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IL-2 delivery by engineered mesenchymal stem cells re-invigorates CD8+ T cells to overcome immunotherapy resistance in cancer

Nature Cell Biology volume 24, pages 1754–1765 (2022)Cite this article

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Abstract

Immune checkpoint blockade (ICB)-based immunotherapy depends on functional tumour-infiltrating lymphocytes (TILs), but essential cytokines are less understood. Here we uncover an essential role of endogenous IL-2 for ICB responsiveness and the correlation between insufficient IL-2 signalling and T-cell exhaustion as tumours progress. To determine if exogenous IL-2 in the tumour microenvironment can overcome ICB resistance, we engineered mesenchymal stem cells (MSCs) to successfully deliver IL-2 mutein dimer (SIL2-EMSC) to TILs. While MSCs have been used to suppress inflammation, SIL2-EMSCs elicit anti-tumour immunity and overcome ICB resistance without toxicity. Mechanistically, SIL2-EMSCs activate and expand pre-existing CD8+ TILs, sufficient for tumour control and induction of systemic anti-tumour effects. Furthermore, engineered MSCs create synergy of innate and adaptive immunity. The therapeutic benefits of SIL2-EMSCs were also observed in humanized mouse models. Overall, engineered MSCs rejuvenate CD8+ TILs and thus potentiate ICB and chemotherapy.

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Fig. 1: Advanced tumour with insufficient IL-2 limits CD8+ TIL expansion, and IL-2 signalling is essential for ICB responsiveness.
Fig. 2: MSC engineering for inducible IL-2 can generate superior anti-tumour activity and overcome ICB resistance without toxicity.
Fig. 3: SIL2-EMSC treatment activates and expands the pre-existing CD8+ TILs for anti-tumour activity.
Fig. 4: SIL2-EMSCs remodel the TME, which re-invigorates exhausted CD8+ TILs.
Fig. 5: Local production of SIL2 effectively generates more TILs that exit through DLN to control distal tumours.
Fig. 6: Engineered MSCs in combination with β-lap chemotherapy create potential synergy for strong innate and adaptive immunity.
Fig. 7: SIL-EMSC treatment inhibits colon cancer growth in a humanized mouse model.

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Data availability

scRNA-seq data that support the findings of this study (Fig. 1a–d and Extended Data Fig. 1) can be accessed through the Gene Expression Omnibus under accession code GSE178881. The human SKCM data were derived from the TCGA Research Network: http://cancergenome.nih.gov/. Cumulative survival rate in patients with SKCM and gene correlation were analysed using Tumor Immune Estimation Resource (TIMER; https://cistrome.shinyapps.io/timer/). Source data are provided with this paper, available online for Figs. 1–7 and Extended Data Figs. 1–10. All other data that support the findings of this study are available from the corresponding author on reasonable request.

Code availability

The scRNA data were processed using Cell Ranger v.2.1.1 (https://www.10xgenomics.com/) and analysed with the R package Seurat v.3.1.2 (https://satijalab.org/seurat/). The R packages fgesa v.1.16.0 (http://bioconductor.org/packages/release/bioc/html/fgsea.html) and msigdbr v.7.2.1 (https://cran.r-project.org/web/packages/msigdbr/index.html) were used to perform the GSEA.

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Acknowledgements

This work was supported by Cancer Prevention and Research Institute of Texas (CPRIT) grant RR150072 given to Y.-X.F. and the NIH/NCI grant R01-CA240952 given to J.Q. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank the Institutional Animal Care and Use Committee Animal Resources Center, and Animal Research Center. SSR#69 plasmid was kindly provided by T. C. He at Chicago University. HIF-1 reporter p2.1 plasmid was kindly provided by W. Luo at UT Southwestern Medical Center. Human cord blood was kindly provided by R. A. Word at Obstetrics and Gynecology Tissue Procurement Facility in UT Southwestern Medical Center, supported by NIH-P01-HD087150. We also thank C. Han, Z. Liu, C. Lu, Y. Liang, X. Cao, C. Dong and B. Moon for providing experiment materials and helpful discussions.

Author information

Author notes

  1. These authors contributed equally to this work: Joonbeom Bae, Longchao Liu, Casey Moore.

Authors and Affiliations

  1. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Joonbeom Bae, Longchao Liu, Casey Moore, Eric Hsu, Anli Zhang, Zhenhua Ren, Zhichen Sun, Xue Wang, Jiankun Zhu, Jian Qiao & Yang-Xin Fu

  2. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Casey Moore & Eric Hsu

  3. Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhichen Sun

  4. Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Jiao Shen

  5. University of Chinese Academy of Sciences, Beijing, China

    Jiao Shen

  6. Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China

    Yang-Xin Fu

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Contributions

Conceptualization, J.B. and Y.-X.F.; methodology, J.B. and Y.-X.F.; investigation, J.B., L.L., C.M., E.H, J.S. and X.W.; writing—original draft, J.B.; writing—review and editing, C.M., E.H., J.Q. and Y.-X.F.; funding acquisition, Y.-X.F. and J.Q.; resources, A.Z., L.L., Z.S., Z.R. and J.Z.; supervision, Y.-X.F.

Corresponding authors

Correspondence to Jian Qiao or Yang-Xin Fu.

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Nature Cell Biology thanks Weiyi Peng, George Coukos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Single cell analysis of CD8+ T cells in early and advanced tumor.

a, Schematic workflow of single cell RNA-Seq data generation. b, Feature plot showing the hallmark genes in each single-cell cluster identified by UMAP. c, UMAP plot showing the distribution of CD8+ T cells in the spleen of day 10 (S10), tumor of day 10 (T10), spleen of day 20 (S20), tumor of day 20 (T20). d, Violin plots showing the expression levels of IL-2 related genes across CD8+ T cell clusters. e, the Gene Set Enrichment Analysis (GSEA) was performed between cluster 1 (advanced tumor specific) and cluster 2 (early tumor specific).

Source data

Extended Data Fig. 2 Advanced tumors contain reduced number of CD8 + TILs and predominantly terminally exhausted cells.

a-c, C57BL/6J mice (T10, n = 6; T20, n = 8) were s.c. inoculated with 1 × 106 MC38 cells. 10 days (T10) or 20 days (T20) after tumor inoculation, TILs were analyzed for the frequency of PD1−TIM3−, PD1 + TIM3−, and PD1+TIM3+ subset (a), stem-like exhausted CD8+ T cells (b), terminally exhausted CD8+ T cells (c). d, C57BL/6 J mice (Control, n = 6; anti-PD-L1, n = 5; anti-PD-L1 + anti-IL15, n = 6; anti-PD-L1 + anti-IL2, n = 5; anti-PD-L1 + anti-IL2Rβ, n = 5) were s.c. inoculated with 1 × 106 MC38 cells and i.p. injected with 150 μg of anti-PD-L1 and/or 200 μg anti-IL2Rβ, 200 μg anti-IL2 or 200 μg anti-IL15 on day 7,10 and 13 (black arrow). Tumor growth was measured twice a week. Data are shown as mean ± s.e.m. from two independent experiments. P value was determined by two-tailed unpaired t test (a-c) or two-way ANOVA (d). Source data are available online.

Source data

Extended Data Fig. 3 Survival, biodistribution and SIL2 production of engineered MSC.

a, Functional activity of WTIL2 and SIL2 were assessed using the HEK-BlueTM IL-2 reporter cell assay (n = 2). b,c, CT26 bearing BALB/c mice (n = 4 per group) were treated with SIL2 (20 μg, i.t.) on day 9. Five days after treatment, the concentration of SIL2 in serum (b), tumor and other tissues (c) were determined by hIgG ELISA. d, Body weight of CT26 bearing BALB/c mice (n = 5 per group) treated with SIL2-EMSC (1 × 106, p.t.) or SIL2 (20 μg, i.t.) on day 9 and 12. e, Surface marker expression of MSC by passage number in primary MSC and immortalized MSC (iMSC). f, Proliferation of CFSE-labeled primary MSC and iMSC assessed by flow cytometry at the indicated time points. g, MC38 bearing C57BL/6 mice (n = 3 per group) were p.t. injected with 5 × 105 luciferase expressing iMSCs on day 9. Luciferase signal was analyzed at the indicated time point. h-j, MC38 bearing mice (n = 4 per group) were p.t. injected with 5 × 105 EMSCs on day 9. The frequency of Ki67+GFP+ cells (h), and CD45−GFP+ cells (i) in tumor were analyzed at the indicated time points. j, 200 μg anti-CD8 was administered 1 day before EMSC injection. Four days after EMSC injection, CD45−GFP+ cells were analyzed in tumor. k, NSG-SGM3 mice were s.c. inoculated with 5 × 105 CT26 tumor cells and 5 × 105 luciferase expressing iMSCs were p.t. treated on day 10. Five days after MSC treatment, organs were extracted and luciferase signal was analyzed. l, Functional activity of SIL2-EMSC culture supernatant was assessed by using HEK-BlueTM IL-2 reporter cell assay (n = 2). Data are shown as mean ± s.e.m. from two independent experiments. P value was determined by two-tailed unpaired t test (b,h,j) or two-way ANOVA (d). Source data are available online.

Source data

Extended Data Fig. 4 Therapeutic effect of IL-2 expressing engineered MSC in vivo.

a, MC38 bearing C57BL/6 mice (Control, n = 7; EMSC (5x), n = 6; SIL2-EMSC (1x), n = 6; SIL2-EMSC (2x), n = 8; SIL2-EMSC, n = 8) were p.t. treated with 1 × 106 EMSC or SIL2-EMSC. Cells were injected every 3 days from day 9 as many times as indicated. b, MC38 bearing C57BL/6 mice (n = 5 per group) were p.t. treated with different number of EMSC or SIL2-EMSC on day 9 and 12 (black arrow). c, B16 bearing C57BL/6 mice (n = 5 per group) p.t. treated with 1 × 106 EMSC or SIL2-EMSC on day 7 and 10 (black arrow). CT26 bearing BALB/c mice (n = 5 per group) p.t. treated with 1 × 106 EMSC or SIL2-EMSC on day 9 and 12 (black arrow). 4T1 bearing BALB/c mice (n = 5 per group) p.t. treated with 1 × 106 EMSC or SIL2-EMSC on day 10 and 13 (black arrow). d,e, Advanced MC38 tumor bearing C57BL/6J mice (EMSC, n = 5; WTIL2-EMSC, n = 6; SIL2-EMSC, n = 6; EMSC + anti-PD-L1, n = 5; WTIL2-EMSC + anti-PD-L1, n = 7; SIL2-EMSC + anti-PD-L1, n = 6) were treated with 100 μg α-PD-L1 (i.p.) in combination with 1 × 106 EMSCs, WTIL2-EMSC or SIL2-EMSCs (p.t.) on day 14 and 17 (black arrow). Tumor growth (d) and survival curve (e) are shown. Tumor growth was measured twice a week. Data are shown as mean ± s.e.m. from two independent experiments. P value was determined by two-way ANOVA (a-d) or log rank test (e). Source data are available online.

Source data

Extended Data Fig. 5 Tumor-targeted production of SIL2 by engineered MSCs prevents systemic toxicity.

a-c, CT26 bearing BALB/c mice (Control, n = 4; SIL2, n = 4; SIL2-EMSC, n = 5) treated with SIL2-EMSC (1 × 106, p.t.) or SIL2 (20 ug, i.t.) on day 9 and 12. Two days after second treatment, serum, lung, and liver were isolated. a, Livers were extracted and fixed in 10% formalin for 7 days, then H&E staining was performed. Representative example of 10x magnification liver staining from each group is shown. b, Serum level of ALT and AST (b) and pulmonary wet weight (c) were examined as described in the Methods. d, CT26 bearing BALB/c mice (Control, n = 5; SIL2, n = 6; SIL2-EMSC, n = 6) treated with SIL2-EMSC (1 × 106, p.t.) or SIL2 (20 ug, i.t.) on day 9 and 12 (black arrow). Tumor growth was measured twice a week. e, CT26 bearing BALB/c mice (n = 5 per group) were treated with SIL2-EMSC (1 × 106, p.t.) or SIL2 (20 ug, i.t.) on day 9. IFNγ and TNFα level in the tumor tissue were determined by CBA at 1, 3, or 5 days after treatment. Data are shown as mean ± s.e.m. from two independent experiments. P value was determined by two-tailed unpaired t test (b,c,e) or two-way ANOVA (d). Source data are available online.

Source data

Extended Data Fig. 6 EMSC and SIL2-EMSC do not affect myeloid cell populations in the TME.

a,b, MC38 bearing mice (Control, n = 4; EMSC, n = 4; SIL2-EMSC, n = 6) were p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on days 9. a, Two days after treatment, the number of macrophages, dendritic cells, and MDSCs were analyzed. b, Cell surface expression level of CD80 and CD86 on dendritic cells were analyzed. c,d, MC38 bearing mice (n = 5) were p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on day 9 and 12. Five days after the second treatment, TILs were analyzed for the number of CD4+ T cells (c) and NK cells (d). e, MC38 bearing C57BL/6J mice (n = 5) were p.t. 1 × 106 EMSC, SIL2-EMSC or WTIL2-EMSC on day 9 and 12. Five days after the second treatment, TILs were analyzed for the CD8/Treg ratio. Data are shown as mean ± s.e.m. from two or three independent experiments. P value was determined by two-tailed unpaired t test (a-e). Source data are available online.

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Extended Data Fig. 7 SIL2-EMSC expands and rejuvenates exhausted CD8+ TILs.

a-c, C57BL/6 J mice (n = 6) bearing MC38 were p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on day 9 and 12. Five days after the last treatment, tumor-infiltrating T cells were analyzed for the fold change of number of PD1−TIM3−, PD1+TIM3−, and PD1+TIM3+ CD44+CD8+ T cell subsets (a) and the frequency of CXCR5+TIM3− subset (b) and CD39+Tcf1− subset (c) in PD1+TOX+CD44+CD8+ T cells in tumor were analyzed. d-f, CT26 bearing BALB/c mice (n = 5) were treated with 1 × 106 EMSC or SIL2-EMSC on day 9 and day 12. Five days after the second treatment, TILs were analyzed for the number of CD8+ T cells (d) and the frequency of PD1−TIM3−, PD1+TIM3−, and PD1+TIM3+ CD8+ T cell subsets (e) and stem-like exhausted CD8+ T cells (f). g-i, C57BL/6J mice (n = 5–6) bearing MC38 were p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on day 9 and 12. Five days after the last treatment, TILs were analyzed for surface expression level of CD25 and CD122 (g, n = 6 per group), the frequency of Ki67+ cells (h, EMSC, n = 5; SIL2-EMSC, n = 6), and the frequency of active caspase 3+ cells (I, EMSC, n = 5; SIL2-EMSC, n = 6) in PD1−TIM3−, PD1+TIM3−, and PD1+TIM3+ CD8+ T cell subsets. j, k, MC38 bearing C57BL/6J mice (n = 4) were i.t. treated with single high dose (20 μg) or prolonged low dose (5 μg, 4 injections) on day 9. Prolonged low dose group were injected with SIL2 every 12 h. CD8+ TILs were analyzed for the frequency of PD1−TIM3−, PD1+TIM3−, PD1+TIM3+ CD8+ T cell subsets and (j) and stem-like CD8+ T cells (k). l, MC38 bearing C57BL/6J mice (n = 4) were treated with SIL2 (10 μg, i.t.) and/or EMSCs (1 × 106, p.t.) and SIL2-EMSC (1 × 106, p.t.) on day 9 and 12. Five days after treatment, CD8+ TILs were analyzed for the frequency of PD1−TIM3−, PD1+TIM3−, PD1+TIM3+ CD8+ T cell subsets. Data are shown as mean ± s.e.m. from two or three independent experiments. P value was determined by two-tailed unpaired t test (a–g,i–l). Source data are available online.

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Extended Data Fig. 8 SIL2-EMSC expands and functionally reinvigorates exhausted CD8+ TILs.

a, CD8+ TILs from MC38 bearing mice (PD1−TIM3−, n = 3; PD1+TIM3−, n = 6; PD1+TIM3+, n = 6) were co-cultured with irradiated MC38 in the presence of WTIL2 or SIL2. Two days later, the frequency of CD8+ TIL subsets were analyzed. b,c, CD8+ TILs from MC38 bearing mice (PD1+TIM3−, n = 4; PD1+TIM3+, n = 5) were sorted out and labeled with CFSE. Cells were co-cultured with irradiated MC38 in the presence or absence of SIL2. Three days later, cell proliferation (b) and apoptosis (c) were determined by flow cytometry. d, IFN-γ reporter mice (n = 5 per group) bearing MC38 were p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on day 9. Two days after treatment, tumor-infiltrating T cells were analyzed to determine the frequency of IFN-γ+ cells in PD1−TIM3−, PD1+TIM3−, and PD1+TIM3+ CD8+ T cell subsets. e, Naive OT-1 splenocytes were activated with OT-1 peptide in the presence of recombinant IL-2. OT-1 peptide primed OT-1 splenocytes were restimulated with dimerized α-CD3 for 2 d. Representative flow plots of the cell surface phenotype after restimulation (left) and PD1+TIM3− or PD1+ TIM3+ CD8+ subsets after sorting (right) are shown. f-h, B16-OVA tumor bearing Rag1 KO mice were adoptively transferred with OTI CD8+ T cells on day 8. Ten days after transfer, PD1+TIM3− or PD1+TIM3+ CD8+ TILs were sorted. B16-OVA tumor bearing C57BL/6 mice (EMSC, n = 5; EMSC + PD1+TIM3−, n = 5; EMSC + PD1+TIM3+, n = 5; SIL2-EMSC, n = 6; SIL2-EMSC + PD1+TIM3−, n = 5; SIL2-EMSC + PD1+TIM3+, n = 5) were adoptively transferred with 10,000 sorted CD8+ TIL subsets on day 5. EMSC or SIL2-EMSC were treated on day 5 and day 8. f, Experiment scheme. Tumor growth (g) and survival curve (h) are shown. Data are shown as mean ± s.e.m. from two to three independent experiments. P value was determined by two-tailed unpaired t test (a-d) or two-way ANOVA (g) or log rank test (h). Source data are available online.

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Extended Data Fig. 9 SIL2-EMSCs are eliminated by administration of β-lap.

a, NQO1 expression in different cancer cell lines and EMSCs was determined by western blotting assay. Representative example from two independent experiments is shown. b, CT26 bearing Balb/c mice were p.t. injected with 5 × 105 luciferase expressing EMSCs on day 9. Two days after MSC injection, mice were i.t. treated with β-lap (5 mg/kg) every other day for four times. Luciferase signal was analyzed at indicated time points. c, CT26 bearing BALB/c mice (EMSC, n = 6; EMSC + β-lap, n = 5; SIL2-EMSC, n = 8; SIL2-EMSC + β-lap, n = 6) were i.t. treated with 1 × 106 EMSC or SIL2-EMSC on day 9 and 12 (black arrow). Two days after MSC injection, mice were i.t. treated with β-lap (15 mg/kg) every other day for four times. Data are shown as mean ± s.e.m. from two independent experiments. P value was determined by two-way ANOVA (c). Source data are available online.

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Extended Data Fig. 10 SIL-EMSC treatment exhibits antitumor responses on humanized mouse model.

a, Cumulative survival in skin cutaneous melanoma (SKCM) patients according to Tcf7 expression level in TCGA database. b, TCGA database RNA-seq analysis of the correlation between the gene expression of Il2 and Tcf7 in patients with SKCM. c, HCT116 bearing humanized mice (Day 5, n = 5; Day 10, n = 3) were p.t. injected with 5 × 105 EMSCs on day 7. The frequency of CD45−GFP+ cells in tumor was analyzed indicated time points. d, Humanized mice (n = 6 per group) were inoculated with 1 × 106 HCT116 cells and p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on day 7 and 10 (black arrow). Tumor growth was measured twice a week. e,f, HCT116 bearing humanized mice (EMSC, n = 3; SIL2-EMSC, n = 4) were p.t. treated with 1 × 106 EMSCs or SIL2-EMSCs on day 7 and 10. Five days after the last treatment, tumor-infiltrating T cells were analyzed for number of CD8+ T cells (e), and TCF1+ stem-like CD8+ T cells (f). g,h, SIL2-EMSCs irradiated with different doses (0–50 Gy). g, Two days after irradiation, cell viability was determined (n = 4). h, Cells were incubated in 1% (hypoxia) O2 for 24 hours. SIL2 levels from cell culture supernatants were determined by ELISA (n = 3). i, C57BL/6 mice (n = 5) were s.c. inoculated with 1 × 106 MC38 cells and p.t. treated with 1 × 106 EMSCs, SIL2-EMSCs, or SIL2-EMSC irradiated with 20 Gy on days 9 and 12 (indicated by black arrow). Tumor growth was measured twice a week. Data are shown as mean ± s.e.m. from two independent experiments. Statistical analysis of TCGA data were performed by log-rank test (a) or Spearman’s rho correlation test (b). P value was determined by two-way ANOVA (d,i) or two-tailed unpaired t test (e, f). Source data are available online.

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Supplementary information

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Supplementary Table

Supplementary Table 1. List of differentially expressed genes from scRNA-seq. Supplementary Table 2. List of antibodies used in this study.

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Bae, J., Liu, L., Moore, C. et al. IL-2 delivery by engineered mesenchymal stem cells re-invigorates CD8+ T cells to overcome immunotherapy resistance in cancer. Nat Cell Biol 24, 1754–1765 (2022). https://doi.org/10.1038/s41556-022-01024-5

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