Lymphoma accelerates T cell and tissue aging

Abstract

The combined effects of aging and cancer on immune cells were investigated in young versus aged mice harboring B cell lymphoma, and in T cells from young and aged B cell lymphoma patients. These analyses revealed that lymphoma alone is sufficient to trigger transcriptional, epigenetic, and phenotypic alterations in young T cells that manifest in aged T cells. In contrast, aged T cells are largely resistant to lymphoma-induced changes. Pathway analyses revealed open chromatin regions and genes controlling iron homeostasis are induced by both lymphoma and aging, and lymphoma-experienced and aged T cells have increased iron pools and are resistant to ferroptosis. Furthermore, both aged and lymphoma-experienced T cells have defects in proteostasis. B cell lymphoma also accelerates aging of other tissues, as evidenced by elevated expression of Cdkn2a and Tnfa. Finally, some lymphoma-induced aging phenotypes are reversible whereas others are fixed, indicating opportunities for improving some cancer-associated aging comorbidities.

Keywords: B cell lymphoma; NK cell; T cell; aging; ferroptosis.

Figure 1.. Lymphoma alters T cell fate and function in young but not aged mice

(A) Experimental design analyzing the effects of B cell lymphoma on young and aged immune systems. (B) Flow cytometry gating strategy for CD4⁺ T cells, CD8⁺ T cells, NK1.1⁺ NK cells, B220^loFSC-A^hi lymphoma cells, and B220^hiFSC-A^lo normal B cells. (C) Total splenocyte number (n = 7). (D) Number of B220^loFSC-A^hi lymphoma cells (n = 7). (E–I) Number of splenic cells (n = 3–7): (E) Ter119⁺ cells; (F) CD8⁺ T cells; (G) CD4⁺ T cells; (H) normal B cells; and (I) NK cells. YC = young control; YL = young with lymphoma, AC = aged control, AL = aged with lymphoma. Data are mean ± SEM and are analyzed by a Student’s t test except for a one-way ANOVA and Tukey’s test (C and E). Data are compiled from at least two independent experiments and each dot represents an individual mouse. **p < 0.01, ***p < 0.001, and ****p < 0.0001, n.s. = not significant.

Figure 2.. Aged T cells and NK cells are refractory to lymphoma-provoked phenotypes

(A) Gating strategy and quantification of naive (N), effector (E), central memory (CM), and CD62L⁻ CD44^lo CD4⁺ T cell populations (n = 5). (B–E) Cell surface expression and representative histograms on CD4⁺ T cells (n = 5): (B) CD39, (C) CD69, (D) KLRG1, and (E) PD-1. Line in flow plots represents where positivity was determined. (F–H) Intracellular expression and representative histograms of transcription factors in the indicated CD4⁺ T cells (n = 3–4) (F) Tbet, (G) Tox, and (H) Foxp3. (I) Gating strategy for CD11b/CD27 NK cell maturation subtypes (n = 3). (J) Cell surface expression and representative histogram of CXCR3 in NK cells (n = 3/group). YC, YL, AC, AL are defined in Figure 1. Data are mean ± SEM and are analyzed by a two-way ANOVA and Sidak’s test (A and I) or Student’s t test (B–H and J). Data are compiled from at least two independent experiments, and each dot represents an individual mouse. *p < 0.05 **p < 0.01, and ***p < 0.001, ****p < 0.0001, n.s. = not significant. See also Figures S1 and S2.

Figure 3.. Lymphoma induces aging-like phenotypes in young T cells

(A) Uniform manifold approximation and projection (UMAP) of T and NK cells identified from scRNA-seq analyses. (B) Distribution of T and NK cells in each experimental group on the common UMAP for the entire dataset. (C) Fold changes induced by aging in major T cell (>1% mean of control T cell population) subpopulations. (D) Fold changes induced by Eμ-Myc lymphoma in major T cell subpopulations in young (red) versus aged (blue) mice. (E) Pearson correlation of fold changes to major T cell populations of young mice induced by aging versus those induced by lymphoma. (F) Pearson correlation of fold changes to NK cell populations of young mice induced by aging versus lymphoma. (G) Genes that are significantly (p < 0.05) increased (yellow) or decreased (blue) by lymphoma in naive/stem-like CD4⁺ T cells. (H) Genes that are significantly (p < 0.05) increased (yellow) or decreased (blue) by lymphoma in CD4⁺ T cells using pseudo-bulk data. (I) Gene set enrichment analysis (GSEA) of major T cell subpopulations comparing the indicated groups. YC, YL, AC, AL are defined in Figure 1. Data in (C) and (D) are mean ± SEM. Data (n = 3/group) are compiled from 3 independent experiments. See also Figures S3 and S4.

Figure 4.. Lymphoma triggers age-related epigenetic reprogramming in young CD4⁺ T cells

(A) Principal-component analysis of bulk ATAC-seq of the indicated CD4⁺ T cells. (B) Fold changes of open chromatin regions (OCRs) with p-adjusted values. Light blue indicates significantly more closed OCRs and yellow indicates significantly more open OCRs (FC < 0.5 or FC > 0.5; p-adj <0.05). (C) Gene set enrichment analysis using HALLMARK pathways of genes associated with OCRs in CD4⁺ T cells. (D) Genes in the heme metabolism pathway around OCRs that are significantly altered in comparisons of YL vs. YC and of AL vs. YL groups. (E) Enrichment of heme metabolism gene expression in lymphoma-experienced young (left) and in aged (right) CD4⁺ T cells, from pseudo-bulk analysis of scRNA-seq, as in Figure 4. (F) ATAC-seq tracks around the Hmox1 locus and the accessibility index for the highlighted regions. (G) Relative openness of OCRs around genes in the Abnormality of Iron Homeostasis Pathway (bars represent normalized read counts, minimum to maximum). YC, YL, AC, AL are defined in Figure 1. Data (n = 3) are compiled from 3 independent experiments. See also Figure S5.

Figure 5.. B cell lymphoma and aging alter iron homeostasis in T cells

(A) Intracellular iron measured by BioTracker Fe²⁺ in CD4⁺ and CD8⁺ T cells (n = 6–14). (B) Intracellular iron measured by BioTracker Fe²⁺ in CD44/CD62L CD4⁺ T cells memory populations (n = 6–11). (C) Intracellular iron measured by BioTracker Fe²⁺ in CD4⁺ T cells from OT-II mice (n = 6). (D) Intracellular iron measured by BioTracker Fe²⁺ in human CD4⁺ and CD8⁺ T cells from young and aged healthy donors or from R/R LBCL patients (n = 5–6). (E) Percent DAPI⁻ viable cells of YC or AC CD4⁺ and CD8⁺ T cells unstimulated or activated overnight ±10 mM ferric ammonium citrate (FAC) (n = 3). (F) Aifm2 and Gpx4 gene expression in young and aged T cells activated overnight (n = 3–4). (G) Percent DAPI⁻ Annexin V⁻ viable cells from human CD4⁺ (left) and CD8⁺ (right) T cells unstimulated or activated for 5 days ±10 mM FAC (n = 3). (H) Percent DAPI⁻ viable cells of YC or YL CD4⁺ and CD8⁺ T cells unstimulated or activated overnight ±10 mM FAC (n = 3). (I) Aifm2 and Gpx4 gene expression in young T cells from control and lymphoma-bearing mice activated overnight (n = 4). (J) ATAC-seq track around the Aifm2 locus and the accessibility index for the highlighted regions. (K) Experimental design for the A20 lymphoma BALB/cJ model. (L) Intracellular iron measured by BioTracker Fe²⁺ in CD4⁺ and CD8⁺ T cells from control and A20 lymphoma-bearing BALB/cJ mice (n = 4–8). (M) Total splenocyte number in control and A20 lymphoma-bearing mice (n = 4–8). (N) Total splenic CD4⁺ and CD8⁺ T cells from control and A20 lymphoma-bearing mice (n = 4–8). YC, YL, AC, AL are defined in Figure 1. Data are mean ± SEM (A–D, L–N) or are mean ± SD (E–I). Data (A–D, K–N) are compiled from at least two independent experiments (2–6) and each solid dot represents a biological replicate; or data (E–I) were repeated at least twice with similar result and each hollow dot represents a technical replicate. Data were analyzed by a Student’s t test (A, C, D, F, I, and L–N). For (B, E, G, and H), data were analyzed by a two-way ANOVA with a Tukey’s test or Sidak’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001; n.s. = not significant. See also Figure S6.

Figure 6.. B cell lymphoma accelerates aging in young T cells and other tissues

(A and B) Cdkn2a and Tnfa expression in aorta and kidney tissues (n = 3–5). (C and D) Cdkn2a expression in the aorta and kidney tissues of young control and PPMBC lymphoma-bearing Rag2^−/− mice (n = 3–5). (E) Gene set enrichment analysis of the SenMayo pathways in young CD4⁺ T cells derived from lymphoma-bearing mice early vs. late following Eμ-Myc lymphoma transplant (GSE183693). (F) Relative gene expression of SenMayo pathway in CD4⁺ T cells from bulk RNA-seq data (GSE183693). (G) Cdkn2a and Grem2 gene expression in CD4⁺ T cells from RNA-seq data (GSE183693). (H) CDKN2A gene expression in human T cells from healthy PBMCs (young, aged) or from apheresis products of young or aged R/R LBCL patients (n = 4–5). (I–M) ATAC-seq tracks of the (I) Cdkn2a, (J) Serpine1, (K) Timp2, (L) Ifng, and (M) Tnfaip2 loci and the accessibility index for the highlighted regions. (N) TNFα production in mouse CD4⁺ and CD8⁺ T cells (n = 3–7). (O) TNFα production in human CD4⁺ T cells and CD8⁺ T cells (n = 5–6). (P and Q) NIAD4 staining in (P) CD4⁺ and CD8⁺ T cells (n = 5–8) or (Q) OT-II CD4⁺ T cells (n = 6). (R and S) ER Tracker in (R) CD4⁺ and CD8⁺ T cells (n = 5–8) or (S) OT-II CD4⁺ T cells (n = 6). YC, YL, AC, AL are defined in Figure 1. YL-E = young with lymphoma early (day 7); YL-L = young with lymphoma late (day 14). Data in (A–D), (G), (H), and (N–S) are mean ± SEM and each solid dot represents a biological replicate. Data in (A–D), (N), and (P–S) are compiled from at least 2 independent experiments. For (A–D), (G), (H), (N–S), statistical significance was determined by Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001; n.s. = not significant. See also Figure S7.

Figure 7.. Depletion of lymphoma cells reverses select age-related markers

(A) Schematic for generating and using Eμ-Myc;Cd19-Cre;iDTR lymphomas that can be depleted by DT treatment of lymphoma-bearing mice. (B) Flow cytometry gating strategy for B220^loFSC-A^hi lymphoma cells from untreated or diphtheria toxin (DT) (300 ng) treated mice. (C) Number of B220^loFSC-A^hi lymphoma cells at endpoint (n = 3–4/group). (D) Total splenocyte number (n = 3–4). (E) Number of splenic CD4⁺ and CD8⁺ T cells from YC, YL, and YL + DT mice (n = 3–4). (F) BioTracker Fe²⁺ in the indicated CD4⁺ and CD8⁺ T cells (n = 3–4). (G) ER Tracker in CD4⁺ and CD8⁺ T cells (n = 3–4). (H) NIAD-4 staining in CD4⁺ and CD8⁺ T cells (n = 3–4). (I) TNFα production in CD4⁺ and CD8⁺ T cells (n = 3–4). (J) IFNγ production in CD4⁺ T cells (n = 3–4). (K) Naive, effector, central memory, and CD62L⁺CD44^lo CD4⁺ T cell populations (n = 3–4). (L and M) Cdkn2a and Tnf expression in aortas and kidneys relative to Gapdh (n = 5–7). YC = young control; YL = young with Eμ-Myc;DTR; YL + DT = young with Eμ-Myc;DTR and treated with 300 ng DT prior to endpoint. Data are mean ± SEM and each point represents a biological replicate. Data are analyzed by a Student’s t test (C), a one-way ANOVA with Dunnett’s multiple comparison test (D–J, L, M), or a two-way ANOVA with Sidak’s multiple comparisons test (K). Data (L and M) are compiled from two experiments or represented of two independent experiment (C–K); each dot represents an individual mouse. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, n.s. = not significant.