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Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism

Abstract

CD8+ T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment1,2,3,4. Reactivating the cytotoxicity of CD8+ T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8+ T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme5, led to potentiated effector function and enhanced proliferation of CD8+ but not CD4+ T cells. This is due to the increase in the plasma membrane cholesterol level of CD8+ T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8+ T cells were better than wild-type CD8+ T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile6,7, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

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Figure 1: Inhibiting cholesterol esterification potentiates CD8+ T-cell effector function.
Figure 2: ACAT1 deficiency potentiates the antitumour activity of CD8+ T cells.
Figure 3: Plasma membrane cholesterol modulates TCR clustering and immunological synapse formation.
Figure 4: Cancer immunotherapies in mice with the ACAT inhibitor avasimibe.

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Acknowledgements

We thank Q. Leng for providing L. monocytogenes, F.-J. Nan for providing K604, and Y. Jiang for some preliminary experiments. We thank H. Gu and D. Li for careful reading of the manuscript. Imaging work was performed at the National Center for Protein Science Shanghai. Chenqi Xu is funded by MOST (2011CB910901 and 2012CB910804), NSFC grants (31370860, 31425009 and 31530022), and CAS grants (Strategic Priority Research Program XDB08020100; KSCX2-EW-J-11). B.L. is funded by MOST (2011CB910901) and NSFC grant 31271377. W.Y. is funded by NSFC grant (31400745) and China Postdoctoral Science Foundation (2014M561533 and 2014T70440). T.-Y.C. and C.C.Y.C. are funded by NIH grant HL 60306.

Author information

Authors and Affiliations

Authors

Contributions

Chenqi Xu conceived the project. B.L., X.L., S.S., B.-.L.S., W.Y. and Y.X. contributed to the design of the project and extensive discussions. P.Z. provided technical help on the tumour models. T.-Y.C. and C.C.Y.C. generated Acat1flox/flox mice. W.Y., Y.B., X.Z., J.Z., X.M. and L.L. performed the ex vivo T-cell experiments and animal experiments. W.Y. and S.C. performed the STORM experiments. Y.B. performed the TIRFM experiments. T.Z. provided human cells. J.Z. and T.Z. performed the human cell experiments. W.L., J.W. and Chenguang Xu helped with the TIRFM setup and data analysis. L.W. helped with the cholesterol staining and quantification. Chenqi Xu, W.Y. and Y.B. wrote the manuscript. Other authors revised the manuscript.

Corresponding authors

Correspondence to Bo-liang Li or Chenqi Xu.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Reprogramming of cellular cholesterol metabolism in activated CD8+ T cells.

a, Filipin III staining (left) and quantification (right) of cellular cholesterol of naive and activated CD8+ T cells stimulated by 5 μg ml−1 plate-bound anti-CD3/CD28 antibodies for 12 h (n = 60). b, Total cellular, plasma membrane and intracellular cholesterol quantified using the cholesterol oxidation-based method (n = 4). c, Relative plasma membrane cholesterol quantified using the biotinylation-based method (n = 4). df, Transcriptional levels of key genes encoding molecules involved in cholesterol synthesis, transport and efflux (n = 3). CD8+ T cells were stimulated with 5 μg ml−1 plate-bound anti-CD3/CD28 antibodies. Results and statistical analysis are relative to quiescent CD8+ T cells. mRNA levels of cholesterol biosynthesis genes, including Srebp1 (also known as Srebf1), Srebp2 (Srebf2), Hmgcr, Hmgcs, Fasn, Acaca and Sqle, were upregulated in activated CD8+ T cells. Ldlr, which encodes the LDL receptor, a major receptor for cholesterol transport, was upregulated in activated CD8+ T cells, whereas, Idol (also known as Mylip), which encodes IDOL, an inducible degrader of the LDL receptor, was downregulated. Cholesterol efflux genes, including Abca1 and Abcg1, were all downregulated in activated CD8+ T cells. gi, Cytokine/granule productions of CD8+ T cells after modulation of cholesterol metabolic pathways (n = 3). Naive CD8+ T cells were pretreated for 6 h with vehicle (DMSO), lovastatin (to inhibit cholesterol biosynthesis) or U18666A (a cholesterol transport inhibitor with pleotropic effects), respectively. Cells were then stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h before intracellular staining. Representative flow cytometric profiles shown in g. Data are representative of two independent experiments, and were analysed by Mann–Whitney test (a) or unpaired t-test (bf, h, i). Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Extended Data Figure 2 ACAT1 deficiency affects the cholesterol metabolism but not the basal energy metabolism of CD8+ T cells.

a, ACAT2 was weakly expressed in mouse CD4+ T cells but was barely detectable in mouse CD8+ T cells. A sample from mouse small intestine was used as a positive control. b, Protein levels of ACAT1 were significantly lower in Acat1CKO (CKO) CD8+ T cells than in wild-type cells, indicating a good knockout efficiency of Acat1 in CD8+ T cells. See Supplementary Fig. 1 for gel source data. c, d, ACAT1 deficiency did not change the transcriptional level of Acat2 in CD4+ and CD8+ T cells of Acat1CKO mice. Cells were stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for the indicated time (n = 3). e, ACAT1 deficiency resulted in significant enhancement of the transcription levels of cholesterol synthesis genes in both naive and activated cells. Transcription levels of cholesterol transport and efflux genes underwent only modest changes in CKO CD8+ T cells. Naive wild-type or CKO CD8+ T cells were stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 for the indicated time (n = 3). f, g, Basal energy metabolism of naive wild-type and CKO CD8+ T cells was measured. No significant difference was observed between wild-type and CKO cells. f, Oxidative phosphorylation was measured by the oxygen consumption rates (OCR) under basal condition, and glycolysis was measured by the extracellular acidification rates (ECAR) under basal condition (n = 5). g, Fatty acid oxidation was measured by the OCR under basal condition and in response to indicated drugs: oligomycin (to block ATP synthesis), FCCP (to uncouple ATP synthesis from the electron transport chain), rotenone and antimycin A (to block complex I and III of the electron transport chain, respectively), and etomoxir (to block mitochondrial fatty acid oxidation). Fatty acid oxidation can be represented by the influence of etomoxir on the OCR. Between wild-type etomoxir and CKO etomoxir: P > 0.05 after FCCP in g (left); P > 0.05 after etomoxir in g (right). Between wild-type and CKO: P > 0.05 after FCCP in g (left): P > 0.05 after control in g (right). No significant difference was observed (n = 3). Data are representative of two (ce, g) or three (f) independent experiments, and were analysed by unpaired t-test (cf) or two-way ANOVA followed with Bonferroni’s multiple comparison tests (g). Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 3 ACAT1 deficiency does not affect thymocyte development and peripheral T-cell homeostasis, but results in enhanced proliferation and reduced apoptosis of CD8+ T cells.

a, b, Flow cytometric analysis of thymocytes and splenic T cells from wild-type and CKO mice (8 weeks old, n = 4). Representative flow cytometric profiles were shown in a. Percentages of CD4 CD8 double negative (DN), CD4+ CD8+ double positive (DP), CD4+ single positive (CD4SP) and CD8+ single positive (CD8SP) cells in total thymocytes were comparable (b, left). Naive (CD44loCD62Lhi), central memory (CD44hiCD62Lhi; CM) and effector/effector memory (CD44hi CD62Llo, effector/EM) of CD4+ and CD8+ T cells from the spleen of wild-type and CKO mice were comparable (b, right). Data were analysed by Mann–Whitney test, and no significant difference was observed. c, Total CD4+ and CD8+ T-cell numbers from the spleen of wild-type and CKO mice (8 weeks old, n = 6) were assessed using flow cytometry. Data were analysed with Mann–Whitney test, and no significant difference was observed. df, Cytokine/granule productions of resting naive (CD62LhiCD44lo) and central memory (CD62LhiCD44hi) CD8+ T cells from the spleen of wild-type and CKO mice (8 weeks old, n = 3). CD8+ T cells were isolated from the spleen and cultured for 4 h in the presence of 5 μg ml−1 brefeldin A. Naive and memory populations were gated by CD62L and CD44 expression. Data were analysed by unpaired t-test, and no significant difference was observed. g, h, Serum levels of IFNγ and auto-antibody anti-dsDNA IgG of wild-type and CKO mice (12 weeks old, WT, n = 7; CKO, n = 6) were assessed using ELISA. Data were analysed by Mann–Whitney test, and no significant difference was observed. i, j, T-cell homeostasis was measured by BrdU labelling and detection. Wild-type and CKO mice (6 weeks old) were injected with a single dose (2 mg) of BrdU intraperitoneally. Peripheral blood was collected at the indicated time and analysed using flow cytometry. Percentages of BrdU+ cells in total peripheral CD4+ and CD8+ T cells of wild-type and CKO mice (n = 6) were plotted. Data were analysed by two-way ANOVA, and no significant difference was observed. k, l, CD8+ T-cell proliferation was measured by CFSE dilution. Cells were stimulated with 1–2 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for the indicated time. Data were analysed by two-way ANOVA (n = 3). m, n, CD8+ T-cell apoptosis was measured by annexin V and propidium iodide (PI) staining. The naive CD8+ cells were isolated from the spleen of wild-type or CKO mice (8 weeks old), and cultured in medium for 24 h without stimulation, or stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. Annexin V and propidium iodide were used to stain early (annexin V+ PI) and late (annexin V+ PI+) cells apoptotic. Apoptotic cells were significantly lower in CKO CD8+ T cells than in wild-type CD8+ T cells. Data were analysed by unpaired t-test (n = 3). Error bars denote s.e.m; *P < 0.05; **P < 0.01.

Extended Data Figure 4 ACAT1 deficiency does not result in significant change of CD4+ T-cell function.

a, b, Cytokine productions of CD4+ T cells (n = 3). Cells were stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for 12 h. Representative flow cytometric profiles are shown in a. ce, Relative transcription levels of Acat1 and Acat2 in naive CD4+ and CD8+ T cells freshly isolated from C57BL/6 mice (n = 3). Acat1 transcription level was significantly higher than Acat2 in CD4+ T cells. Acat1 transcription levels were comparable between CD4+ and CD8+ T cells, whereas the Acat2 transcription level in CD4+ T cells was significantly higher than that in CD8+ T cells. Acat2 transcription level in CD4+ T cells was set as 1 in c. Acat1 and Acat2 transcription levels in CD8+ T cells were set as 1 in d and e. f, Filipin III staining to analyse cellular cholesterol distribution in naive and activated CD4+ T cells from wild-type and CKO mice. Data were analysed by unpaired t-test (be) or Mann–Whitney test (f). Error bars denote s.e.m; *P < 0.05; ***P < 0.001.

Extended Data Figure 5 ACAT1 deficiency promotes CD8+ T-cell response but does not result in autoreactivity to self-antigens.

ae, Listeria monocytogenes was used to infect wild-type and CKO mice to induce a strong T-cell response. a, IFNγ production of CD8+ T cells from wild-type and CKO mice infected (day 7 after infection) with Listeria monocytogenes (LM) that exogenously express OVA antigen, or were uninfected (UI). The splenocytes were re-stimulated with PMA (50 ng ml−1) plus ionomycin (1 μM) for 4 h in the presence of 5 μg ml−1 brefeldin A. CD8+ IFNγ+ cells were gated for analysis (UI, n = 3; LM, n = 4). b, IFNγ production of OVA-specific CD8+ T cells from the wild-type and CKO mice infected (day 7 after infection) with Listeria monocytogenes that exogenously express OVA antigen or uninfected. Splenocytes were stimulated with OVA257–264 peptide for 24 h. CD8+ IFNγ+ cells were gated for analysis (UI, n = 5; LM, n = 6). c, IFNγ levels in serum of infected or uninfected mice were assessed by ELISA (UI, n = 5; LM WT, n = 8; LM CKO, n = 6). d, Liver Listeria monocytogenes titre was analysed at day 6 after infection (n = 6). e, Percentages of IFNγ+ cells in CD4+ cells from the spleens of wild-type and CKO mice were assessed as in a (WT, n = 4; CKO, n = 5). f, g, Effect of ACAT1 deficiency on CD8+ T-cell responses to different antigens. f, Naive wild-type OT-I or Acat1CKO OT-I (CKO OT-I) CD8+ T cells were stimulated with autologous splenocytes pulsed with foreign antigen (N4, A2, T4 or G4), positive-selection-supporting antigen (R4) or self-antigen (Catnb). Flow cytometry was used to measure IFNγ production (n = 4). g, Splenocytes from wild-type OT-I or CKO OT-I mice were stimulated with OVA257–264 to generate mature CTLs. CTLs were incubated with EL-4 cells pulsed with different antigens for 4 h, and LDH release was measured to assess cytotoxic efficiency (n = 4). Data are representative of three (f, g) independent experiments, and were analysed by Mann–Whitney test (ae) or unpaired t-test (f, g). Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 6 ACAT1 deficiency promotes antitumour response of CD8+ T cells in different tumour models.

ac, B16F10 melanoma cells (2 × 105) were subcutaneously injected into wild-type (n = 6) or CKO (n = 8) mice to induce skin melanoma. On day 7, CD8+ T cells were isolated from draining lymph nodes of wild-type and CKO mice. Flow cytometry was used to analyse surface expression of activation marker CD44 and IFNγ production of CD8+ T cells, as well as CD8+ T-cell number and CD8/CD4 T-cell ratio. Data are representative of three independent experiments, and were analysed by Mann–Whitney test. di, B16F10 melanoma cells (2 × 105) were intravenously injected into wild-type or CKO mice to induce melanoma with lung metastasis. d, e, On day 20, lungs were isolated to count tumour numbers (WT, n = 9; CKO, n = 8). Data are representative of three independent experiments, and were analysed by Mann–Whitney test. f, On day 14, lung sections were stained with haematoxylin and eosin to assess the infiltration of melanoma into lung. g, Survival was analysed by log-rank (Mantel–Cox) test (WT, n = 11; CKO, n = 14). h, i, On day 20, lung infiltrating CD8+ T cells were isolated and flow cytrometry was used to measure the granule and cytokine productions as well as surface expression of the activation marker CD44. Data were analysed by Mann–Whitney test. jl, Lewis lung carcinoma cells (2 × 106) were intravenously injected into wild-type or CKO mice to induce lung cancer. On day 35, lungs were isolated to count tumour numbers. Tumour multiplicity data were analysed by Mann–Whitney test (k, n = 7). Survival was analysed by log-rank (Mantel–Cox) test (l, WT, n = 11; CKO, n = 13). Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 7 Cholesterol level of the plasma membrane directly affects CD8+ T-cell function.

To reduce the cholesterol level of the plasma membrane, CD8+ T cells were treated with MβCD at different doses for 5 min. To increase the cholesterol level, CD8+ T cells were treated with MβCD-coated cholesterol (chol) at different doses for 15 min. a, b, Measurements of plasma membrane cholesterol level of CD8+ T cells by biotinylation-based method. MβCD-coated cholesterol treatment increased whereas MβCD decreased plasma membrane cholesterol. a, Wild-type OT-I CTLs were treated with 10 μg ml−1 MβCD-coated cholesterol, and CKO OT-I CTLs were treated with 1 mM MβCD. WT OT-I, n = 8; CKO OT-I, n = 6. b, Naive wild-type polyclonal CD8+ T cells were treated with 10 μg ml−1 MβCD-coated cholesterol (n = 4). c, d, TCR levels of CD8+ T cells after MβCD or MβCD-coated cholesterol treatment. MβCD treatment reduced whereas MβCD-coated cholesterol treatment did not change the surface TCR level. OT-I CTLs (c) and naive wild-type polyclonal CD8+ T cells (d) were treated as in a and b, respectively. IC, isotype control. eg, TCR clustering after treatment of naive wild-type CD8+ T cells with 10 μg ml−1 MβCD-coated cholesterol. Super-resolution STORM images of TCR were acquired and analysed as Fig. 3g–i. Increasing plasma membrane cholesterol of CD8+ T cells promoted the clustering of TCR. g, WT, n = 73; WT + chol, n = 62. h, TCR signalling after treatment of wild-type OT-I CTLs with 10 μg ml−1 MβCD-coated cholesterol, measured by immunoblotting. Cells were then stimulated with 2 μg ml−1 anti-CD3 and anti-CD28 antibodies for indicated time at 37 °C. Increasing plasma membrane cholesterol of CD8+ T cells promoted the signalling of TCR. See Supplementary Fig. 1 for gel source data. i, j, Cytokine/granule productions of CD8+ T cells treated with MβCD (i) or MβCD-coated cholesterol (j) (n = 3). Cells were stimulated with 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h at 37 °C. k, l, Cytotoxicity of CD8+ T cells treated with MβCD (k) or MβCD-coated cholesterol (l) (n = 3). CTLs were then incubated with EL-4 cells pulsed with OVA257–264 for 4 h. LDH release was measured to assess cytotoxic efficiency. Data were analysed by unpaired t-test (a, b, il) or Mann–Whitney test (g). Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 8 Homing of naive wild-type or CKO OT-I T cells to secondary lymphoid organs in the B16-OVA melanoma-bearing mice.

Naive OT-I T cells were isolated from wild-type or CKO OT-I TCR transgenic mice, and labelled with CTDR (cell tracker deep red dye) or CFSE (carboxyfluorescein succinimidyl ester), respectively. Labelled wild-type and CKO cells were mixed at 1:1 ratio, and the mixture (107 cells) was intravenously injected into the B16-OVA melanoma bearing C57BL/6 mice. After 12 h, the indicated tissues from the mice were isolated and the percentages of the labelled cells were assessed using flow cytometry. a, Flow cytometric analysis of the homing receptor CCR7 and CD62L surface level of naive wild-type and CKO OT-I T cells. No significant difference was observed. b, The percentages of transferred cells in total CD8+ T cells were assessed using flow cytometry. Data were analysed with Mann–Whitney test (n = 11). Data are representative of two independent experiments. Error bars denote s.e.m; *P < 0.05; **P < 0.01.

Extended Data Figure 9 Avasimibe treatment leads to enhanced TCR clustering and signalling, as well as more efficient formation of immunological synapse.

a, Cytokine/granule productions of CD8+ T cells after avasimibe treatment (n = 3). The naive cells were pretreated for 6 h with avasimibe or vehicle (DMSO) and then stimulated by 5 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. Data were analysed by t-test. b, CTL cytotoxicity after avasimibe treatment measured by the LDH assay (n = 3). OT-I CTLs were pretreated with avasimibe or vehicle for 6 h and then incubated with EL-4 cells pulsed with OVA257–264 peptide for 4 h. Data were analysed by t-test. c, An MTS-based cell viability assay was performed to assess the toxicity of avasimibe to B16F10 cells (n = 6). Data were analysed by one-way ANOVA, and no significant difference was observed. d, e, Filipin III staining to analyse cellular cholesterol distribution in naive CD8+ T cells treated with avasimibe or vehicle. d, Representative images. e, Data were analysed by Mann–Whitney test. 0, n = 217; 0.5, n = 139; 1, n = 133. fh, Super-resolution STORM images of TCR in naive CD8+ T cells treated with avasimibe or vehicle. f, Representative images. g, Ripley’s K-function was used to analyse TCR molecules distribution. h, The r value at the maximal L(r) − r value of Ripley’s K-function curves, and data were analysed by Mann–Whitney test. 0, n = 41; 0.5, n = 60; 1, n = 66. i, Representative TIRFM images of immunological synapses of CD8+ T cells treated with avasimibe (1 μM) or vehicle for 6 h. Cells were stimulated by PLB-bound anti-CD3 for the indicated time and fixed by 4% PFA before imaging. j, Areas of the immunological synapses (n > 60 cells). The formation and contraction of the immunological synapses of CD8+ T cells treated with avasimibe were more rapid than those treated with vehicle. Data were analysed by two-way ANOVA. k, TCR proximal and downstream signalling was assessed using immunoblotting of protein phosphorylation. OT-I CTLs were treated with 1 μM avasimibe or vehicle for 6 h and then stimulated with 2 μg ml−1 soluble anti-CD3 and anti-CD28 for the indicated time. See Supplementary Fig. 1 for gel source data. Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 10 Avasimibe treatment leads to potentiated effector function and enhanced proliferation of tumour-infiltrating CD8+ T cells.

C57BL/6 mice bearing B16F10 melanoma were treated with avasimibe (15 mg kg−1) or same dose of DMSO in PBS for four times by intraperitoneal injection. The mice were euthanized at day 18 and the tumours were isolated. a, The CD44 surface expression and cytokine/granule productions of tumour-infiltrating CD8+ T cells were assessed using flow cytometry (n = 6). b, CD8+ T-cell number, CD8+/CD4+ T-cell ratio and Ki-67 level of tumour-infiltrating CD8+ T cells were assessed using flow cytometry (n = 7). c, Percentages of CD8+ central memory (CD44hiCD62Lhi) and CD8+ effector/effectory memory (CD44hi CD62Llo) were assessed using flow cytometry (control, n = 12; avasimibe, n = 11). d, Surface levels of PD-1, CTLA-4 and TCR of tumour-infiltrating CD8+ T cells. PD-1 level was indicated by the percentage of PD-1hi cells. CTLA-4 and TCR levels were indicated by median fluorescence intensity. PD-1 and CLTA-4 staining, n = 7; TCR staining, n = 5. e, Treg cell (FoxP3+ CD4+) and myeloid-derived suppressor cell (MDSC) (Gr1+ CD11b+) percentages in the tumour microenvironment were assessed using flow cytometry (n = 7). Data were analysed by Mann–Whitney test. Error bars denote s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary information

Supplementary Figure 1

This file contains the gel source data for Figure 3f and Extended Data Figures 2a, b 7h, 9k. (PDF 748 kb)

Imaging of TCR during the formation of immunological synapse of Acat1CKO or WT CD8+ T cells

A CD8+ T cell from spleen of Acat1CKO or littermate WT mouse, labeled with Alexa568 α-mTCRβ Fab, was imaged at the interface with planar lipid bilayer containing α-mCD3ε by total internal reflection fluorescence microscopy. The acquisition rate of the image is 0.33 frame per second (fps), and the playback rate is 30 fps. (MOV 5651 kb)

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Yang, W., Bai, Y., Xiong, Y. et al. Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism. Nature 531, 651–655 (2016). https://doi.org/10.1038/nature17412

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