Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An engineered IL-2 partial agonist promotes CD8+ T cell stemness

Abstract

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Differential effects of H9T versus IL-2 and H9 on CD8+ T cells.
Fig. 2: Transcriptional profile and epigenetic landscape of H9T-expanded CD8+ T cells.
Fig. 3: Altered metabolism in H9T-expanded CD8+ T cells.
Fig. 4: Increased anti-tumour activity of H9T-expanded CD8+ T cells.

Data availability

Our ATAC-seq, ChIP–seq and RNA-seq data are available at the NCBI Gene Expression Omnnibus (GEO) under the accession number GSE138698. Publicly available previously generated ChIP–seq56 (GSE36890) and ATAC-seq22 (GSE88987) data were also used in this study. Source data are provided with this paper.

References

  1. 1.

    Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62–68 (2015).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Shah, N. N. & Fry, T. J. Mechanisms of resistance to CAR T cell therapy. Nat. Rev. Clin. Oncol. 16, 372–385 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Crompton, J. G., Sukumar, M. & Restifo, N. P. Uncoupling T-cell expansion from effector differentiation in cell-based immunotherapy. Immunol. Rev. 257, 264–276 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Pipkin, M. E. et al. Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Immunity 32, 79–90 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Kalia, V. et al. Prolonged interleukin-2Rα expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity 32, 91–103 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Gattinoni, L., Speiser, D. E., Lichterfeld, M. & Bonini, C. T memory stem cells in health and disease. Nat. Med. 23, 18–27 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Gattinoni, L., Klebanoff, C. A. & Restifo, N. P. Paths to stemness: building the ultimate antitumour T cell. Nat. Rev. Cancer 12, 671–684 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Silva, D. A. et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature 565, 186–191 (2019).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Spolski, R., Li, P. & Leonard, W. J. Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat. Rev. Immunol. 18, 648–659 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Leonard, W. J., Lin, J. X. & O’Shea, J. J. The γc family of cytokines: basic biology to therapeutic ramifications. Immunity 50, 832–850 (2019).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Kinter, A. L. et al. The common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands. J. Immunol. 181, 6738–6746 (2008).

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Mujib, S. et al. Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway. J. Immunol. 188, 3745–3756 (2012).

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Jin, H. T. et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc. Natl Acad. Sci. USA 107, 14733–14738 (2010).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Sakuishi, K. et al. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J. Exp. Med. 207, 2187–2194 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Levin, A. M. et al. Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533 (2012).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Mitra, S. et al. Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps. Immunity 42, 826–838 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Wang, X., Rickert, M. & Garcia, K. C. Structure of the quaternary complex of interleukin-2 with its α, β, and γc receptors. Science 310, 1159–1163 (2005).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Liu, D. V., Maier, L. M., Hafler, D. A. & Wittrup, K. D. Engineered interleukin-2 antagonists for the inhibition of regulatory T cells. J. Immunother. 32, 887–894 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    McLane, L. M., Abdel-Hakeem, M. S. & Wherry, E. J. CD8 T cell exhaustion during chronic viral infection and cancer. Annu. Rev. Immunol. 37, 457–495 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Im, S. J. et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 537, 417–421 (2016).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Scott-Browne, J. P. et al. Dynamic changes in chromatin accessibility occur in CD8+ T cells responding to viral infection. Immunity 45, 1327–1340 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Wang, R. & Green, D. R. Metabolic checkpoints in activated T cells. Nat. Immunol. 13, 907–915 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Sukumar, M. et al. Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function. J. Clin. Invest. 123, 4479–4488 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    van der Windt, G. J. et al. Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. Immunity 36, 68–78 (2012).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  26. 26.

    Manjunath, N. et al. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108, 871–878 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Hinrichs, C. S. et al. IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy. Blood 111, 5326–5333 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Onishi, M. et al. Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation. Mol. Cell Biol. 18, 3871–3879 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Blank, C. U. et al. Defining ‘T cell exhaustion’. Nat. Rev. Immunol. 19, 665–674 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Yang, Q. et al. TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow. Nat. Immunol. 16, 1044–1050 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Liu, X. et al. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11, 179–186 (1997).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Udy, G. B. et al. Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc. Natl Acad. Sci. USA 94, 7239–7244 (1997).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Vodnala, S. K. et al. T cell stemness and dysfunction in tumors are triggered by a common mechanism. Science 363, eaau0135 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Hanada, K. I., Yu, Z., Chappell, G. R., Park, A. S. & Restifo, N. P. An effective mouse model for adoptive cancer immunotherapy targeting neoantigens. JCI Insight 4, e124405 (2019).

    PubMed Central  Article  PubMed  Google Scholar 

  35. 35.

    Zhu, E. F. et al. Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2. Cancer Cell 27, 489–501 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Sockolosky, J. T. et al. Selective targeting of engineered T cells using orthogonal IL-2 cytokine–receptor complexes. Science 359, 1037–1042 (2018).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Bijl, J., Sauvageau, M., Thompson, A. & Sauvageau, G. High incidence of proviral integrations in the Hoxa locus in a new model of E2a–PBX1-induced B-cell leukemia. Genes Dev. 19, 224–233 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Jacoby, E. et al. Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD. Blood 127, 1361–1370 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Yamamoto, T. N. et al. T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy. J. Clin. Invest. 129, 1551–1565 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Ya, Z., Hailemichael, Y., Overwijk, W. & Restifo, N. P. Mouse model for pre-clinical study of human cancer immunotherapy. Curr. Protoc. Immunol. 108, 20.21.21–20.21.43 (2015).

    Article  Google Scholar 

  41. 41.

    Kochenderfer, J. N., Yu, Z., Frasheri, D., Restifo, N. P. & Rosenberg, S. A. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood 116, 3875–3886 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    van der Windt, G. J., Chang, C. H. & Pearce, E. L. Measuring bioenergetics in T cells using a seahorse extracellular flux analyzer. Curr. Protoc. Immunol. 113, 3.16B.1–13.16B.14 (2016).

    Google Scholar 

  43. 43.

    Hermans, D. et al. Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8+ T cell stemness and antitumor immunity. Proc. Natl Acad. Sci. USA 117, 6047–6055 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Sukumar, M. et al. Mitochondrial membrane potential identifies cells with enhanced stemness for cellular therapy. Cell Metab. 23, 63–76 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Lin, J. X. et al. Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells. Nat. Commun. 8, 1320 (2017).

    ADS  PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46.

    Li, P. et al. BATF–JUN is critical for IRF4-mediated transcription in T cells. Nature 490, 543–546 (2012).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Buenrostro, J. D., Wu, B., Chang, H. Y. & Greenleaf, W. J. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.21–21.29.29 (2015).

    Article  Google Scholar 

  48. 48.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  49. 49.

    Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Wherry, E. J. et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27, 670–684 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Doering, T. A. et al. Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory. Immunity 37, 1130–1144 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Wu, T. et al. The TCF1–Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness. Sci. Immunol. 1, eaai8593 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Lin, J. X. et al. Critical role of STAT5 transcription factor tetramerization for cytokine responses and normal immune function. Immunity 36, 586–599 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank R. Ahmed for critical comments; W. Yang and C. Wu for discussions; and the NHLBI DIR Flow Cytometry Core, DNA Sequencing and Genomics Core for assistance with cell sorting and DNA sequencing. The work was supported by the Division of Intramural Research at the National Heart, Lung, and Blood Institute, and by the Division of Intramural Research at the National Cancer Institute (NCI). K.C.G. was supported by National Institutes of Health (NIH) grant AI51321, the Parker Institute of Cancer Immunotherapy, the Ludwig Foundation, the Mathers Foundation and the Howard Hughes Medical Institute. J.D.P was supported by the NIH (R01AI07761, P41EB028239-01) and The Bloomberg–Kimmel Institute for Cancer Immunotherapy. N.P.R was supported by the Intramural Research Program of the NCI and the Cancer Moonshot Program for the Center for Cell-Based Therapy at the NCI, NIH. L.G. was supported by the Intramural Research Program of the US NIH, NCI, Center for Cancer Research (ZIABC011480).

Author information

Affiliations

Authors

Contributions

F.M., Z.Y., J.O., L.Z., C.R.G., T.N.Y., D.H. and S.M. designed and performed experiments, and analysed data. F.M., J.O., Y.C., F.M.G., S.S.M., L.K.P. and M.R. purified protein. P.L. and F.M. analysed the bioinformatics data. R.S., W.L.,  X.Z., J.-X.L. and L.G. analysed data and edited the paper. J.D.P., N.P.R., K.C.G. and W.J.L. supervised the project and analysed data. F.M. and W.J.L. wrote the paper.

Corresponding authors

Correspondence to Nicholas P. Restifo, K. Christopher Garcia or Warren J. Leonard.

Ethics declarations

Competing interests

W.J.L. K.C.G. and S.M. are inventors on patents and patent applications that include H9T. L.G. is an inventor on a patent that describes methods for the generation and isolation of stem-cell memory T (Tscm) cells. L.G. has consulting agreements with Lyell Immunopharma, AstraZeneca, Turnstone Biologics, Xcelcyte and Advaxis Immunotherapies. L.G. is on the scientific advisory board of Poseida Therapeutics and Kiromic, and is a stockholder of Poseida Therapeutics.

Additional information

Peer review information Nature thanks Greg Delgoffe, Stephen Jameson and E. Wherry for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of H9T-expanded CD8+ T cells.

a, Pre-activated mouse CD8+ T cells were rested overnight and cultured with PBS, IL-2, H9, or H9T as indicated for 10 min, and western blotted for phospho-STAT5 (pSTAT5) and pERK. Total STAT5 and ERK were included as controls (on the same gel of pSTAT5 and pERK). Data are representative of two independent experiments. Relative densitometry is shown below each panel (normalized to the IL-2 condition). For gel source data, see Supplementary Fig. 1b. b, Pre-activated CD8+ T cells were rested overnight and stimulated with 10 nM IL-2 or H9T, lysed, and immunoprecipitated with anti-STAT5A or anti-STAT5B antibodies followed with western blotting analysis for pSTAT5 and STAT5A or STAT5B as loading controls (on the same gel). Data are representative of two independent experiments. Relative densitometry is shown below each panel (normalized to the IL-2 condition). For gel source data, see Supplementary Fig. 1c. Data are from two independent repeats. c, Pre-activated CD8+ T cells were rested overnight, cultured with PBS, IL-2, H9, or H9T for 6 days, fixed, permeabilized and intracellularly stained for pSTAT5; n = 4 mice. Data are presented as mean values ± SEM, one-way ANOVA test with Dunnett’s correction. Data are from two independent repeats. d, Pre-activated mouse CD8+ T cells were rested overnight and then cultured in medium containing serial dilutions of IL-2, H9, or H9T for 8 days and analysed for TIM-3 expression. Data are representative of three independent experiments. eh, Pre-activated CD8+ T cells were cultured with 10 nM IL-2, H9, or H9T for 8 days, and surface expression of TIM-3 (e), PD-1 (f), LAG-3 (g), and 2B4 (h) was analysed by flow cytometry. Data are representative of four independent experiments

Source data.

Extended Data Fig. 2 Functional analysis of CD8+ T cells expanded with IL-2, H9 or H9T.

a, b, Expression of CD62L and the percentage of memory-like cells (CD62L+CD44+). Pre-activated mouse CD8+ T cells were cultured with 10 nM IL-2, H9, or H9T for 8 days, and stained for CD44 and CD62L. Data are mean values ± SEM, n = 6 mice, one-way ANOVA test with Dunnett’s correction. Data are representative of three independent experiments. c, Expression of CD62L in 8 day IL-2- or H9T-cultured cells or cells cultured for 6 days in H9T-containing medium and then switched to IL-2-containing medium for 2 days. Pre-activated CD8+ T cells were cultured with 10 nM IL-2 or H9T, and on day 6, a fraction of the H9T-expanded cells was washed and subsequently cultured with 10 nM IL-2. Cells were collected two days later and analysed by flow cytometry. Data are presented as mean values ± SEM, n = 4 mice, one-way ANOVA test with Dunnett’s correction. Data are representative of two independent experiments. dg, Cytokine production and memory population in CD8+ T cells expanded with IL-2, H9, or H9T. Pre-activated CD8+ T cells were expanded for 8 days with IL-2, H9, or H9T, stimulated with 100 nM gp100 or control peptide for 1 h, and then treated with 5 µg/ml Brefeldin A for 5 h. Cells were then fixed for intracellular staining of IFN-γ, n = 5 mice (d); TNF, n = 5 mice (e); IL-2, n = 3 mice (f); and IL-10, n = 5 mice (g); Data are presented as mean values ± SEM, one-way ANOVA test with Dunnett’s correction. Data are representative of two independent experiments

Source data.

Extended Data Fig. 3 RNA-seq and ATAC-seq analysis of CD8+ T cells expanded with IL-2, H9 or H9T.

a, Volcano plots of RNA-seq data from pre-activated mouse CD8+ T cells that were expanded for 6 days with IL-2, H9, or H9T. Shown are gene expression differences between cells expanded with IL-2 versus H9T (a) or H9 versus H9T (b). Data are representative of two independent experiments. c, Differentially expressed genes for CD8+ T cells expanded for 6 days with H9 versus H9T. Data are representative of two independent experiments. d, GSEA analysis of RNA-seq data compared with endogenous memory versus exhausted populations of cells, with Kolmogorov–Smirnov test. Data are representative of two independent experiments. eg, Pre-activated CD8+ T cells were expanded for 8 days with IL-2, H9, or H9T and then either permeabilized for intracellular staining of TCF-1 (e) and BLIMP-1 (f) or analysed for surface expression of CXCR3 (g). Data are representative of three independent experiments. h, i, Expression of CD62L and TIM-3 in control or Tcf7-deficient CD8+ T cells. CD8+ T cells were isolated from Tcf7 conditional knock-out mice (Tcf7 −/−)) or control littermates (Tcf7fl/fl) and activated for 2 days, rested overnight, followed with expansion in IL-2- or H9T-containing medium. Surface staining of CD62L (h) and TIM-3 (i) is shown. Data are mean ± SEM, n = 3 mice, two-sided t-test. Data are representative of two independent experiments. j, Differentially expressed genes from RNA-seq were aligned to ATAC-seq plots. Data are representative of two independent experiments. k, l, ATAC-seq data from CD8+ T cells expanded for 6 days were aligned with in vivo generated effector, exhausted, and memory populations; the ATAC-seq parts of k are also shown in Fig. 2j with ChIP–seq data. Shown is chromatin accessibility at the Havcr2 (k) and Il10 (l) loci. Data are representative of two independent experiments

Source data.

Extended Data Fig. 4 Metabolic profiling of IL-2-, H9- and H9T-expanded CD8+ T cells.

a, Photograph showing the medium colour of mouse CD8+ T cells expanded for 8 days with IL-2, H9, or H9T. Data are representative of two independent experiments. b, c, CD8+ T cells were isolated and expanded for 8 days with IL-2, H9, or H9T, and 5 million cells were collected for metabolomics analysis; data correspond to this of Fig. 3a. Relative levels of glucose (b) and lactate (c) are presented as mean values ± SEM, one-way ANOVA test with Dunnett’s correction, n = 3 mice. Data are representative of two independent experiments. d, 8 day IL-2, H9, or H9T expanded CD8+ T cells were incubated with or without 2-NBDG to assess glucose uptake. Data are presented as mean values ± SEM, one-way ANOVA test with Dunnett’s correction, n = 4 mice. Data are representative of two independent experiments. e, Eight-day IL-2, H9, or H9T expanded CD8+ T cells were incubated with or without TMRM to assess mitochondrial membrane potential. Data are presented as mean values ± SEM, one-way ANOVA test with Dunnett’s correction, n = 8 mice. Data are representative of two independent experiments. fh, Pre-activated CD8+ T cells were treated with control or 1 mM 2-DG in the presence of IL-2 for 2 days. Cells were subsequently stained with antibodies to TCF-1 (n = 3 mice), CD62L (n = 3 mice), or pSTAT5 (n = 6 mice). Data are presented as mean values ± SEM, with two-tailed, paired t-test. Data are representative of two independent experiments. i, PCA plot of RNA-seq data from 2-DG and control treated cells. Pre-activated CD8+ T cells were treated with 10 nM IL-2 with or without 2-DG for 2 days, and RNA was extracted for library preparation. Data are from three mice

Source data.

Extended Data Fig. 5 Comparison of actions of H9T versus natural cytokines in CD8+ T cells.

a, b, Pre-activated mouse (a) or human (b) CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 6 days, and cell density was counted by beads-based flow cytometry. Data are mean ± SEM, one-way ANOVA test with Dunnett’s correction. a, n = 6 mice; b, n=6 donors. Data are representative of two independent experiments. c, Pre-activated mouse CD8+ cells were rested and cultured with 10 nM of the indicated cytokines for 6 days, and TIM-3 expression was examined by flow cytometry. Data are mean ± SEM, one-way ANOVA test with Dunnett’s correction, n = 4 mice. Data are representative of two independent experiments. df, Pre-activated mouse CD8+ T cells were rested overnight and cultured with 10 nM of the indicated cytokines for 0, 0.5, 1, 2, or 4 h. Cells were then fixed, permeabilized, and stained for STAT5-pY694 (d), AKT-pS473 (e) and ERK-pT202/pY204 (f). Data are mean ± SEM, n = 6 mice, Data are representative of two independent experiments. g, h, Pre-activated mouse CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 1 day and collected for RNA-seq. PCA analysis (g) and selected gene expression (h) are shown. Data are combined from two biological repeats

Source data.

Extended Data Fig. 6 Comparison of H9T with natural cytokines in human CD8+ T cells.

a, Pre-activated human CD8+ cells were rested and cultured with 10 nM indicated cytokines for 6 days, and TIM-3 expression was examined. Data are mean ± SEM, one-way ANOVA test with Dunnett’s correction, = 6 donors. Data are from two independent experiments. bd, Pre-activated human CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 0, 0.5, 1, 2 and 4 h and stained with anti-pSTAT5, pAKT, and pERK. Data are mean ± SEM, = 6 donors. Data are from two independent experiments. ei, Pre-activated human CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 24 h and cells were collected for RNA-seq. Selected genes expression (e), PCA analysis (f) and GSEA were shown, Kolmogorov–Smirnov test. Data are from three donors. jl, Pre-activated human CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 6 days, and stained with anti-CD27, CCR7 and Granzyme B antibodies. Data are mean ± SEM, one-way ANOVA test with Dunnett’s correction, = 6 donors. Data are from two independent experiments. m, Pre-activated human CD8+ cells were rested and cultured with 10 nM of the indicated cytokines for 6 days, followed by TMRM analysis. Data are mean ± SEM, one-way ANOVA test with Dunnett’s correction, n = 5 donors. Data are from two independent experiments. n, Pre-activated human CD8+ T cells were rested and cultured with 1 nM IL-2 alone or in the presence of 1, 10 and 100 nM H9T or IL-15. TIM-3 expression was analysed by flow cytometry after 2 days. Data are mean ± SEM, one-way ANOVA test with Dunnett’s correction, n = 5 donors. Data are from two independent experiments

Source data.

Extended Data Fig. 7 Dose response of IL-2, IL-15 and H9T in CD8+ T cells.

ac, Dose response of IL-2, IL-15, and H9T in human CD8+ T cells. Pre-activated human CD8+ cells were rested and cultured with 0-100 nM of IL-2, IL-15, or H9T for 6 days and stained for surface expression of TIM-3 (a) or permeabilized and stained for intracellular granzyme B (b). Cell expansion rate (c) was assessed using flow cytometry based counting beads. Data are mean ± SEM, n=2 donors. Data are representative of two independent experiments. df, mRNA levels of TCF7, CD27 and SLC2A1 in human CD8+ T cells. Pre-activated human CD8+ cells were rested and stimulated with 1 nM of IL-2, IL-15, or H9T for 24 h. Cells were collected and mRNA extracted for qPCR analysis of TCF7 (d), CD27 (e) and SLC2A1 (f). The mRNA expression was normalized to that of RPLP0. Data are representative of two independent experiments

Source data.

Extended Data Fig. 8 Effects of H9T on human CD4+ T cells.

ad, Phenotypic analysis of expanded human CD4+ T cells. Pre-activated human CD4+ cells were cultured with 10 nM H9T, IL-2, IL-15, or IL-7 + IL-15 for 6 days, and mitochondrial membrane potential (a) and surface expression of TIM-3 (b), CCR7(c), and CD27 (d) were examined by flow cytometry analysis after staining. Data are presented as mean values ± SEM, n = 5 donors, with paired two-sided t-test. Data are representative of two independent experiments

Source data.

Extended Data Fig. 9 Effects of STAT5 activation on T cell exhaustion and stemness.

a, ChIP-seq analysis of STAT5A- and STAT5B-binding sites at the Havcr2 locus from previously published datasets (GSE36890). b, c, TIM-3 expression on CD8+ T cells from Stat5a (b) and Stat5b (c) knock-out mice versus wild-type littermate controls. Cells were expanded for 8 days with 10 nM of IL-2 as described above, and TIM-3 expression analysed by flow cytometry. Data are mean ± SEM, n = 4 mice, two-sided t-test. Data are from two independent repeats. d, ChIP–seq analysis of STAT5-binding sites at HAVCR, GZMB, TCF7, SLC2A1 and SLC2A3 loci. Human CD8+ T cells were pre-activated with anti-CD3/anti-CD28 beads for 2 days, rested overnight, incubated with 10 nM IL-2 or H9T for 2 h, and then fixed and lysed for ChIP–seq. Data are from two independent experiments. eh, Three days after retroviral transduction, mouse CD8+ T cells expressing empty vector (EV)-GFP or STAT5A-1*6 vector-GFP were sorted and cultured in H9T-containing medium for an additional four days prior to staining with the indicated antibodies. Data are representative of two independent experiments. im, RNA-seq analysis of the effects of STAT5A-1*6 expression. Mouse CD8+ T cells were treated as above, and cells were collected for RNA-seq. PCA analysis (i) and expression of selected genes (j) are shown. GSEA analysis of IL-2-STAT5 signalling (k), PI3K-AKT-mTOR signalling (l) and exhaustion versus memory (m) are also shown, Kolmogorov–Smirnov test. Data are from two biological repeats. np, ATAC-seq analysis of the effect of STAT5A-1*6 expression. Mouse CD8+ T cells were treated as described above, and cells were collected for ATAC-seq. Shown are PCA analysis to compare IL-2-, H9-, and H9T-expanded cells (n), and ATAC-seq data at the Havcr2 (o) and Tcf7 (p) loci

Source data.

Extended Data Fig. 10 Efficacy of H9T in adoptive cell immunotherapy.

a, Tumour growth after transfer of pmel-1 cells that expanded with IL-2, H9 or H9T for 8 days into B16 melanoma-bearing mice, with PBS as a control. n = 14 for H9 group and n = 15 mice for all other groups; data are from three independent repeats. b, Blood cells from mice cured of B16 melanoma tumour after adoptive transfer of H9T-expanded CD8+ pmel-1 cells were stained for CD8 and CD90.1. Data are from two independent experiments. c, d, IL-2- or H9T-expanded CD8+ pmel-1 cells were transferred into B16 melanoma-bearing mice, with PBS as a control. Mice were irradiated one day before cell transfer but not injected i.p. with IL-2 after cell transfer (no IP) or not irradiated but injected with 180,000 IU IL-2 i.p. daily for 3 days beginning on the day of transfer (no IR). Data are mean ± SEM, n = 5 mice. Data are from two independent repeats. eg, TIM-3 and PD1 profiling of pmel-1 cells in tumour and draining lymph nodes 7 days after adoptive transfer. Data are mean ± SEM, n = 5 mice, one-way ANOVA test with Dunnett’s correction. Gating strategy is shown (g). Data are from three independent repeats. h, B16 tumour size 8 days after pmel-1 CD8+ T cells infusion. Data are mean ± SEM, n = 5 mice. Data are from three independent repeats. i, j, Phenotype of pmel-1 cells in tumour and draining lymph nodes 5 or 10 days after adoptive transfer. Data are from two independent experiments. km, Seven days after adoptive transfer, CD8+CD90.1+ cells was sorted from draining lymph nodes and analysed by RNA-seq. Selected gene expression (k, l) and GSEA analysis of memory versus exhausted cells (m) with Kolmogorov–Smirnov test are shown. Data are from two independent repeats

Source data.

Supplementary information

Supplementary Figures

This file contains Supplementary Figs. 1 and 2. Supplementary Fig. 1 contains the uncropped western blots and Supplementary Fig. 2 shows the flow cytometry gating strategies.

Reporting Summary

Supplementary Table 1

All RefSeq genes from RNA-seq data related to Fig. 2a.

Supplementary Table 2

Differentially expressed genes between IL-2 and H9T. Related to Fig. 2b, c, and Extended Data Fig. 3a.

Supplementary Table 3

Differentially expressed genes between H9 and H9T. Related to Fig. 2c and Extended Data Fig. 3b, c.

Supplementary Table 4

ATAC-seq peaks of mouse CD8+ T cells expanded for 6 days as indicated. Related to Fig. 2h–j.

Supplementary Table 5

Significant metabolites. Related to Fig. 3a.

Supplementary Table 6

All detected metabolites. Related to Fig. 3b.

Supplementary Table 7

RNA-seq of mouse CD8+ T cells treated with IL-2 or IL-2 plus 2-DG for 2 days. Related to Fig. 3m.

Supplementary Table 8

RNA-seq of mouse CD8+ T cells treated with H9T, IL-2, IL-15 or IL-7 + 15 for 24 hours. Related to Extended Data Fig. 5h.

Supplementary Table 9

RNA-seq of human CD8+ T cells treated with H9T, IL-2, IL-15 or IL-7 + 15 for 24 hours. Related to Extended Data Fig. 6e.

Supplementary Table 10

RNA-seq of mouse CD8+ T cells overexpressing STAT5A-1*6 or a control vector. Related to Extended Data Fig. 9j.

Supplementary Table 11

ATAC-seq of mouse CD8+ T cells overexpressing STAT5A-1*6 or a control vector. Related to Extended Data Fig. 9n.

Supplementary Table 12

RNA-seq of CD90.1+ mouse CD8+ T cells purified from draining lymph nodes 7 days after transfer. Related to Extended Data Fig. 10k.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mo, F., Yu, Z., Li, P. et al. An engineered IL-2 partial agonist promotes CD8+ T cell stemness. Nature 597, 544–548 (2021). https://doi.org/10.1038/s41586-021-03861-0

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing