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Research ArticleAgingImmunology
Open Access | 
10.1172/jci.insight.187271
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Nelson, C. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Podestà, M. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Gempler, M. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Lee, J. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Batty, C. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Mathenge, P. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Sainju, A. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Chang, M. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Ke, H. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Tullius, S. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
Find articles by Abdi, R. in: JCI | PubMed | Google Scholar
1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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1Transplantation Research Center, Division of Renal Medicine, Department of Medicine; and
2Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
3Unit of Nephrology, Dialysis, and Renal Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
4Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.
5Division of Transplant Surgery, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
6Ragon Institute of Mass General Brigham, MIT, and Harvard, Cambridge, Massachusetts, USA.
7Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Peter T. Sage, 221 Longwood Avenue, EBRC 3rd Floor, Boston, Massachuetts, 02115, USA. Phone: 617.525.8002; Email: psage@bwh.harvard.edu.
Authorship note: CSN and MAP contributed equally to this work.
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Published March 4, 2025 - More info
Published in Volume 10, Issue 8 on April 22, 2025
AbstractHumoral immunity is orchestrated by follicular helper T (Tfh) cells, which promote cognate B cells to produce high-affinity, protective antibodies. In aged individuals, humoral immunity after vaccination is diminished despite the presence of Tfh cells, suggesting defects after initial Tfh cell formation. In this study, we utilized both murine and human systems to investigate how aging alters Tfh cell differentiation after influenza vaccination. We found that young Tfh cells underwent progressive differentiation after influenza vaccination, culminating in clonal expansion of effector-like cells in both draining lymph nodes and blood. In aging, early stages of Tfh cell development occurred normally. However, aging rewired the later stages of development in Tfh cells, resulting in a transcriptional program reflective of cellular senescence, sustained pro-inflammatory cytokine production, and metabolic reprogramming. We investigated the extent to which this rewiring of aged Tfh cells is due to the age-associated inflammatory (“inflammaging”) microenvironment and found that this setting was sufficient to both block the transition of Tfh cells to a post-effector resting state and skew Tfh cells toward the age-rewired state. Together, these data suggest that aging dampens humoral immunity by cytokine-mediated rewiring of late effector Tfh cell differentiation into an activated, yet less functional, cellular state.
Graphical Abstract
Introduction
The average age of the US population continues to rise, with approximately 1 in 5 US citizens currently exceeding 65 years of age. It is established that the adaptive immune system changes with age and frailty. While there is a lack of consensus regarding the relationship between age and vaccine responses, some studies suggest that the magnitude of protective antibodies elicited by both primary vaccination and vaccine boosting (e.g., secondary/repeat vaccination) is attenuated in aged individuals (1–5). In addition, aged individuals exhibit a reduced durability of vaccine-elicited antibodies, with titers that wane more rapidly than those in young individuals (6, 7). There is increasing recognition that aged lymphocytes possess unique cellular programming and are not simply attenuated versions of their youthful counterparts. The aging microenvironment contains increased concentrations of some pro-inflammatory cytokines produced by both immune and stromal cells, which creates a distinctive “inflammaging” microenvironment (8–10). However, the precise mechanisms by which aging alters antibody-mediated immunity after vaccination are poorly understood because of the complexity of the aging process.
The goal of vaccination is to elicit protective antibodies that persist for long periods; however, in some circumstances, such as influenza vaccination, protection wanes rapidly (11). T follicular helper (Tfh) cells stimulate B cells to undergo affinity maturation in germinal centers (GCs), eventually culminating in the development of memory B cells that respond upon antigen reexposure or plasma cells that produce high-affinity antibodies (12, 13). Newer data suggest that Tfh cells undergo progressive differentiation, a process that is required to generate effector Tfh cells that promote the GC reaction (14). Some effector Tfh cells can partially downregulate the effector program but remain epigenetically poised, called a TfhEx state. Furthermore, a subset of Tfh cells assume a memory-like phenotype, exit the GC, and can be found circulating in the peripheral blood (15). Since the magnitude of circulating Tfh (cTfh) cells correlates with the quality, quantity, and longevity of antibody responses in some settings, a fundamental goal of vaccination is to induce sustained cTfh responses (16–18). Conversely, T follicular regulatory (Tfr) cells dampen Tfh-mediated B cell activation and antibody production, while optimizing affinity maturation (19).
The current paradigm suggests the GC reaction is altered in aged individuals. B cells exhibit age-related functional changes, including decreased class switch recombination, bimodal somatic hypermutation, decreased plasma cell differentiation, and enhanced expression of the transcription factor T-bet (19, 20). Furthermore, aged Tfh cells exhibit altered tissue localization as well as transcriptional programming (21, 22). Intriguingly, the total frequency of Tfh cells is higher in aged individuals (23), which is accompanied by an increase in the prototypical Tfh cytokine IL-21 in the serum of some aged individuals (24). Therefore, aging does not limit the presence of Tfh cells, per se, but may regulate the tissue localization and transcriptional profile of Tfh cells. However, how the complex physiology of aging alters Tfh differentiation is poorly understood.
In this study, we assessed how aging alters Tfh differentiation in response to seasonal influenza vaccination, utilizing both murine models and human clinical samples. In both mice and humans, Tfh cells underwent progressive Tfh development in response to influenza vaccination. Settings of aging were able to support normal early stages of Tfh development in lymph nodes and blood, including development of stem-like progenitor Tfh cells. However, aging induced rewiring in the later stages of Tfh development, culminating in transcriptionally distinct effector Tfh cells with features of augmented effector states, cytokine production, and cellular senescence. Extrinsically, the inflammaging microenvironment was sufficient to induce some of these changes in late effector Tfh differentiation, including the inability to transition to a resting state, pro-inflammatory cytokine production, and a cellular senescence-like state. These studies demonstrate that humoral immunity is uniquely wired in aging rather than simply an attenuated version of a youthful immune system.
ResultsTfh cells clonally expand in lymph nodes and blood after influenza vaccination. Differentiation of Tfh cells has recently been shown to require sequential developmental stages in lymphoid organs, such as the draining lymph node (dLN), after vaccination (14). Some Tfh cells can leave the dLN and enter the circulation before full differentiation, but the relationship between these populations and those that reside in LNs is not fully understood. Thus, we first assessed the clonal relationships of Tfh cells in dLNs and blood of influenza-vaccinated mice. We administered an unadjuvanted quadrivalent influenza vaccine (Afluria Influenza A+B, 2010–2011 formulation) to 8-week-old C57BL/6 mice and 10 days later sorted total follicular T cells (gated as CD4+CXCR5+CD19–; Tfh and Tfr together) from the dLN and blood (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.187271DS1). Cells were processed for single-cell RNA-Seq (scRNA-Seq) with matched TCR-Seq (Figure 1A). The 6,752 follicular T cells that passed quality control filters separated into 10 distinct clusters in uniform manifold approximation and projection (UMAP) space (Figure 1B). Tissue origin was a dominant factor for the positioning of cells in clusters, since follicular T cells from the dLN separated from the blood (Figure 1C). Only a single cluster, cluster 6, contained cells from both the dLN and blood. However, even within this cluster, we observed evidence of segregation. We then separated the follicular T cells into Tfh and Tfr cells by assigning cells that expressed Foxp3 or a T regulatory cell–dominant (Treg-dominant) transcriptional profile as Tfr cells (Supplemental Figure 1, B and C). Tfr cells were found in cluster 2 of the blood, as well as clusters 4 and 5 of the dLN, whereas Tfh cells were found in all clusters (Figure 1D). Notably, the tissue of origin was a stronger overall driver of transcriptional programs than Tfh versus Tfr cell type, consistent with previous findings (25).
Figure 1Expansion and clonal overlap of murine Tfh cells from LNs and blood after influenza vaccination. (A) Experimental schematic. Wild-type mice (8–12 weeks of age) were administered the Afluria quadrivalent influenza vaccine (total 1.5 μg HA protein). After 10 days, CD4+CXCR5+ cells were sorted from both dLN and peripheral blood, and single-cell RNA sequencing (scRNA-Seq) with matched TCR-Seq was performed. (B–D) UMAPs of n = 6,752 cells by unsupervised cluster assignment (B), by tissue (C), and by cell type (D). (E) (Left) TCRβ-V and J gene usage. Percentage indicates frequency of use in dataset. (Right) Clonal sharing between the top 25 TCR clones by prevalence in LN and blood. Connecting lines indicate shared clones. (F) (Left) TCRβ V-J gene segment usage and (right) clonal sharing between Tfh and Tfr cells. (G) Clonal expansion (<0.01% “rare,” 0.01%–0.033% “small,” 0.033%–0.067% “medium,” 0.067%–0.1% “large,” and >0.1% “hyperexpanded”) for LN and blood cells. (H) TCR complementarity-determining region 3 amino acid sequence and predicted influenza specificity of the top 10 clones shared between LN and blood Tfh cells. Each clone is color coded. (I) TCRβ -V gene usage for clones in H. (J) UMAP of clones from H, including annotation of cluster 8 from B. (K) Module score for a Tfh Effector module (derived from ref. 14) expressed as a feature plot (left) or violin plot (right). Violin plot displays the Tfh Effector feature score 95% confidence interval for cells in indicated clusters, with the shape indicating the probability density and horizontal line denoting the median value. (L) (Left) Differential gene expression between cluster 8 and clusters 3, 4, 5, and 9 of the LN. (Right) Density plots for indicated genes. Data are from a single scRNA-Seq experiment of 2 individual mice concatenated.
Reclustering of the 5,352 Tfh and 1,391 Tfr cells independently revealed separation of dLN and blood populations for both cell types (Supplemental Figure 1, E–K). Analysis of differentially expressed genes (DEGs) between dLN and blood Tfh/Tfr cells identified 103 tissue-specific genes in common between Tfh and Tfr, suggesting similar tissue-specific transcriptional programming (Supplemental Figure 1K). Genes enriched in the blood compartment included some consistent with a quiescent state (Klf3, Dusp1). In contrast, genes similarly enriched in dLN included genes associated with effector Tfh cells (Bcl6, Pdcd1/PD-1, Id3), as well as costimulatory molecules (Tnfrsf4/OX40, Tnfrsf9/4-1BB).
Next, we compared TCR sequences to determine clonal overlap of Tfh cells in the dLN and blood after influenza vaccination. Overall, TCRβ chain V-J usage was similar in follicular T cell clones between dLN and blood, suggesting clonal overlap (Figure 1E). Moreover, 0.5% of all, and 16% of the most abundant, clones were shared with expanded Tfh clones in the blood, further supporting clonal overlap of Tfh cells after influenza vaccination (Figure 1E). Although the vast majority of Tfr cells are thought to originate from natural Tregs, some studies have suggested a small portion of Tfr cells can originate from non-Tregs as “induced” Tfr cells (26). However, consistent with prior reports (27), we found that TCRβ gene usage was distinct between Tfh and Tfr cells. Furthermore, we observed minimal overlap of Tfh clones with dLN or blood Tfr cells, with only 0.08% of Tfr clones overlapping with Tfh cells (Figure 1F). Notably, all Tfr clones were singletons, and there was no evidence of clonal proliferation (Figure 1F).
The lower amount of clonal expansion in blood, compared with dLN, Tfh cells suggests circulating cells have more clonal diversity (Figure 1G). Despite differences in the extent of clonal expansion, a small subset of Tfh clones were expanded in both the dLN and blood. Of the top 10 most expanded clones in both tissues, all were predicted to have reactivity for influenza A virus. The most common predicted influenza epitopes included HA, RNA polymerase, and matrix proteins (Figure 1H), though the top predicted specificity for a few expanded clones was for other viruses or autoantigens (Supplemental Figure 1L). Expanded Tfh clones made use of a diversity of TCRβ V-gene segments; however, V20 was the most common (Figure 1I). These Tfh clones were found in clusters 8 and 9 of the dLN as well as most Tfh clusters of the blood (Figure 1J). Cluster 8 had enrichment of a Tfh effector gene module (derived from TfhFull vs. Tfh progenitor-like, or TfhProg, cells) (14), suggesting this population is a fully differentiated effector Tfh population (Figure 1K). Consistent with this, Tfh effector genes Il21, Maf, Bcl6, Il4, and Cd40lg were among the most differentially expressed genes in this cluster (Figure 1L) (14). Together, these data indicate that Tfh cells undergo sequential development in the dLN after influenza vaccination, culminating in an effector Tfh state, and that some of these expanded clones gain access to the blood to recirculate.
Tfh cells from aged mice undergo transcriptional rewiring during later stages of development to induce a cellular senescence-like program. Aging can affect the quality of the antibody response elicited by influenza vaccination (1–5). Passive infusion of serum from young, influenza-vaccinated mice was protective against severe influenza infection, whereas infusion of serum from aged, vaccinated mice was not (Supplemental Figure 2). To investigate the mechanism of this observed deficiency, we vaccinated both young and 80-week-old (aged) mice to assess how settings of aging alter progressive Tfh differentiation after influenza vaccination utilizing scRNA-Seq with matched TCR-Seq (Figure 2A). Consistent with previous reports (19, 28), the frequency of total CD4+CXCR5+ follicular T cells as determined by flow cytometry was higher in aged versus young vaccinated mice (Figure 2B). The number of DEGs between young and aged follicular T cells was calculated separately for each tissue. We found a greater number of DEGs (corrected P < 0.01; fold-change > 2) in the blood compared with the dLN (Figure 2C). We focused further analysis on blood Tfh cells, since 1) we found stronger age-related transcriptional changes in blood Tfh cells, 2) we found more diverse phenotypes of expanded Tfh cells in the blood of young mice, and 3) most human Tfh studies have access to blood only for analysis. We combined 7,360 CD4+CXCR5+ follicular T cells from the blood of young or aged mice. These cells segregated into 11 unique clusters in UMAP space (Figure 2D and Supplemental Table 1). There were clear differences in the distribution of young and aged cells in clusters, with miloR neighborhood analysis revealing that follicular T cells from aged mice disproportionately occupied clusters 2, 4, 8, and 10 (Figure 2, E and F). In contrast, cluster 3, and to a lesser extent cluster 6, were disproportionately occupied by young folli
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