Priming with LSD1 inhibitors promotes the persistence and antitumor effect of adoptively transferred T cells

doi:10.1038/s41467-024-48607-4

. 2024 May 21;15(1):4327.

doi: 10.1038/s41467-024-48607-4.

Priming with LSD1 inhibitors promotes the persistence and antitumor effect of adoptively transferred T cells

Fengqi Qiu^#¹, Peishan Jiang^#^{1

2}, Guiheng Zhang^{1

2}, Jie An², Kexin Ruan¹, Xiaowen Lyu³, Jianya Zhou⁴, Wanqiang Sheng^{5

6}

Affiliations

¹ Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
² Institute of Immunology and Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou, China.
³ State Key Laboratory of Cellular Stress Biology, Fujian Provincial Key Laboratory of Reproductive Health Research, School of Medicine, Xiamen University, Xiamen, China. xiaowenlyu@xmu.edu.cn.
⁴ Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. zhoujy@zju.edu.cn.
⁵ Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. wanqiang_sheng@zju.edu.cn.
⁶ Institute of Immunology and Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou, China. wanqiang_sheng@zju.edu.cn.

^# Contributed equally.

PMID: 38773088
PMCID: PMC11109160
DOI: 10.1038/s41467-024-48607-4

Priming with LSD1 inhibitors promotes the persistence and antitumor effect of adoptively transferred T cells

Fengqi Qiu et al. Nat Commun. 2024.

. 2024 May 21;15(1):4327.

doi: 10.1038/s41467-024-48607-4.

Authors

Fengqi Qiu^#¹, Peishan Jiang^#^{1

2}, Guiheng Zhang^{1

2}, Jie An², Kexin Ruan¹, Xiaowen Lyu³, Jianya Zhou⁴, Wanqiang Sheng^{5

6}

Affiliations

¹ Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
² Institute of Immunology and Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou, China.
³ State Key Laboratory of Cellular Stress Biology, Fujian Provincial Key Laboratory of Reproductive Health Research, School of Medicine, Xiamen University, Xiamen, China. xiaowenlyu@xmu.edu.cn.
⁴ Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. zhoujy@zju.edu.cn.
⁵ Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. wanqiang_sheng@zju.edu.cn.
⁶ Institute of Immunology and Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou, China. wanqiang_sheng@zju.edu.cn.

^# Contributed equally.

PMID: 38773088
PMCID: PMC11109160
DOI: 10.1038/s41467-024-48607-4

Abstract

The antitumor efficacy of adoptively transferred T cells is limited by their poor persistence, in part due to exhaustion, but the underlying mechanisms and potential interventions remain underexplored. Here, we show that targeting histone demethylase LSD1 by chemical inhibitors reshapes the epigenome of in vitro activated and expanded CD8⁺ T cells, and potentiates their antitumor efficacy. Upon T cell receptor activation and IL-2 signaling, a timely and transient inhibition of LSD1 suffices to improve the memory phenotype of mouse CD8⁺ T cells, associated with a better ability to produce multiple cytokines, resist exhaustion, and persist in both antigen-dependent and -independent manners after adoptive transfer. Consequently, OT1 cells primed with LSD1 inhibitors demonstrate an enhanced antitumor effect in OVA-expressing solid tumor models implanted in female mice, both as a standalone treatment and in combination with PD-1 blockade. Moreover, priming with LSD1 inhibitors promotes polyfunctionality of human CD8⁺ T cells, and increases the persistence and antitumor efficacy of human CD19-CAR T cells in both leukemia and solid tumor models. Thus, pharmacological inhibition of LSD1 could be exploited to improve adoptive T cell therapy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. LSD1 inhibition by GSK elevates cytokine production and decreases inhibitory receptor expression in in vitro activated CD8⁺ T cells.**
a Principal component analysis (PCA) of RNA-seq data of mouse CD8⁺ T cells activated and expanded in vitro for 5 days with the treatment of GSK2879552 (GSK) or vehicle control (Veh) (n = 3 mice per group). b Volcano plot showing differential gene expression in RNA-seq analysis of GSK- versus Veh-treated CD8⁺ T cells (n = 3, [Fold change] > 1.5 and q-value < 0.05 as the cutoff]). c Dot map showing the top 10 terms in the GO analysis of the upregulated genes in the comparison of GSK- versus Veh-treated CD8⁺ T cells. P-values were calculated by one-sided Fisher’s exact test and adjusted by the Benjamini-Hochberg method. d Heatmaps showing differential gene expression of effector molecules, chemokines, inhibitory receptors, and transcription factors (q < 0.05, marked in bold). Flow cytometry of cytokines (e), GzmB (f), inhibitory receptors (g), and SLAMF6 (h) expressed in GSK- or Veh-treated CD8⁺ T cells (n = 3). MFI, mean fluorescence intensity. i Flow cytometry analysis of frequencies of CD44⁺CD62L⁺ cells in GSK- or Veh-treated CD8⁺ T cells (n = 3). Data in (e–i) are presented as mean ± standard deviation (SD) and are representative of three independent experiments. Statistical significance was determined by two-sided unpaired t-test (e–i). Source data are provided as a Source Data file.

**Fig. 2. GSK treatment reshapes chromatin landscapes of CD8⁺ T cells.**
a Heatmaps of ChIP-seq signals at LSD1-bound promoters (TSS ± 1 kb) in GSK- or Veh-treated CD8⁺ T cells, generated by K-means clustering on H3K4me2 signals. Heatmaps of ChIP-seq signals at defined enhancers (Peak ± 1 kb) with (b) or without (c) LSD1 binding, generated by K-means clustering on H3K4me1 signals. d Heatmaps of ChIP-seq signals at promoters (TSS ± 1 kb) of upregulated effector and cytokine genes and downregulated inhibitory receptor genes. IGV snapshots showing ChIP-seq tracks at genomic loci of *Tnf* (e), *Slamf6* (f), and *Pdcd1* (g). Differential peaks were determined by p < 0.05, with changes over 20% up/down. The presented data are from one of two repeated experiments (a–g).

**Fig. 3. LSD1 responds to both TCR and IL-2 signaling to induce IR expression.**
a Flow cytometry analysis of PD-1 expression in CD8⁺ T cells activated with different concentrations (μg/ml) of anti-CD3/anti-CD28 for 2 days and expanded with IL-2 for 3 days (n = 3). b Flow cytometry analysis of PD-1 expression in unstimulated CD8⁺ T cells cultured with IL-2 and IL7 for 5 days in the presence or absence of GSK (n = 3). c Flow cytometry analysis of inhibitory receptors (IRs) and SLAMF6 in CD8⁺ T cells after 2-day activation with anti-CD3/anti-CD28 and 6-day expansion with IL-2, during which GSK was added as indicated (n = 3). Immunoblot of LSD1 (d) and flow cytometry analysis of IR expression (e, n = 3) in *Rosa26*^Cre-ERT2Lsd1^f/f CD8⁺ T cells on day 5, treated with 4-OHT during the 2-day TCR activation period (4OHT-A) or the 3-day IL-2 expansion period (4OHT-E). f Flow cytometry analysis of Ca²⁺ influx in GSK- or Veh-treated CD8⁺ T cells in response to PMA stimulation (n = 3). ns, not statistically significant. g Flow cytometry of CD44 in GSK- or Veh-treated CD8⁺ T cells on day 5 (n = 3). h Flow cytometry of PD-1 in CD8⁺ T cells after 2-day activation and 3-day expansion with different concentrations of IL-2 (n = 3). i Flow cytometry of PD-1 in CD8⁺ T cells treated with anti-IL-2, tofacitinib, or vehicle control (Mock) (n = 3). j Immunoblot of p-STAT5 in CD8⁺ T cells treated with GSK and/or tofacitinib (Tofa). k, l Flow cytometry of PD-1 and SLAMF6 in wild-type (k, n = 2), *Stat5*^f/f, and *Cd4*^CreStat5^f/f (*Stat5*^cKO) (l, n = 3) CD8⁺ T cells with the indicated treatments for 5 days. m Immunoblots of IL-2 signaling proteins in CD8⁺ T cells treated with GSK or Veh for 5 days. n Flow cytometry of EOMES in GSK or Veh-treated CD8⁺ T cells (n = 3). o Flow cytometry of PD-1 in Rosa26-Cas9 CD8⁺ T cells transduced with sgScramble or sgEOMES and treated with GSK or Veh for 5 days (n = 3). Data in this figure are presented as mean ± SD and are representative of three (a–g, n, o) or two (h–m) independent experiments. Statistical significance in this figure was determined by two-sided unpaired t-test. Source data are provided as a Source Data file.

**Fig. 4. LSD1 inhibition impedes the in vitro induction of T-cell exhaustion.**
a Experimental design for in vitro induction of exhaustion by repeated anti-CD3 stimulation and functional assessment of exhausted CD8⁺ T cells. Flow cytometry of PD-1 (b), TIM-3 (c), and CD39 (d) in CD8⁺ T cells after repeated stimulation (Tex) or continuous IL-2 expansion (Teff) for 10 days with or without GSK treatment (n = 3). e Percentages of TIM-3⁺CD39⁺ cells among PD-1⁺ Teff and Tex cells (n = 3). f Flow cytometry analysis of cytokine production in GSK- or Veh-treated Teff and Tex cells (n = 3). g Cytotoxicity of GSK- or Veh-treated OT1 cells after repeated stimulation, assessed by antigen-dependent killing assay (n = 4). E:T, effector to target ratio. h Percentages of CD25⁺CD62L⁺ cells among GSK- or Veh-treated Teff and Tex cells (n = 3). i Experimental design for in vitro induction of exhaustion in OT1 cells by co-culture. Flow cytometry analysis of IR (j) and cytokine (k) expression in exhausted OT1 (OT1ex) cells with or without GSK treatment (n = 3). l Percentages of CD44⁺CD62L⁺ cells among GSK- or Veh-treated OT1ex cells (n = 3). Data in this figure are presented as mean ± SD and are representative of three (b–f, h) or two (g, j–l) independent experiments. Statistical significance in this figure was determined by two-sided unpaired t-test. Source data are provided as a Source Data file.

**Fig. 5. LSD1 inhibitor-primed OT1 cells show reduced exhaustion and enhanced antitumor efficacy.**
Tumor growth and survival curves of mice subcutaneously inoculated with B16-OVA (a, b) or Yumm1.7-OVA (c, d) tumor cells and transferred with 1 million GSK-, ORY1001- or Veh-primed OT1 cells, or PBS as indicated (n = 6 mice per group). e Abundance of transferred CD45.2 OT1 cells in B16-OVA tumors, detected by flow cytometry on day 5 post transfer (n = 3 mice per group). Flow cytometry analysis of PD-1 (f), CD39 (g), and TIM-3 (h) expression in B16-OVA tumor-infiltrating OT1 cells (TILs) on day 5 post transfer (n = 3 mice per group). i Percentages of CD44⁺CD62L⁺ cells in OT1 TILs (n = 3 mice per group). Tumor growth (j) and survival curves (k) of B16-OVA tumor-bearing mice transferred with 1 million GSK- or Veh-primed OT1 cells or PBS, and injected with anti-PD1 or isotype control as indicated (n = 6 mice per group). l Tumor growth curves of B16-OVA tumor-bearing mice transferred with PBS or *Rosa26*^Cre-ERT2Lsd1^f/f OT1 cells with vehicle or 4-OHT treatment during the 2-day activation period (4-OHT at Activation) or the 3-day expansion period (4-OHT at Expansion) (n = 5–6 mice per group as indicated). m Tumor growth curves of B16-OVA tumor-bearing mice transferred with 5 million OT1 cells primed with GSK for different time durations (n = 6–8 mice per group as indicated). n Flow cytometry of PD-1, SLAMF6, and CD62L in OT1 cells treated with GSK for the indicated times during in vitro activation and expansion and analyzed on day 6 (n = 3). o CETSA assay detecting soluble LSD1 in activated CD8⁺ T cells collected after treatment with 0.5 μM GSK for the indicated hours and heated at 53 °C. p CETSA assay detecting soluble LSD1 in activated CD8⁺ T cells treated with 0.5 μM GSK for the indicated time durations and collected after 6 days of total culture. Data are representative of three (a, b) or two (c–l, n–p) independent experiments and are presented as mean ± SEM (a–m) or mean ± SD (n). Statistical significance was determined by two-sided unpaired t-test (e–i, n), two-way ANOVA (a, c, j, l, m), or log-rank test (b, d, k). Source data are provided as a Source Data file.

**Fig. 6. Transient priming with GSK improves the in vivo persistence of OT1 cells.**
Percentages of Ki-67⁺ (a) and Annexin V⁺ (b) cells in transferred OT1 TILs or endogenous CD8⁺ TILs (c) isolated from B16-OVA tumors and analyzed by flow cytometry on day 5 post transfer (n = 5 mice per group). d Experimental design for co-transfer of Veh- and GSK-primed OT1 cells to B16-OVA tumor-bearing mice and TIL analysis by flow cytometry. e Frequencies of Veh-primed CD45.1/CD45.2 and GSK-primed CD45.2 OT1 cells in B16-OVA tumors analyzed by flow cytometry on day 5 (n = 5 mice per group) and day 8 (n = 3 mice per group) after co-transfer. Flow cytometry analysis of percentages of Annexin V⁺ cells (f) and PD-1 expression (g) in OT1 TILs in the co-transfer assay (n = 3 mice per group). h Tumor weights of B16-WT tumor-bearing mice transferred with GSK- or Veh-primed OT1 cells (n = 5 mice per group). Frequencies of GSK- or Veh-primed OT1 cells in B16-WT tumors (i) and tumor-draining lymph nodes (TdLNs, j) analyzed by flow cytometry on day 5 post transfer (n = 5 mice per group). Frequencies (k) and phenotypes (l) of Veh-primed CD45.1/CD45.2 and GSK-primed CD45.2 OT1 cells in the spleens of mice immunized with OVA_257–264/poly(I:C) on day 1 and analyzed by flow cytometry on day 5 after co-transfer (n = 3 mice per group). m Frequencies of GSK- or Veh-primed OT1 cells in peripheral blood of OVA_257–264/poly(I:C)-immunized mice, analyzed by flow cytometry on day 14 and day 28 after co-transfer (n = 3 mice per group). Data in this figure are presented as mean ± SEM and represent three (a–c) or two (e–g, k–m) independent experiments. Statistical significance was determined by two-sided unpaired t-test (a–c, h–j) or paired t-test (e–g, k–m). Source data are provided as a Source Data file.

**Fig. 7. Pharmacological LSD1 inhibition enhances the antitumor potency of human CAR T cells.**
Flow cytometry analysis of cytokine production (a) and multiple-cytokine producers (b) in human peripheral CD8⁺ T cells activated and expanded in vitro for 10 days with or without GSK treatment (n = 9 donors). c Flow cytometry analysis of GzmB and Ki-67 expression in GSK- or Veh-treated human CD8⁺ T cells (n = 6 donors). d Flow cytometry analysis of FMC63-scFv and EGFP expression in human CD8⁺ T cells transduced with a lentiviral plasmid encoding CD19-CAR and EGFP and treated with or without GSK. Cytotoxicity of GSK- or Veh-treated CD19-CAR T cells or unmodified mock T cells in tumor killing assays using CD19⁺ Nalm6-lucif-EGFP (e, f) or Raji (g) cells as targets and single-stimulated (e) or repeatedly stimulated (f, g) CD19-CAR T cells as effectors (n = 3 for e and g, and n = 4 for f). Representative bioluminescence images (h), quantitative bioluminescence imaging data (i), and survival curves (j) of NCG mice intravenously injected with 10⁶ Nalm6-lucif-EGFP cells and infused with 2 × 10⁵ CD19-CAR or mock T cells 7 days later (n = 5 mice per group). Representative flow plots (k), frequencies (l), and cell numbers (m) of human mock CD8⁺ T cells and GSK- or Veh-primed CD19-CAR CD8⁺ T cells in peripheral blood of NCG recipient mice on day 12 post transfer (n = 4 mice for mock T and CAR T Veh, and n = 3 mice for CAR T GSK). n Tumor growth curves of NCG mice subcutaneously injected with 10⁶ A375-CD19 cells and infused with 2 × 10⁵ CD19-CAR T or mock T cells 8 days later (n = 5 mice per group). Data are pooled from three independent experiments (a–c) or represent two independent experiments (d–m) and are presented as mean ± SD (e–g) or mean ± SEM (l–n). Statistical significance was determined by paired t-test (a, c), two-sided unpaired t-test (e–g, l, m), two-way ANOVA (n), or log-rank test (j). Source data are provided as a Source Data file.

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References

1. Brentjens RJ, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 2013;5:177ra138. doi: 10.1126/scitranslmed.3005930. - DOI - PMC - PubMed
1. Maude SL, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N. Engl. J. Med. 2018;378:439–448. doi: 10.1056/NEJMoa1709866. - DOI - PMC - PubMed
1. Park JH, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N. Engl. J. Med. 2018;378:449–459. doi: 10.1056/NEJMoa1709919. - DOI - PMC - PubMed
1. Marofi F, et al. CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res. Ther. 2021;12:81. doi: 10.1186/s13287-020-02128-1. - DOI - PMC - PubMed
1. Baulu E, Gardet C, Chuvin N, Depil S. TCR-engineered T cell therapy in solid tumors: state of the art and perspectives. Sci. Adv. 2023;9:eadf3700. doi: 10.1126/sciadv.adf3700. - DOI - PMC - PubMed

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[1] Brentjens RJ, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 2013;5:177ra138. doi: 10.1126/scitranslmed.3005930. - DOI - PMC - PubMed

[2] Brentjens RJ, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 2013;5:177ra138. doi: 10.1126/scitranslmed.3005930. - DOI - PMC - PubMed

[3] Maude SL, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N. Engl. J. Med. 2018;378:439–448. doi: 10.1056/NEJMoa1709866. - DOI - PMC - PubMed

[4] Maude SL, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N. Engl. J. Med. 2018;378:439–448. doi: 10.1056/NEJMoa1709866. - DOI - PMC - PubMed

[5] Park JH, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N. Engl. J. Med. 2018;378:449–459. doi: 10.1056/NEJMoa1709919. - DOI - PMC - PubMed

[6] Park JH, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N. Engl. J. Med. 2018;378:449–459. doi: 10.1056/NEJMoa1709919. - DOI - PMC - PubMed

[7] Marofi F, et al. CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res. Ther. 2021;12:81. doi: 10.1186/s13287-020-02128-1. - DOI - PMC - PubMed

[8] Marofi F, et al. CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res. Ther. 2021;12:81. doi: 10.1186/s13287-020-02128-1. - DOI - PMC - PubMed

[9] Baulu E, Gardet C, Chuvin N, Depil S. TCR-engineered T cell therapy in solid tumors: state of the art and perspectives. Sci. Adv. 2023;9:eadf3700. doi: 10.1126/sciadv.adf3700. - DOI - PMC - PubMed

[10] Baulu E, Gardet C, Chuvin N, Depil S. TCR-engineered T cell therapy in solid tumors: state of the art and perspectives. Sci. Adv. 2023;9:eadf3700. doi: 10.1126/sciadv.adf3700. - DOI - PMC - PubMed

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Priming with LSD1 inhibitors promotes the persistence and antitumor effect of adoptively transferred T cells

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Priming with LSD1 inhibitors promotes the persistence and antitumor effect of adoptively transferred T cells

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