log(median ratio) = 1, P = 0.03; log(median ratio) = 1.32, P = 0.03; and log(median ratio) = 1.46, P = 0.03; respectively]”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The number of RECs was highly variable among individual mice.

    “In general, the number of RECs was highly variable among individual mice, possibly representing the individual aging process.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Tregs marker CD25 was similarly expressed by aTregs and rTregs.

    “The Tregs marker CD25 was similarly expressed by aTregs and rTregs (fig. S5B).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Single-cell resolution studies showed elevated cell-to-cell transcriptional variability in the activation program of CD4 T cells and in leukocytes from lungs of old mice.

    “Only recently, studies performed at a single-cell resolution showed elevated cell-to-cell transcriptional variability in the activation program of CD4 T cells (16) and in leukocytes from lungs of old mice (17).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [16] Martinez-Jimenez C. P., Eling N., Chen H.-C., Vallejos C. A., Kolodziejczyk A. A., Connor F., Stojic L., Rayner T. F., Stubbington M. J. T., Teichmann S. A., de la Roche M., Marioni J. C., Odom D. T., Aging increases cell-to-cell transcriptional variability upon immune stimulation. Science 355, 1433– 1436 (2017). 28360329 PMC5405862 source

      Not checked in the free run.

    • cites [17] Angelidis I., Simon L. M., Fernandez I. E., Strunz M., Mayr C. H., Greiffo F. R., Tsitsiridis G., Ansari M., Graf E., Strom T.-M., Nagendran M., Desai T., Eickelberg O., Mann M., Theis F. J., Schiller H. B., An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nat. Commun. 10, 963 (2019). 30814501 PMC6393476 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    Cells did not cluster topologically by depth of sequencing, experimental batch, or individual mouse.

    “Cells did not cluster topologically by depth of sequencing, experimental batch, or individual mouse (fig. S2, D and E).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Increased variation of chromatin modifications was observed in immune cells from elderly individuals.

    “In line with these findings, increased variation of chromatin modifications was recently observed in immune cells from elderly individuals (18).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Cheung P., Vallania F., Warsinske H. C., Donato M., Schaffert S., Chang S. E., Dvorak M., Dekker C. L., Davis M. M., Utz P. J., Khatri P., Kuo A. J., Single-cell chromatin modification profiling reveals increased epigenetic variations with aging. Cell 173, 1385– 1397.e14 (2018). 29706550 PMC5984186 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Previous studies on classical CD4 T cell subsets described reduced proliferation, lower IL-2 secretion, and accumulation of metabolic defects, along with higher production of pro-inflammatory mediators.

    “For example, previous studies performed on classical CD4 T cell subsets described reduced proliferation, lower secretion of interleukin-2 (IL-2) cytokine (7, 8), and accumulation of metabolic defects (9), along with higher production of pro-inflammatory mediators (10).”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [7] Li G., Yu M., Lee W.-W., Tsang M., Krishnan E., Weyand C. M., Goronzy J. J., Decline in miR-181a expression with age impairs T cell receptor sensitivity by increasing DUSP6 activity. Nat. Med. 18, 1518– 1524 (2012). 23023500 PMC3466346 source

      Not checked in the free run.

    • cites [8] Haynes L., Eaton S. M., Burns E. M., Randall T. D., Swain S. L., CD4 T cell memory derived from young naive cells functions well into old age, but memory generated from aged naive cells functions poorly. Proc. Natl. Acad. Sci. U.S.A. 100, 15053– 15058 (2003). 14657384 PMC299903 source

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      Not checked in the free run.

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      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    These old-cell marker genes were recently reported to be up-regulated under chronic inflammatory conditions.

    “which were recently reported to be up-regulated under chronic inflammatory conditions (23–25).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [23] Andrés Cerezo L., Šumová B., Prajzlerová K., Veigl D., Damgaard D., Nielsen C. H., Pavelka K., Vencovský J., Šenolt L., Calgizzarin (S100A11): A novel inflammatory mediator associated with disease activity of rheumatoid arthritis. Arthritis Res. Ther. 19, 79 (2017). 28446208 PMC5405489 source

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      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Systemic low-grade chronic inflammation that develops with age, referred to as inflammaging, appears to affect the phenotype and function of CD4 T cells.

    “In addition, systemic low-grade chronic inflammation that develops with age, referred to as “inflammaging”, also appears to affect the phenotype and function of CD4 T cells (14, 15).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14] Tsukamoto H., Senju S., Matsumura K., Swain S. L., Nishimura Y., IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age. Nat. Commun. 6, 6702 (2015). 25850032 PMC4396369 source

      Not checked in the free run.

    • cites [15] Furman D., Chang J., Lartigue L., Bolen C. R., Haddad F., Gaudilliere B., Ganio E. A., Fragiadakis G. K., Spitzer M. H., Douchet I., Daburon S., Moreau J.-F., Nolan G. P., Blanco P., Déchanet-Merville J., Dekker C. L., Jojic V., Kuo C. J., Davis M. M., Faustin B., Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states. Nat. Med. 23, 174– 184 (2017). 28092664 PMC5320935 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Aging is associated with deterioration of the immune system, rendering the elderly more prone to infections, chronic inflammatory disorders, and vaccination failure.

    “One of the key hallmarks of aging is the deterioration of the immune system, rendering the elderly more prone to infections, chronic inflammatory disorders, and vaccination failure (1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Goronzy J. J., Weyand C. M., Successful and maladaptive T cell aging. Immunity 46, 364– 378 (2017). 28329703 PMC5433436 source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Aging is associated with a change in the structure of the CD4 T cell compartment, where accumulating dysfunctional subsets contribute to immune failure.

    “Overall, aging is associated with a change in the structure of the CD4 T cell compartment, where accumulating dysfunctional subsets contribute to immune failure.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The Naïve_Isg15 population possibly indicates an intermediate state of T cell receptor-mediated activation or direct IFN signaling.

    “which possibly indicate an intermediate state of T cell receptor–mediated activation or direct IFN signaling (28).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Szabo P. A., Levitin H. M., Miron M., Snyder M. E., Senda T., Yuan J., Cheng Y. L., Bush E. C., Dogra P., Thapa P., Farber D. L., Sims P. A., A single-cell reference map for human blood and tissue T cell activation reveals functional states in health and disease. bioRxiv, 555557 (2019). PMC6797728 31624246

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    In young rodents and humans, CD4 T cells comprise a high frequency of naïve cells.

    “In young rodents and humans, CD4 T cells comprise a high frequency of naïve cells, reflecting the ability of the immune system to encounter new antigens, respond to them effectively, and generate immune memory (4).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4] Nikolich-Žugich J., The twilight of immunity: Emerging concepts in aging of the immune system. Nat. Immunol. 19, 10– 19 (2018). 29242543 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    These genes imply a strong relationship between the inflammatory state and the identity of CD4 T cells in aging and can be used as general markers for aged CD4 T cells.

    “These genes imply a strong relationship between the inflammatory state and the identity of CD4 T cells in aging and can be used as general markers for aged CD4 T cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Regulatory T cells appear to accumulate with age and contribute to reduced responsiveness of effector T cells.

    “Furthermore, regulatory T cells (Tregs) appear to accumulate with age and further contribute to reduced responsiveness of effector T cells (11, 12).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] Han G.-M., Zhao B., Jeyaseelan S., Feng J.-M., Age-associated parallel increase of Foxp3+CD4+ regulatory and CD44+CD4+ memory T cells in SJL/J mice. Cell. Immunol. 258, 188– 196 (2009). 19482268 PMC2706307 source

      Not checked in the free run.

    • cites [12] Haynes L., Maue A. C., Effects of aging on T cell function. Curr. Opin. Immunol. 21, 414– 417 (2009). 19500967 PMC3800142 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    With aging, the naïve subset shrinks and highly differentiated memory cells accumulate, often showing dysregulated properties.

    “With aging, the naïve subset shrinks along with the accumulation of highly differentiated memory cells, which often show dysregulated properties (5, 6).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Akbar A. N., Henson S. M., Lanna A., Senescence of T lymphocytes: Implications for enhancing human immunity. Trends Immunol. 37, 866– 876 (2016). 27720177 source

      Not checked in the free run.

    • cites [6] Tsukamoto H., Clise-Dwyer K., Huston G. E., Duso D. K., Buck A. L., Johnson L. L., Haynes L., Swain S. L., Age-associated increase in lifespan of naive CD4 T cells contributes to T-cell homeostasis but facilitates development of functional defects. Proc. Natl. Acad. Sci. U.S.A. 106, 18333– 18338 (2009). 19815516 PMC2759371 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Each of the three RECs uses defined sets of genes and TFs, which together promote an extreme immune phenotype that can hinder immunity in old age.

    “Together, these results demonstrate that each of the three RECs uses defined sets of genes and TFs, which together promote an extreme immune phenotype that can hinder immunity in old age (Fig. 5E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    While differentiation pathways, antigen specificity, function, and accumulation of cytotoxic CD4 cells in aging are yet to be revealed, their correlation with increased levels of circulating inflammatory cytokines suggests they may mark a stage in aging with robust immune failure and chronic inflammation.

    “While the differentiation pathways, antigen specificity, function, and accumulation of cytotoxic CD4 cells in aging are yet to be revealed, their correlation with increased levels of circulating inflammatory cytokines suggests that they may mark a stage in aging with a robust immune failure and chronic inflammation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    After quality control, 24,007 cells were retained for further analysis.

    “Together, 24,007 cells were kept for further analysis after quality control.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Aging is marked by a complex landscape of CD4 T cells, with increased frequencies of subsets with effector (TEM, exhausted, cytotoxic) and regulatory (aTregs) signatures.

    “Overall, these results demonstrate that aging is marked by a complex landscape of CD4 T cells, with increased frequencies of subsets with effector (including TEM, exhausted, and cytotoxic cells; fig. S4B) and regulatory (aTregs) signatures.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    After FACS sorting, CD4 T cells in all samples reached greater than 99% purity.

    “After sorting, CD4 T cells in all samples reached >99% purity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In activated CD8 T cells, these TFs regulate production of cytokines and lytic proteins such as TNF, IFNγ, GzmB, and perforin.

    “In activated CD8 T cells, these TFs regulate the production of cytokines and lytic proteins, such as tumor necrosis factor (TNF), IFNγ, granzyme B (GzmB), and perforin (48).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Pearce E. L., Mullen A. C., Martins G. A., Krawczyk C. M., Hutchins A. S., Zediak V. P., Banica M., DiCioccio C., Gross D. A., Mao C.-a., Shen H., Cereb N., Yang S. Y., Lindsten T., Rossant J., Hunter C. A., Reiner S. L., Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302, 1041– 1043 (2003). 14605368 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    t-SNE projections showed similar shapes across a range of perplexity values (10, 30, 50, 100, and 150).

    “Perplexity of the t-SNE projection was set to 10, 30 (default), 50, 100, and 150, and showed similar shapes of projections.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Both aTregs and exhausted cells have been directly implicated in the tumor microenvironment as cells that can suppress tumor immunity, and thus a decline in T cell effector functions along with chronic inflammation and increased frequencies of RECs may play a key role in age-related onset of cancer.

    “For example, both aTregs and exhausted cells have been directly implicated in the tumor microenvironment as cells that can suppress tumor immunity (29, 30), and thus a decline in T cell effector functions along with chronic inflammation and increased frequencies of RECs may play a key role in age-related onset of cancer.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [29] Zheng C., Zheng L., Yoo J.-K., Guo H., Zhang Y., Guo X., Kang B., Hu R., Huang J. Y., Zhang Q., Liu Z., Dong M., Hu X., Ouyang W., Peng J., Zhang Z., Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169, 1342– 1356.e16 (2017). 28622514 source

      Not checked in the free run.

    • cites [30] Guo X., Zhang Y., Zheng L., Zheng C., Song J., Zhang Q., Kang B., Liu Z., Jin L., Xing R., Gao R., Zhang L., Dong M., Hu X., Ren X., Kirchhoff D., Roider H. G., Yan T., Zhang Z., Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat. Med. 24, 978– 985 (2018). 29942094 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The gene signatures of aTregs and cytotoxic CD4 T cells indicate enhanced regulatory and effector cytotoxic profiles, suggesting these subsets play a key role in deterioration of the immune system with age.

    “The gene signatures of the aTregs and the cytotoxic CD4 T cells indicate their enhanced regulatory and effector cytotoxic profiles, respectively, suggesting that these subsets play a key role in the deterioration of the immune system with age.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning

    In all processing steps, wide-bore pipette tips were used, and centrifugation condition did not exceed 400g, to minimize physical damage to the cells caused by shearing forces.

    “In all processing steps, wide-bore pipette tips were used, and centrifugation condition did not exceed 400g, to minimize physical damage to the cells caused by shearing forces.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The mechanisms underlying induction of the old-cell marker genes in old CD4 T cells are unclear, but together with increased circulating cytokines and chemokines, they may serve as biomarkers for immune senescence.

    “Whereas the mechanisms underlying the induction of these genes in old CD4 T cells is yet unclear, together with the increased levels of circulating cytokines and chemokines, they may serve a useful set of biomarkers for immune senescence.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    RECs accumulation with time predominantly occurs in non-lymphatic immune compartments, as opposed to lymphatic sites, which exhibit a relatively preserved T cell composition.

    “Together, these findings indicate that the RECs’ accumulation with time predominantly occurs in non-lymphatic immune compartments, as opposed to lymphatic sites, which exhibit a relatively preserved T cell composition.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The first 20 principal components used for t-SNE were all statistically significant (P < 7.2 × 10−18).

    “To create the t-SNE projections, the first 20 PCs (all of which were significant, P < 7.2 × 10−18) were used via RunTSNE function by the elbow method (fig. S2C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Higher frequency of cells producing IL-21 and IL-17a was observed within cytotoxic cells, probably due to active Rora regulon.

    “We also observed higher frequency of cells producing IL-21 and IL-17a within cytotoxic cells (fig. S7A), probably due to the active Rora regulon [Fig. 5B; (49)].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Yang X. O., Pappu B. P., Nurieva R., Akimzhanov A., Kang H. S., Chung Y., Ma L., Shah B., Panopoulos A. D., Schluns K. S., Watowich S. S., Tian Q., Jetten A. M., Dong C., T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28, 29– 39 (2008). 18164222 PMC2587175 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All surgical and experimental procedures were approved by the Institutional Animal Care and Use committee of Ben-Gurion University of the Negev, Israel.

    “All surgical and experimental procedures were approved by the Institutional Animal Care and Use committee of Ben-Gurion University of the Negev, Israel.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Clustering results were robust to the number of principal components used (10, 19, or 21), yielding similar clustering patterns on the t-SNE projection.

    “We repeated the clustering process based on the first 10, 19, and 21 PCs. These led to similar clustering patterns on the t-SNE projection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The frequency of these subsets was associated with elevated levels of inflammatory cytokines in the sera (primarily IL-27, IFNβ, and IL-6), suggesting a link between inflammaging and the fate of CD4 T cells in aging.

    “Notably, the frequency of these subsets was associated with elevated levels of inflammatory cytokines in the sera (primarily IL-27, IFNβ, and IL-6), suggesting a link between the process of inflammaging (35) and the fate of CD4 T cells in aging.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [35] Franceschi C., Campisi J., Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. J. Gerontol. A Biol. Sci. Med. Sci. 69 (suppl. 1), S4– S9 (2014). 24833586 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All samples in the flow cytometry experiment excluded dead cells, clumps, and debris.

    “All the samples in the experiment excluded dead cells, clumps, and debris.”
    Quote in the paperNot a substantial claim
48 of 48

  • The authors state this as an assumption.

    Age-related changes in CD4 T cells result from age-related thymus involution, repeated antigen encounters, and intrinsic cellular senescence processes.

    “These changes are assumed to result from age-related thymus involution, repeated antigen encounters, and intrinsic cellular senescence processes (4, 13).”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Tregs gradually accumulate with age, and the percentage of CD4+CD25+ Tregs in the peripheral blood of elderly mice significantly increased.

    “Moreover, Tregs gradually accumulate with age [131–133] and the percentage of CD4+CD25+ Tregs in the peripheral blood of elderly mice significantly increased [134].”

    From Immunosenescence: a key player in cancer development (2020) · cites this paper as [131]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Different multiple-comparison corrections specified for Fig. 4

    Minor

    The Fig. 4 legend specifies Tukey correction, while the statistical-analysis section says comparisons among more than two groups used Bonferroni correction. These describe different corrections for the reported analysis.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. P. A. Szabo, H. M. Levitin, M. Miron, M. E. Snyder, T. Senda, J. Yuan, Y. L. Cheng, E. C. Bush, P. Dogra, P. Thapa, D. L. Farber, P. A. Sims, A single-cell reference map for human blood and tissue T cell activation reveals functional states in health and disease. bioRxiv, 555557 (2019).
  2. E. Mereu, G. Iacono, A. Guillaumet-Adkins, C. Moutinho, G. Lunazzi, C. P. Santos, I. Miguel-Escalada, J. Ferrer, F. X. Real, I. Gut, H. Heyn, matchSCore: Matching single-cell phenotypes across tools and experiments. bioRxiv, 314831 (2018).

Cited by

Sorted by citations

Authors
Elyahu, Y., Hekselman, I., Eizenberg-Magar, I., Berner, O., Strominger, I., Schiller, M., Mittal, K., Nemirovsky, A., Eremenko, E., Vital, A., Simonovsky, E., Chalifa-Caspi, V., Friedman, N., Yeger-Lotem, E., & Monsonego, A.
Affiliations
  • Ben-Gurion University of the Negev (IL)
  • Weizmann Institute of Science (IL)
  • Technion – Israel Institute of Technology (IL)
  • National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Corresponding author
  • Elyahu, Yehezqel
  • Hekselman, Idan
  • Monsonego, Alon
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2019-08-02
Volume
5
Issue
8
Pages
eaaw8330-eaaw8330
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.aaw8330
PMC
PMC6703865
OpenAlex
W2969244270
Cited by
324
References
60
Citations by year
2026: 52 · 2025: 52 · 2024: 69 · 2023: 53 · 2022: 38 · 2021: 36 · 2020: 24
FWCI
9.72
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • Israel Science Foundation ((# 684/14))
  • Litwin and Gural Foundations
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Life Sciences › Immunology and Microbiology › Immunology › Psoriasis: Treatment and Pathogenesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
Keywords
CD4+ T cells, aging, regulatory T cells, effector T cells, chronic inflammation, immunosenescence, age-related diseases, T cell subsets
MeSH terms
Aging, Animals, Phenotype, CD4-Positive T-Lymphocytes, T-Lymphocyte Subsets, Sequence Analysis, RNA, Mice, Immunomodulation, Single-Cell Analysis, High-Throughput Nucleotide Sequencing

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

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