Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling

Abstract

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion—they can also shape the fate and function of CD8+ T cells.

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Fig. 1: FRCs upregulate both immunosuppressive and stimulatory molecules in response to activated T cells.
Fig. 2: FRCs enhance CD8+ T cell cytokine production independently of Nos2 expression.
Fig. 3: FRC-derived IL-6 is necessary and sufficient for the enhanced production of CD8+ T cell cytokines.
Fig. 4: FRCs or IL-6 alter enhancers in activated CD8+ T cells that regulate distinct patterns of gene expression.
Fig. 5: FRCs or IL-6 amplify metabolic and survival pathways in activated CD8+ T cells via epigenetic and transcriptional alterations.
Fig. 6: FRCs enhance the bioenergetic state of activated CD8+ T cells.
Fig. 7: FRC-derived signals extend the longevity of virus-specific CD8+ T cells during influenza infection.

Data availability

All data that relate to or support the observations described in this study are available from the corresponding authors upon reasonable request. The ATAC-seq and RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE136905. The Microarray data are accessible through GEO accession no. GSE136958.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant no. RO1 5R01DK074500 to S.J.T. and W.N.H.; no. P01AI108545 to A.H.S; no. T32 CA207021 to M.W.L) and by a Howard Hughes Medical Institute Gilliam Fellowship for Advanced Study (to F.D.B). We thank members of the Turley, Haining and Sharpe laboratories for scientific insights and discussions. We also thank A. E. Mayfield for critical reading of the manuscript.

Author information

F.D.B. conceived and conducted most of the experiments, analyzed and interpreted data and wrote the manuscript. D.R.S. conducted experiments and analyzed and interpreted data. J.G., M.W.L., V.L.-K., F.A.S., H-J.K., K.B.Y., S.J.H.R., K.B. and V.N.K. conducted experiments and interpreted data. J.D.T. and I.A.S. discussed data and provided technical assistance. V.C., N.N.D. and B.D.M. discussed and interpreted data. A.H.S., W.N.H. and S.J.T. directed the study, analyzed and interpreted results and wrote the manuscript.

Correspondence to Arlene H. Sharpe or W. Nicholas Haining or Shannon J. Turley.

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Competing interests

V.N.K. and S.J.T. are employees of Genentech. W.N.H. is an employee of Merck. V.C. is an employee of Novartis. F.D.B. is an employee of Neon Therapeutics. The authors declare no competing interests.

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Peer review information L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 FRCs dampen IFN-γ production yet enhance the production of IL-2 and TNF in newly activated CD8+ T cells.

(a) Gating strategy to highlight CD8+ T cells within whole splenocyte mixtures (24 h post anti-CD3/CD28 activation). (b) ICS of IFN-γ in CD8+ T cells activated in splenocyte mixtures via anti-CD3/CD28 for 24 h or activated via anti-CD3/CD28 for 24 h in the presence of wild-type FRCs. (c) Summary of results in b (n = 4 [Activated T cells] and n = 5 [Activated T cells + WT FRCs] biologically independent cell cultures per group). ****P < 0.0001 (two-tailed Student’s t-test). Data in c are composite of two biologically independent experiments (mean ± s.d). (d) ICS for IL-2 (top) and TNF (bottom) in purified CD8+ T cells activated via plate-bound anti-CD3 and soluble anti-CD28 for 24 h or activated via plate-bound anti-CD3 and soluble anti-CD28 in the presence of WT FRCs. (e) Summary of results in d as percentage of cytokine positive cells (n = 4 biologically independent cell cultures per group) **P = 0.0053 (IL-2), **P = 0.0018 (TNF) (two-tailed Student’s t-test) Data in e are a composite of two biologically independent experiments (mean ± s.d).

Supplementary Figure 2 CD8+ T cells activated alone or near Nos2−/− FRCs experience the same degree of initial activation and proliferation.

(a) CD25 expression on resting CD8+ T cells (resting), those activated in splenocyte mixtures via soluble anti-CD3/CD28 for 24 h (activated) or activated via anti-CD3/CD28 for 24 h in the presence of Nos2−/− FRCs. Top panel, representative plots; bottom panel, summary (n = 2 biologically independent cell cultures per group). (b) CFSE dilution in same CD8+ T cell conditions as a at 48 h post activation. Top panel, representative plots; bottom panel, summary (n = 3 biologically independent cell cultures per group). ns P = 0.2829 (two-tailed Student’s t-test). Data are representative of two biologically independent experiments (mean ± s.d).

Supplementary Figure 3 Characteristics of chromatin accessible regions, RNA transcripts and epigenetic signatures in CD8+ T cells.

(a) Scatterplot displaying correlation in peak intensity between biological replicates for each CD8+ T cell condition. The conditions were CD8+ T cells sorted following activation in whole splenocyte mixture via soluble anti-CD3/CD28 for 48 h (Act), activated with anti-CD3/CD28 in presence of Nos2−/− FRCs (FRC) or activated with anti-CD3/CD28 plus 100ng/ml recombinant IL-6 (IL-6). (b) Principal component analysis of biological replicates for CD8+ T cells under each condition across all 81, 821 ChARs. (c) ATAC-seq fragment sizes for all three conditions (n = 3 biologically independent cell cultures per group). (d) Fold enrichment of regions annotated for histone marks and enhancer states for all three conditions. Data represent one experiment with three biologically independent cell cultures per group. (e) Scatterplot displaying correlation in gene expression between biological replicates for each CD8+ T cell condition. The conditions were CD8+ T cells sorted following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 for 48 h (Act), activated with anti-CD3/CD28 in presence of Nos2−/− FRCs (FRC) or activated with anti-CD3/CD28 plus 100ng/ml recombinant IL-6 (IL-6). (f) Principal component analysis of biological replicates for CD8+ T cells under each condition. (n = 3 biologically independent cell cultures [FRC] and 2 biologically independent cell cultures [Act and IL-6]). (g) Display of gene expression using hierarchical clustering of replicates by condition with Pearson correlation. Data represent one experiment (n = 3 biologically independent cell cultures [FRC] and 2 biologically independent cell cultures [Act only and Act + IL-6]). (h) Overlap of H3K4me3 peaks identified in naive, terminal effector and memory precursor cells (Gray et al. 2017) with the FRC, IL-6 and Act conditions. (i) Overlap of ATAC-seq profiles corresponding to naive, effector and memory T cells (Sen et al. 2016) with the FRC, IL-6 and Act conditions.

Supplementary Figure 4 IL-6 enhances phospho S6 levels and neutral lipid accumulation in activated T cells.

(a) Phospho flow cytometry of p-S6 levels in resting CD8+ T cells or those activated in whole splenocyte mixtures via anti-CD3/CD28 for 48 h or activated via anti-CD3/CD28 for 48 h in the presence of recombinant IL-6 (100ng/ml). Left panel, representative plot; right panel, summary (n = 6 biologically independent cell cultures per group) ****P < 0.0001 (two-tailed Student’s t-test) (b) Bodipy staining in CD8+ T cells either solely activated for 48 h with anti-CD3/CD28 or activated via anti-CD3/CD28 in the presence of recombinant IL-6 (100ng/ml). Left panel, representative plot; right panel, summary (n = 4 [Activated T cells] and 5 [Activated T cells + IL-6] biologically independent cell cultures) ns P = 0.0720 (two-tailed Student’s t-test) Data are a composite of two biologically independent experiments (mean ± s.d).

Supplementary Figure 5 FRCs extend the longevity of activated CD8+ T cells in vitro and during acute LCMV infection.

(a) Frequency of viable CD8+ T cells activated in splenocyte mixtures via soluble anti-CD3/CD28 for 48 h versus CD8+ T cells activated via anti-CD3/CD28 for 48 h in the presence of Nos2−/− FRCs. (b) Summary of results in a (n = 2 biologically independent cell cultures per group). (c) Frequency of viable T cells from a after being sorted and allowed to rest in alpha-mem media (supplemented with 10% fetal bovine serum and 1% penicillin streptomycin) for three days. (d) Summary of results in c (n = 2 biologically independent cell cultures per group). Data in b and d are representative of two biologically independent experiments. (e) Competitive frequencies of pre-activated P14 T cells before transfer (Day 0) and 7 days following transfer into wild type recipients infected with LCMV Armstrong. Transferred T cells were recovered from the lungs of infected animals. Top panel, representative plots; bottom panel, summary (n = 3 mice per group) *P = 0.0185 (two-tailed ratio paired t-test). (f) Frequency of competitive mixes at Day 0 and 7 days post LCMV infection. Transferred T cells were recovered from the spleen of infected animals. Top panel, representative plots; bottom panel, summary (n = 10 mice per group).****P < 0.0001 (two-tailed ratio paired t-test). Data in f are representative of three biologically independent experiments. (g) BrdU incorporation in transferred P14 T cells 8 days after the onset of influenza infection. Transferred T cells were recovered from the lungs of infected animals. (h) Summary of results in g (n = 5 mice per group). ns P = 0.4011 (two-tailed paired t-test) (mean ± s.d).

Supplementary Figure 6 Neutralizing IL-6 prior to adoptive transfer decreases the absolute number of recovered IL-2+ and TNF+ P14 T cells.

(a) Competitive frequencies of P14 T cells activated in whole splenocyte mixture with anti-CD3/CD28 in the presence of Nos2−/− FRCs and 10 μg/ml anti-IL-6 blocking antibody versus P14 T cells activated under the same conditions with 10 μg/ml of isotype control antibody. Il6−/− recipients were used for the transfers in a. Top panel, representative plots; bottom panel, summary. (n = 7 mice per group) ***P = 0.0004 (two-tailed ratio paired t-test) (b) Representative plots of ICS for IL-2 (left) and TNF (right) from CD8+ T cells recovered from the lungs of infected mice 9 days post influenza infection (n = 7 mice per group) (c) Summary of results in b (n = 7 mice per group) ns P = 0.0562 (IL-2), ns P = 0.6148 (TNF) (two-tailed paired t-test). (d) Absolute number of recovered cytokine positive P14 T cells. (n = 7 mice per group) *P = 0.0375 (IL-2), *P = 0.0265 (TNF) (two-tailed paired t-test), data in c shown as mean ± s.d. For b-d, the transferred cells were re-challenged in vitro with gp33 peptide (KAVYNFATC) for 6 h in the presence of Brefeldin A, followed by ICS staining to measure cytokine production.

Supplementary Figure 7 Generation and infection of bone marrow chimeras.

(a) Percent chimerism (following tail vein bleed) in C57BL/6J (WT) or Il6−/− host 6-12 weeks after transfer of donor cells. Representative plots shown in a. (b) Left panel, summary of data in a; right panel, hematopoietic cell lineage reconstitution in irradiated recipients. n = 10 mice per group (mean ± s.d) (c) Frequency of CD45 lymph node stromal cells. Top panel, representative plots; bottom panel, graphic display (n = pooled popliteal lymph nodes from 3 mice). (d) ICS for IL-6 in stromal populations from popliteal lymph nodes. The lymph nodes were harvested on day 8 following CFA/OVA immunization and OT-1 T cell injection. 100 μg Brefeldin A was injected intravenously (tail vein) to block protein transport 5 h prior to lymph node harvest and digest. Single cell suspensions were placed in Brefeldin A for an additional 2 h prior to ICS staining. Top panel, representative plots; bottom panel, graphic display. Data are representative of two biologically independent experiments with pooled popliteal lymph nodes from 3 wild-type C57BL/6J mice. (e) Frequency of CD44+, CD8+ T cells within the lungs of WT and Il6−/− bone marrow chimeras. Analysis conducted in animals that successfully cleared influenza infection from Fig. 7h (n = 6 of 10 mice for WT recipients and n = 1 of 10 mice for Il6−/− recipients) (mean ± SEM).

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Brown, F.D., Sen, D.R., LaFleur, M.W. et al. Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling. Nat Immunol 20, 1668–1680 (2019). https://doi.org/10.1038/s41590-019-0515-x

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