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Radiation-activated secretory proteins of Scgb1a1+ club cells increase the efficacy of immune checkpoint blockade in lung cancer

Abstract

Radiation therapy (RT) in combination with an immune checkpoint inhibitor (ICI) represents a promising regimen for non-small cell lung cancer (NSCLC); however, the underlying mechanisms are poorly characterized. We identified a specific dose of RT that conferred tumor regression and improved survival in NSCLC models when combined with ICIs. The immune-modulating functions of RT were ascribed to activated lung-resident Scgb1a1+ club cells. Notably, mice with club-cell-specific knockout of synaptosome-associated protein 23 failed to benefit from the combination treatment, indicating a pivotal role of club cell secretome. We identified eight club cell secretory proteins that inhibited immunosuppressive myeloid cells, reduced pro-tumor inflammation and enhanced antitumor immunity. Notably, CC10, a member of club cell secretome, was increased in plasma of patients with NSCLC responding to the combination therapy. By revealing an immunoregulatory role of club cells, our studies have the potential to guide future clinical trials of ICIs in NSCLC.

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Fig. 1: A specific dose of RT enhances efficacy of PD-1 inhibition in HKP1 NSCLC model.
Fig. 2: Does of 4 Gy-RT increases T-cell infiltration and activation in HKP1-bearing lungs.
Fig. 3: Treatment with 4 Gy-RT activates club cells in the lung microenvironment.
Fig. 4: Club cells contribute to the efficacy of the combination treatment.
Fig. 5: Club cell secretome contributes to the efficacy of the combination treatment.
Fig. 6: Club cells contribute to the immune landscape in HKP1 mice treated with combination therapy.
Fig. 7: Club secretory proteins reduce immunosuppressive functions of MDSCs.
Fig. 8: Intranasal administration of club cocktail improves therapeutic efficacy of PD-1 antibody in vivo.

Data availability

Bulk RNA-seq and scRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE157883. Source data have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Xiang of the Genomics Resources Core Facility and J. McCormick of the Flow Cytometry Core Facility for their professional advice. We thank the Formenti and Demaria labs for providing expertise and guidance on the use of focal tumor radiation. The small animal irradiator was obtained with the support of NIH S10 RR027619-01. We thank C. Spinelli, J. Gakuria, A. Nasar and M. Malbari for clinical data support. We also thank X. Lin of Rutgers University for statistical consultation. This work was supported in part by National Cancer Institute grant National Institutes of Health (NIH) U01 CA188388 (V.M. and S.T.W.), 1UG3 CA244697 (V.M and N.K.A). Y.B. was supported by NIH R01 CA244413. G.J.M. was supported by postdoctoral fellowships of National Cancer Institute T32 CA203702, NIH CTSC KL2-TR-002385 and PhRMA Foundation 2020 Postdoctoral fellowship in Translational Medicine. This work was also supported by funds from The Neuberger Berman Foundation Lung Cancer Research Center; a generous gift from Jay and Vicky Furman; and generous funds donated by patients in the Division of Thoracic Surgery to N.K.A. The funding organizations played no role in experimental design, data analysis or manuscript preparation.

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Contributions

Y.B., D.G. and V.M. designed the experiments. Y.B. and Y.Z. performed the experiments. G.J.M., S.L., J.K. and D.R. provided suggestions and technical support for experiments. G.J.M. supervised statistics analyses. V.M., D.G. and N.K.A. supervised this study. D.G., J.S. and S.W. performed bulk and scRNA-seq analyses. Y.B. wrote the manuscript. V.M., D.G., G.J.M. and N.K.A. edited the manuscript with input from other authors. All authors discussed the results and conclusions drawn from the studies.

Corresponding authors

Correspondence to Nasser K. Altorki, Dingcheng Gao or Vivek Mittal.

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The authors declare no competing interests.

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Peer review information Nature Cancer thanks Georgios Stathopoulos, Ralph Weichselbaum 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 Representative tiled immunofluorescent images of HKP1 lungs.

a, Stitched microscopy images (10x) showing impact of 4Gy-RT on the infiltration of CD8+ T cells in tumor islets 1 day after last dose of RT. Tiled images (1.6mm X 1.6mm) are shown. CD8 (Green) and E-cadherin (Red) were stained. Tumor (T) and Adjacent lung tissue (A) are separated by dashed lines. n=9 sections from 4 mice/treatment were evaluated. b. Example of gating strategy of flow-cytometric analyses of T cells. Flow plots were sequentially gated as cells, single cells, CD3+ cells, CD4+ or CD8+ ; in CD4+ or CD8+ sub-gate, cytokine+ cells were gated based on unstained controls, followed by applying same gates to all the samples. This gating strategy was employed for Fig. 2b–f, Fig. 4c (middle and right panels), Fig. 5c (middle and right panels), Extended Data Fig. 2e, Extended Data Fig. 3c, Extended Data Fig. 6d and Extended Data Fig. 8d.

Extended Data Fig. 2 FTY720 abrogated immune-activating responses induced by 4Gy-RT.

a, Histogram plots (right) and quantitation (left) showing an increase in % CD3+ T cells in HKP1-bearing lungs in comparison to naïve lungs. Representative of n=3 individual mice b, Cytokine production (IFNγ and TNF-α) by CD4+ T cells in TME at Day 7,13, and 21 after tumor implantation. Representative of n=3 lungs. c, Treatment and analysis scheme for HKP1 mice. d, Quantification of circulating T cells. Blood samples were obtained via submandibular bleeding, and lymph nodes collected from same mice served as controls. Cell counts/ 100,000 single flow events. Representative of n=3 mice. Unpaired two-tailed Student’s t-test, ****P< 0.0001. e, Flow-cytometric analyses of cytokine productions by T cells. Right, representative flow-cytometric plots/histogram. Left, quantitation of % cytokine+ T cells in the HKP1 lungs. Representative of n=3 lungs. One-way ANOVA with Sidak multicomparison, ****P< 0.0001. f, Tumor growth curves of mouse cohorts treated with PBS or FTY720 as indicated. All the mice (n=5) in f were also received 4 Gy-RT and α-PD-1 antibody. Two-way ANOVA with Sidak’s post hoc test. ****P< 0.0001.

Source data

Extended Data Fig. 3 Sustained effector phenotypes of T cells after combination treatment.

a, Histograms of PD-1 expression by cytokine+ vs. cytokine- T cells (left: CD4+ and right: CD8+) in HKP1-bearing lungs. Representative of n=5 mice. b, Representative Immunofluorescent images of HKP1 lungs showing infiltration of CD8+ T cells into tumor islets at Day7 post-RT in combination with IgG or α-PD-1 antibody. CD8 (Green) and E-cadherin (Red) were stained. Tumor (T) and Adjacent lung tissue (A) are separated by dashed lines. Scale bar: 20µM. n=9 sections from 3 mice/treatment. c, Flow cytometry plots showing the production of IFNγ and TNF-α by CD4+ T cells (left) and GzmB expression by CD8+ T cells (right) in HKP1 lungs in response to therapies at Day7 post-RT. Representative of n=5 mice. d, Flow cytometry analysis of central memory T cells in lymphoid tissues. Spleen and tumor-draining lymph nodes were collected from mice treated with mock, 4Gy-RT or 4Gy-RT in combination with α-PD1 antibody. Cells were stained with a-CD44, a-CD62L and a-CCR7 antibodies for central memory T cells. Upper:Flow events were gated sequentially as cells, single cells, CD3+, CD44+/CD62L+, and CCR7+. Lower: Data presented as cell counts/100,000 single flow events. Represetative of n=4 mice. **P=0.0039, ****P<0.0001. Two-way ANOVA with Tukey’s post hoc test.

Source data

Extended Data Fig. 4 4Gy-RT elicited neither extensive tumor cell death nor increased cross-presenting DCs in bronchial lymph nodes.

a. Representative tiled immunofluorescent images of radiated HKP1 lungs showing the impact of various doses of RT on tumor cell death. Cleaved caspase-3 (white) and E-cadherin+ tumor islets (red) were stained. Scale Bar: 100µM. Right panel: quantification of total fluorescence arear of cleaved caspase-3. (n = 6 lung sections from 3 mice/treatment. Data are mean ± SEM. ns: non-significant, **** P ≤ 0.0001. One-way ANOVA with Sidak’s post hoc test. b. Representative flow-cytometric plots showing the impact of 4Gy-RT on CD103+ DCs in bronchial lymph nodes (BrLNs). BrLNs were collected, processed to single cell suspension, and stained with antibodies as indicated. Flow events were gated as cells, single cells, Epcam-/CD3-, CD8a+, and MHCII+. Flow contour plots were representative of 2 independent experiments. Data are pooled sample from 3 mice/treatment.

Source data

Extended Data Fig. 5 Differential gene expression in the HKP1 lungs receiving various doses of RT with or without α-PD1 antibody.

a. 144 genes specifically upregulated by 4Gy-RT. b. Volcano plots showing genes upregulated by 4Gy-RT when compared to 0Gy and 8Gy-RT in both the IgG (left) and α-PD1 antibody cohort (right) (P<0.01, Fold change≥2, LSMean≥5). c. RT-PCR analyses of club cell feature genes. Different cellular compartments, immune (CD45+), tumor (cherry+), club (EpcamhighCD24low) and other stromal (CD45-Epcam-) cells were sorted by flow cytometry. Expression of club cell feature genes (Scgb1a1, Scgb3a1, Cyp2f2, Hp and Scgb3a2) were analyzed by RT-PCR. CD45+ cells served as control and was set as 1. Data are mean ± SEM. n=4 mice.0Gy vs. 4Gy-RT (Relative expression in club cells): *P= 0.0102 (Scgb1a1); **P= 0.0036 (Scgb1c1); *P= 0.0328 (Cyp2f2); *P= 0.0492 (Hp); *P= 0.0156 (Scgb3a2).two-way ANOVA with Sidak’s post hoc test.

Source data

Extended Data Fig. 6 4Gy-RT activates club cells to mediate immune activation.

a, Flow cytometry analysis of club cells (CD45- /Epcamhi /CC10+) in naive lungs after different doses of RT. Flow events were gated sequentially as cells and single cells first as described in Extended data Fig. 1b. Representative of n = 5 mice. b, Club cell counts/100,000 single cell events. n=5 mice, ****P< 0.0001, one-way ANOVA with Tukey’s post hoc test. c, Representative histograms showing the increased proliferation rate (Ki67+) of club cells after 4Gy-RT. Representative of n = 5 mice. d. Cytokine production by T cells in response to 4 Gy-RT relied on club cells. Representative flow cytometry plots showing the IFNγ+ /TNF-α+ /CD4+ T cells and GzmB+/CD8+ T cells upon 4Gy-RT in HKP1-bearing lungs of mice that were pharmacologically (naphthalene) or genetically (DT) depleted of club cells. Representative of n = 4 mice. 0Gy- and vehicle-treated mice served as controls.

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Extended Data Fig. 7 4Gy-RT enhances efficacy of PD-1 inhibition by activating club cells.

a, Upper: schematic depicting treatment strategy. CMT-167-bearing mice were treated with IgG or α-PD1 antibody (0.1 mg/mouse, i.p.) at day 11, 14, and 17. Mice also received mock RT or 4Gy-RT at day 11,12 and 13. Lower: tumor growth curves of CMT-167 mice described in upper panel. n=5 mice. IgG vs.4Gy-RT+ α-PD1: P=0.019; IgG vs.α-PD1: NS; IgG vs.4Gy-RT: NS. two-way ANOVA with Tukey’s post hoc test. b, Upper: schematic depicting treatment strategy. CMT-167-bearing mice were treated with naphthalene (200mg/kg, i.p.) at Day 10 to deplete club cells before radiation. From Day11, mice were treated as described in a. lower: tumor growth curves of CMT-167 mice described in upper panel. Vehicle (n=5 mice) vs. naphthalene (n=4 mice): **P=0.0031. Two-way ANOVA with Holm-Sidak’s multiple comparisons test. c. Upper: schematic depicting treatment strategy. Mice bearing HKP1 subcutaneous tumor were treated with IgG or α-PD1 antibody (0.2 mg/mouse, i.p.) at day 14, 17, and 21. Mice also received mock RT or 4Gy-RT at day 14,15 and 16. Lower: tumor growth curves of HKP1 mice described in upper panel. Data are mean ± SEM. n=5 mice. α-PD1 vs.4Gy-RT+ α-PD1: P=0.99, ns, non-significant. Two-way ANOVA with Tukey’s post hoc test. d, HKP1 subcutaneous tumor weights. HKP1 subcutaneous tumors were dissected at Day30. Data are mean ± SEM. IgG and 4Gy-RT: n=4; α-PD1 and 4Gy-RT+α-PD1: n=5 mice. α-PD1 vs.4Gy-RT+α-PD1: P=0.99(ns). One-way ANOVA with Sidak’s multiple comparison test.

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Extended Data Fig. 8 Analyses of radiation-activated club cells and the role of their secretion.

a, Flow cytometry sorting strategy of club cells. Single cell suspensions were prepared from HKP1-bearing lungs of animals treated with or without 4Gy-RT. Flow events were gated sequentially as cells and single cells as described in Extended Data Fig. 1b. Club cells (Epcamhigh/CD24low) were sorted by flow cytometry. b, Representative electron microscopy images of club cells, n= 29 cells. Significant increase of secreting vesicles were observed in 4Gy-RT treated-club cells when compared to 0Gy control. Scale bar, 2µM. c, Lung sections stained for E-cadherin (red) and CC10 (yellow), or Haemotoxylin and Eosin (H&E). Representative images of n=10 sections from 3 mice/treatment. d, Cytokine production by T cells in response to 4Gy-RT relies on the secretory function of club cells. Representative flow cytometry plots showing IFNγ+ /TNF-α+/CD4+ T cells and GzmB+ /CD8+ T cells in 0Gy- or 4Gy-RT-treated HKP1 lungs of Snap23-wt and Snap23-fl/fl mice. Representative of n = 6 mice.

Extended Data Fig. 9 The t-SNE plots of scRNA-seq of HKP1-bearing lungs.

The mean expression values of the genes encoding inflammatory and immunosuppressive mediators are highlighted in various cell clusters. (data are pooled samples from 4 mice).

Extended Data Fig. 10 Expression of club secretory proteins and evaluation of their immunomodulatory functions.

a. RNA-seq heatmap showing the top 8 candidate genes. RNA-seq analyses of HKP1 lungs treated with RT or RT combined with α-PD1 antibody (each sample pooled from 3 mice, 5–6 samples/RT dose).Top genes upregulated specifically by 4Gy-RT (compared to 0Gy and 8Gy-RT) and encoding club secretory proteins were showed in the heatmap (two-sided adjusted P value <0.05, Fold change ≥ 2). b. Western blot analyses of 8 secretory club proteins. HEK293T cells were transfected individually with pCMV6 plasmid containing cDNA of Sftpd, Sftpa1, Sftpb, Hp, Scgb1a1, Scgb3a1, Scgb3a2, and Scgb1c1. 2µL of concentrated supernatant were subject to immunoblotting against their common Flag tag. Representative of n = 3 independent experiment. c. Schematic depicting MDSC culture and treatment strategies. d. Suppression of secretory club proteins on expression of Arg1 and iNOS in MDSCs. Representative IF images of Arg1 (left) and iNOS (right) of MDSCs treated with GM-CSF/IL-6 (positive control), PBS (negative control) or HKP1 supernatant with mock or club cocktail. n=3 field of 2 independent experiments, Scale Bar: 50µM. e. Tumor growth curves of mice which had survived HKP1 tumor upon the combination therapy of club cocktail and α-PD1 antibody. Naïve mice served as controls. n=3 mice. ****P<0.0001. Two-way ANOVA Šídák’s multiple comparisons test.

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Ban, Y., Markowitz, G.J., Zou, Y. et al. Radiation-activated secretory proteins of Scgb1a1+ club cells increase the efficacy of immune checkpoint blockade in lung cancer. Nat Cancer 2, 919–931 (2021). https://doi.org/10.1038/s43018-021-00245-1

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