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Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells

Nature Immunology volume 22, pages 1042–1051 (2021)Cite this article

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Abstract

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

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Fig. 1: Ad vectors target human fibroblasts.
Fig. 2: Ccl19-cre+ FSCs mediate induction of inflationary memory CD8+ T cells.
Fig. 3: Ccl19-cre+ FSCs collaborate with BATF3+ cross-presenting DCs for the induction of LacZ-specific CD8+ T cell responses.
Fig. 4: Pulmonary Ccl19-cre+ FSCs support inflating memory CD8+ T cells.
Fig. 5: Ad-LacZ/FlexON activates pulmonary Ccl19-expressing, immune-stimulatory FSCs.
Fig. 6: Pulmonary FSC-derived IL-33 preserves metabolic and functional fitness of inflationary memory CD8+ T cells.

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Data availability

scRNA-seq data are available in the ArrayExpress database (accession numbers E-MTAB-9558 and E-MTAB-9580). Ensembl GRCm38.94 was used as a reference to build index files for alignments in scRNA-seq analysis. Further information and requests for resources should be directed to and will be fulfilled by the lead contacts B.L. (burkhard.ludewig@kssg.ch) and P. Klenerman (paul.klenerman@medawar.ox.ac.uk). Source data are provided with this paper.

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Acknowledgements

We thank S. Caviezel-Firner and C. Engetschwiler for excellent technical support. This study received financial support from the Swiss National Science Foundation (grants 166500 and 159188 to B.L.), Swiss Cancer Research (KFS-4162-02-2017-R to P. Krebs), the Wellcome Trust (109965MA to P. Klenerman) and research fellowship grants from the British Infection Association (to J.M.C.) and the Wellcome Trust (099897/Z/12/A to J.M.C.).

Author information

Author notes

  1. These authors contributed equally: Paul Klenerman, Burkhard Ludewig.

Authors and Affiliations

  1. Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland

    Jovana Cupovic, Sandra S. Ring, Lucas Onder, Mechthild Lütge, Hung-Wei Cheng, Angelina De Martin, Lukas Flatz, Elke Scandella & Burkhard Ludewig

  2. Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany

    Jovana Cupovic

  3. Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK

    Julia M. Colston, Nicholas M. Provine & Paul Klenerman

  4. Department of Dermatology, University Hospital Zurich, Zurich, Switzerland

    Lukas Flatz

  5. Institute of Microbiology, ETH Zurich, Zurich, Switzerland

    Annette Oxenius

  6. Institute of Pathology, University of Berne, Berne, Switzerland

    Philippe Krebs

  7. Department of Urology, Kantonsspital St. Gallen, St. Gallen, Switzerland

    Daniel Engeler

  8. Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland

    Burkhard Ludewig

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  1. Jovana Cupovic
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Contributions

B.L., P. Klenerman and J.C. designed the study, discussed data and wrote the paper. J.C., J.M.C., S.S.R., L.O., A.D.M., H.-W.C. and D.E. conducted experiments and discussed data. M.L. performed bioinformatic analyses and discussed data. N.M.P. discussed data and provided reagents. P. Krebs and A.O. discussed data and provided reagents. L.F. and E.S. discussed data.

Corresponding authors

Correspondence to Paul Klenerman or Burkhard Ludewig.

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Competing interests

L.F. is a cofounder and shareholder of Hookipa Pharma. B.L., L.O. and H.-W.C. are cofounders and shareholders of Stromal Therapeutics. S.S.R. and H.-W.C. are part-time employees of Stromal Therapeutics. The remaining authors declare no competing interests.

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Peer review information Nature Immunology thanks Stephen Jameson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Adenoviral vectors ChAdOx1 and HuAd5 transduce human fibroblasts.

a, Preparation of sliced tissue cultures from human palatine tonsils following infection with adenoviral vectors. b, Flow cytometry-based gating strategy for PBMCs analysis. c and d, Frequency of CD3+ T cells, CD14+ HLA-DR+ monocytes or CD11c+ dendritic cells (DCs) within GFP+ cells following infection with (c) ChAdOx1-GFP or (d) HuAd5-GFP. e, Flow cytometry-based gating strategy for cultured tonsillar tissue stromal cell analysis. f and g, Frequencies of PDPN+ and CD31+ cells within GFP+ cultured tonsillar tissue stromal cells (TSC) following infection with (f) ChAdOx1-GFP or (g) HuAd5-GFP. h to m, Infection of cultured (h to j) skin- or (k to m) lung-derived stromal cells with ChAdOx1-GFP or HuAd5-GFP with representative FACS plots. (i and l) Frequency of GFP+ cells after infection with indicated adenoviral vectors at different multiplicity of infection (MOI). (j and m) Frequency of PDPN+ or CD31+ cells within GFP+ cells after infection with indicated adenoviral vectors. Pooled data from n = 3 PBMC samples [(c) and (d)] and n = 3 TSC samples [(f) and (g)]. Data from one experiment with single skin [(h-j)] or lung tissue donor [(k-m)]. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test [(c) and (d)] and unpaired two-tailored Student`s t test [(f) and (g)], with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

Source data

Extended Data Fig. 2 Induction of inflationary memory CD8+ T cells following Ad5-LacZ immunization.

a to c, B6 mice were immunized i.v. with Ad-LacZ. (a) Kinetics of the frequency of bgal96 or bgal497 tetramer+ CD8+ T cells in blood. Frequency of bgal96 or bgal497 tetramer+ CD8+ T cells in indicated organs on (b) day 21 or (c) day 50. d, Flow cytometry-based gating strategy for tetramer+ CD8+ T cell analysis. e, Ubi-Cre ERT2 mice were immunized i.v. with Ad-LacZ/FlexON. Frequency of bgal96 or bgal497 tetramer+ CD8+ T cells in blood after application of Tamoxifen. (f to g) LysM-Cre mice were immunized i.v. with Ad-LacZ/FlexON. f, Kinetics of bgal96 tetramer+ CD8+ T cell response with representative FACS plots. g, Kinetics of non-inflationary bgal497 tetramer+ CD8+ T cell response with representative FACS plots. Cre-negative mice were used as controls (Ctrl). Pooled data from 2 independent experiments with n = 4-8 mice [(a)]; n = 4 mice per group [(b) and (c)], representative of two experiments with n = 2 per group [(e)], n = 8 (LysM-Cre+) and n = 8 (Ctrl) [(f) and (g)]. Values indicate mean±s.e.m. for each time point. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Bonferroni multiple comparison test [(f) to (g)] with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

Source data

Extended Data Fig. 3 Cross-talk between Ccl19-Cre+ cells and professional antigen presenting cells during the induction of LacZ-specific T cell responses.

The indicated bone marrow chimeric mice were generated and vaccinated i.v. with Ad-LacZ/FlexON. (a and b) The frequency of bgal497 tetramer+ CD8+ T cells was monitored in blood. Values indicate mean±s.e.m. for each time point. Pooled data from 2 independent experiments with n = 10 (Ctrl to Ccl19-Cre) to 8 (H2-Kb−/− to Ccl19-Cre) mice [(a)] and n = 5 (Ctrl to Ccl19-Cre) and 5 (Batf3−/− to Ccl19-Cre) mice [(b)]. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Bonferroni multiple comparison test [(a) and (b)] *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

Source data

Extended Data Fig. 4 4. Location of Ccl19-Cre+ cells that contribute to induction and maintenance of memory inflating CD8+ T cells.

a, Ccl19-EYFP mice were splenectomized and immunized i.v. with Ad-LacZ/FlexON. The frequency of tetramer+ CD8+ T cells was monitored in blood or the indicated organs. b to g, Ccl19-EYFP/iDTR mice were vaccinated i.v. with Ad-LacZ/FlexON and DT or PBS were given intranasal (in) or intraperitoneal (sys) on day 3 and 5. (c) Enumeration of EYFP+ cells in the lungs of day 7 Ad-LacZ/FlexON-vaccinated mice, following i.n. DT or PBS application. (d) Representative confocal microscopy image of EYFP+ FSCs in the liver and spleen after systemic or intranasal DT injection. Scale bar equals 30 μm. (e) Representative FACS plots of bgal96 tetramer+ CD8+ T cells in blood, lung, liver and spleen on day 21. (f) Frequency of bgal497 tetramer+ CD8+ T cells in the indicated organs. (g) Frequency of bgal497 tetramer+ CD8+ T cells in the blood at the indicated time points. h, Flow cytometry-based gating strategy for lung hematopoietic cells analysis used in Fig.4e. Dots represent individual mice. Bar graphs indicate mean ±s.e.m. Data from one [(a)] or pooled data from 2 independent experiments [(b) to (g)] with n = 2 mice per group [(a)]; n = 4 (DT intranasal) and n = 5 (PBS intranasal) [(c)]; representative data from n = 3 (DT systemic), n = 7 (DT intranasal) and n = 5 (PBS control) [(d)]; and n = 5 (8 blood) (DT systemic), n = 9 (DT intranasal) and n = 14 (PBS control) [(f)], n = 5 (days 7 and 50), 10 mice (day 14) and 8 (day 21) [(g)]. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Bonferroni multiple comparison test [(a)], unpaired two-tailored Student`s t test [(c) and (g)] or one-way ANOVA with Tukey’s multiple comparison test [(f)] with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

Source data

Extended Data Fig. 5 Phenotypic changes in Ccl19-Cre+ cells after Ad-LacZ/FlexON immunization.

a to f, Ccl19-EYFP mice were immunized i.v. with Ad-LacZ/FlexON. On day 21 pulmonary FSCs were analyzed. (a) Gating for analysis of EYFP+ cells. (b to f) Representative FACS histograms with gMFI/MFI and frequencies of (b) PDPN+, (c) ICAM1+, (d) PDGFRa+, (e) PDGFRb+ or (f) VCAM1+ cells within EYFP+ cells. Dots represent individual mice and bar graphs indicate mean ±s.e.m. Pooled data from 2 independent experiments with n = 5 mice per group [(e)] and n = 9 mice per group [(f)]. Statistical analysis was performed using unpaired two-tailored Student`s t test [(e) and (f)] with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

Source data

Extended Data Fig. 6 Ad-LacZ/FlexON activates pulmonary Ccl19-expressing immune-stimulatory FSCs.

a, Averaged expression of cluster specific marker genes for pulmonary FSC clusters in merged naive and Ad-LacZ/FlexON-immunized Ccl19-EYFP mice. b, Heatmaps displaying indicated genes across all Lumhigh and Il33high FSCs in EYFP+ FSCs from Ad-LacZ/FlexON-immunized or naive mice. c, Prediction of differentiation dynamics based on RNA velocity analysis of EYFP+ FSCs constructed based on recent changes in the transcriptional rate of a gene to predict the future state of individual cells. d, Representative flow cytometric analysis of Sca-1+CD34+ cell populations within EYFP+ cells. e, Frequency Sca-1+ CD34+ cell within EYFP+ cells. f, Quantitative real-time PCR analysis of Il33 expression in pulmonary EYFP+ FSCs. g, Quantitative real-time PCR analysis of Il33 expression in EYFP+ pulmonary FSC populations. h, Quantitative real-time PCR analysis of LacZ mRNA copy numbers per 1,000 cells in the indicated EYFP+ pulmonary FSC populations. i, Reconstruction of IL-33 staining in lung EYFP+ cells of Ccl19-EYFP mice at indicated time points after Ad-LacZ/FlexON immunization, based on images shown in Fig. 5j. Scale bar equals 30 μm. Dots represent individual mice and bar graphs indicate mean ±s.e.m. Pooled data from two independent experiments with n = 8 naive and 9 AdLacZFlexON immunized [(e)]; n = 5 [(f)], n = 6 [(h)] mice; one experiment n = 6 mice [(g)]. Representative data from n = 4 mice per group (i). Statistical analysis was performed using unpaired two-tailored Student’s t test [(e)], Mann Whitney [(f), (g)], one-way ANOVA with Turkey`s multiple comparison test [(h)] with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

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Extended Data Fig. 7 Bronchus-associated lymphoid tissues in the lungs of Ad-LacZ/FlexON immunized Ccl19-EYFP mice.

a, Overview image of three lung lobes (Sup, superior; Inf, inferior; Mid, middle) of the right lung. Dashed line in the schematic indicates approximate sagittal plane of sectioning. Arrowheads indicate lymphoid cell aggregates in the hilus region. Scale bar equals 2 mm. b, Images show hilus regions in lungs of Ccl19-EYFP mice. Scale bar equals 100 μm. Representative of 2 independent experiments, n = 4 mice per condition.

Extended Data Fig. 8 Pulmonary FSC-derived IL-33 preserves metabolic and functional fitness of inflationary memory CD8+ T cells.

(a to d, Ccl19-Cre-EYFP/iDTR mice were immunized i.v. with Ad-LacZ/FlexON and DT or PBS was given intranasally on day 3 and 5. Single cell RNA-seq analysis of bgal96 tetramer+ CD8+ T cells isolated from lungs on day 21. (a) Network plots displaying most significantly enriched gene ontologies based on transcriptional differences in bgal96 tetramer+ CD8+ T cells from DT vs PBS treated Ccl19-Cre/iDTR EYFP mice and with enriched genes annotated. (b) Violin plots show expression of Atp5b, Cox5a, Cox8a, Tomm20. (c) Representative FACS histograms of Ki67 staining on pulmonary bgal96 tetramer+ CD8+ T cells (left) and frequency of Ki67+ bgal96-specific CD8+ T cells (right). (d) Representative FACS dot-plot of live-dead stain negative pulmonary bgal96 tetramer+ CD8+ T cells (left) and frequency of Aqua-negative bgal96-specific CD8+ T cells (right). e, Quantitative real-time PCR analysis of Sdha, Uqcrc2 and Cox4i2 expression in bgal96-specifc CD8+ T cells sorted from the lung of Ccl19-Cre+ Il33fl/fl and Ccl19-Cre+ Il33+/+ mice, day 21 after Ad-LacZ/FlexON immunization. Dots represent individual mice. Bar graphs indicate mean ±s.e.m. (c-e) Pooled data from 2 independent experiments with n = 8 mice per group [(c)]; n = 7 mice [(d)]; and n = 4 mice [(e)]. Statistical analysis was conducted using unpaired two-tailored Student’s t test with *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are provided in the Source Data.

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Extended Data Fig. 9 Kinetics and tissue distribution of inflationary memory CD8+ T cells under conditions of Ccl19-Cre+ lung FSC depletion and Il33-ablation in Ccl19-Cre+ cells.

Ccl19-Cre-EYFP/iDTR mice and Ccl19-Cre Il33fl/fl mice (Il33fl/fl) were immunized i.v. with Ad-LacZ/FlexON and CD8+ T cells were analyzed on day 21. Ccl19-Cre-EYFP/iDTR mice were treated i.n. with DT or PBS on day 3 and 5 after immunization. a, Number of bgal96 tetramer+ CD8+ T cells in lung and spleen. b, Frequency of bgal497 tetramer+ CD8+ T cells in blood, lung and spleen. c, Frequency of KLRG1+/CX3CR1+ bgal497 tetramer+ CD8+ T cells in the lungs. d, IFN-g- and TNF-producing pulmonary bgal497-specific CD8+ T cells. Dots represent individual mice. Bar graphs indicate mean ±s.e.m. Pooled data from 2 independent experiments with n = 9 (DT intranasal), n = 9 (Ccl19-Cre Il33fl/fl) and n = 19 (Ccl19-EYFP/iDTR PBS i.n. and Ccl19-Cre Il33+/+) mice [(a)]; n = 9 (DT intranasal), n = 8 (Ccl19-Cre Il33fl/fl) and n = 19 (Ccl19-EYFP/iDTR PBS i.n. and Ccl19-Cre Il33+/+) mice [(b)]; n = 6 (DT intranasal), n = 9 (Ccl19-Cre Il33fl/fl) and n = 16 (Ccl19-EYFP/iDTR PBS i.n. and Ccl19-Cre Il33+/+) mice [(c)]; n = 7 (DT intranasal), n = 8 IFNg, n = 6 TNF (Ccl19-Cre Il33fl/fl) and n = 18 (Ccl19-EYFP/iDTR PBS i.n. and Ccl19-Cre Il33+/+) mice [(d)]. Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparison test with *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001. Exact P values are provided in the Source Data.

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Cupovic, J., Ring, S.S., Onder, L. et al. Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells. Nat Immunol 22, 1042–1051 (2021). https://doi.org/10.1038/s41590-021-00969-3

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