Abstract
The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3,4,5,6,7,8,9,10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The scRNA-seq datasets from Ag OFF, Ag ON and Ag ON/CPI mice are available from the Gene Expression Omnibus (GEO) database under accession number GSE228586. The scRNA-seq datasets from LCMV Armstrong- and LCMV clone 13-infected mice are available from the GEO database under accession numbers GSE182509 and GSM5530565. The scRNA-seq datasets from human donors are available from the GEO database under accession number GSE229279. Source data are provided with this paper.
Code availability
The custom code used in this article for quantification of epidermal thickness can be accessed at https://github.com/dakwok/The-Histological-Inflammation-Computation-software. The custom code used in this article for analysis of scRNA-seq data with PHATE (scprep package) can be accessed at https://github.com/krishnaswamylab/scprep. The custom code used in this article for analysis of scRNA-seq with MAGIC can be accessed at https://github.com/KrishnaswamyLab/magic. The CellPhoneDB algorithm can be accessed at https://www.cellphonedb.org/. The custom code used in this article for analysis of scRNA-seq data with the MELD package can be accessed at https://github.com/KrishnaswamyLab/MELD.
References
Maeda, Y. et al. Detection of self-reactive CD8+ T cells with an anergic phenotype in healthy individuals. Science 346, 1536–1540 (2014).
Yu, W. et al. Clonal deletion prunes but does not eliminate self-specific αβ CD8+ T lymphocytes. Immunity 42, 929–941 (2015).
Nishimura, H., Nose, M., Hiai, H., Minato, N. & Honjo, T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 11, 141–151 (1999).
Liang, S. C. et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur. J. Immunol. 33, 2706–2716 (2003).
Probst, H. C., McCoy, K., Okazaki, T., Honjo, T. & van den Broek, M. Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4. Nat. Immunol. 6, 280–286 (2005).
Keir, M. E. et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. J. Exp. Med. 203, 883–895 (2006).
Martin-Orozco, N., Wang, Y. H., Yagita, H. & Dong, C. Cutting edge: programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens. J. Immunol. 177, 8291–8295 (2006).
Keir, M. E., Freeman, G. J. & Sharpe, A. H. PD-1 regulates self-reactive CD8+ T cell responses to antigen in lymph nodes and tissues. J. Immunol. 179, 5064–5070 (2007).
Pauken, K. E. et al. Cutting edge: identification of autoreactive CD4+ and CD8+ T cell subsets resistant to PD-1 pathway blockade. J. Immunol. 194, 3551–3555 (2015).
Nelson, C. E. et al. Reprogramming responsiveness to checkpoint blockade in dysfunctional CD8 T cells. Proc. Natl Acad. Sci. USA 116, 2640–2645 (2019).
Postow, M. A., Sidlow, R. & Hellmann, M. D. Immune-related adverse events associated with immune checkpoint blockade. N. Engl. J. Med. 378, 158–168 (2018).
ElTanbouly, M. A. & Noelle, R. J. Rethinking peripheral T cell tolerance: checkpoints across a T cell’s journey. Nat. Rev. Immunol. 21, 257–267 (2021).
Redmond, W. L. & Sherman, L. A. Peripheral tolerance of CD8 T lymphocytes. Immunity 22, 275–284 (2005).
Probst, H. C., Lagnel, J., Kollias, G. & van den Broek, M. Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8+ T cell tolerance. Immunity 18, 713–720 (2003).
Parish, I. A. et al. The molecular signature of CD8+ T cells undergoing deletional tolerance. Blood 113, 4575–4585 (2009).
Johnson, D. B. et al. Fulminant myocarditis with combination immune checkpoint blockade. N. Engl. J. Med. 375, 1749–1755 (2016).
Shi, V. J. et al. Clinical and histologic features of lichenoid mucocutaneous eruptions due to anti-programmed cell death 1 and anti-programmed cell death ligand 1 immunotherapy. JAMA Dermatol. 152, 1128–1136 (2016).
Luoma, A. M. et al. Molecular pathways of colon inflammation induced by cancer immunotherapy. Cell 182, 655–671.e622 (2020).
Yasuda, Y. et al. CD4+ T cells are essential for the development of destructive thyroiditis induced by anti-PD-1 antibody in thyroglobulin-immunized mice. Sci. Transl. Med. 13, abb7495 (2021).
Reschke, R. et al. Checkpoint blockade-induced dermatitis and colitis are dominated by tissue-resident memory T cells and Th1/Tc1 cytokines. Cancer Immunol. Res. 10, 1167–1174 (2022).
Khan, S. & Gerber, D. E. Autoimmunity, checkpoint inhibitor therapy and immune-related adverse events: a review. Semin. Cancer Biol. 64, 93–101 (2020).
Medetgul-Ernar, K. & Davis, M. M. Standing on the shoulders of mice. Immunity 55, 1343–1353 (2022).
Damo, M. et al. Inducible de novo expression of neoantigens in tumor cells and mice. Nat. Biotechnol. 39, 64–73 (2021).
Ventura, A. et al. Restoration of p53 function leads to tumour regression in vivo. Nature 445, 661–665 (2007).
Kaur, H., Nikam, B. P., Jamale, V. P. & Kale, M. S. Lichen Planus Severity Index: a new, valid scoring system to assess the severity of cutaneous lichen planus. Indian J. Dermatol. Venereol. Leprol. 86, 169–175 (2020).
Pircher, H., Bürki, K., Lang, R., Hengartner, H. & Zinkernagel, R. M. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 342, 559–561 (1989).
Moon, K. R. et al. Visualizing structure and transitions in high-dimensional biological data. Nat. Biotechnol. 37, 1482–1492 (2019).
Garcia-Diaz, A. et al. Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Cell Rep. 19, 1189–1201 (2017).
Efremova, M., Vento-Tormo, M., Teichmann, S. A. & Vento-Tormo, R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand–receptor complexes. Nat. Protoc. 15, 1484–1506 (2020).
Hofmann, M. & Pircher, H. E-cadherin promotes accumulation of a unique memory CD8 T-cell population in murine salivary glands. Proc. Natl Acad. Sci. USA 108, 16741–16746 (2011).
Burkhardt, D. B. et al. Quantifying the effect of experimental perturbations at single-cell resolution. Nat. Biotechnol. 39, 619–629 (2021).
Hudson, W. H. et al. Proliferating transitory T cells with an effector-like transcriptional signature emerge from PD-1+ stem-like CD8+ T cells during chronic infection. Immunity 51, 1043–1058.e1044 (2019).
Kaech, S. M., Hemby, S., Kersh, E. & Ahmed, R. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111, 837–851 (2002).
Schaberg, K. B. et al. Immunohistochemical analysis of lichenoid reactions in patients treated with anti-PD-L1 and anti-PD-1 therapy. J. Cutan. Pathol. 43, 339–346 (2016).
Bouneaud, C., Kourilsky, P. & Bousso, P. IImpact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13, 829–840 (2000).
Zehn, D. & Bevan, M. J. T cells with low avidity for a tissue-restricted antigen routinely evade central and peripheral tolerance and cause autoimmunity. Immunity 25, 261–270 (2006).
Marchingo, J. M. & Cantrell, D. A. Protein synthesis, degradation, and energy metabolism in T cell immunity. Cell Mol. Immunol. 19, 303–315 (2022).
Zinselmeyer, B. H. et al. PD-1 promotes immune exhaustion by inducing antiviral T cell motility paralysis. J. Exp. Med. 210, 757–774 (2013).
Fife, B. T. et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal. Nat. Immunol. 10, 1185–1192 (2009).
Vella, J. et al. Dendritic cells maintain anti-tumor immunity by positioning CD8 skin-resident memory T cells. Life Sci. Alliance 4, e202101056 (2021).
Miller, B. C. et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol. 20, 326–336 (2019).
Im, S. J. et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 537, 417–421 (2016).
Yost, K. E. et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 25, 1251–1259 (2019).
Dammeijer, F. et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell 38, 685–700.e688 (2020).
Francis, D. M. et al. Blockade of immune checkpoints in lymph nodes through locoregional delivery augments cancer immunotherapy. Sci. Transl. Med. 12, aay3575 (2020).
Im, S. J., Konieczny, B. T., Hudson, W. H., Masopust, D. & Ahmed, R. PD-1+ stemlike CD8 T cells are resident in lymphoid tissues during persistent LCMV infection. Proc. Natl Acad. Sci. USA 117, 4292–4299 (2020).
Tattersall, I. W. & Leventhal, J. S. Cutaneous toxicities of immune checkpoint inhibitors: the role of the dermatologist. Yale J. Biol. Med. 93, 123–132 (2020).
Nüssing, S. et al. Efficient CRISPR/Cas9 gene editing in uncultured naive mouse T cells for in vivo studies. J. Immunol. 204, 2308–2315 (2020).
van Dijk, D. et al. Recovering gene interactions from single-cell data using data diffusion. Cell 174, 716–729.e727 (2018).
Dunlap, G. S. et al. Single-cell transcriptomics reveals distinct effector profiles of infiltrating T cells in lupus skin and kidney. JCI Insight 7, e156341 (2022).
Acknowledgements
The authors thank Joshi laboratory members for helpful discussions and reviewing the manuscript; M. Huelsmeyer for tool supply. We also thank the Yale Flow Cytometry Core, Yale School of Medicine Comparative Pathology Research Core, and the Yale Center for Genome Analysis. The Yale Flow Cytometry Core is supported in part by NIH grant P30CA016359 and S10OD026996. This work was supported by grants from The G. Harold & Leila Y. Mathers Foundation (YAL182 to N.S.J.), the Melanoma Research Alliance (569588 to N.S.J.), the Yale SPORE in Skin Cancer (2P50CA196530-06 to N.S.J.), the Yale Liver Center Morphology Core (NIH grant P30DK034989), and the American Cancer Society (Research Scholar Award to N.S.J). N.S.J. is a recipient of the Mark Foundation Emerging Leader Award and of the Pershing Square Sohn Prize. M.D. was supported by the Leslie H. Warner Post-Doctoral Research Fellowship. N.I.H. was supported by the Dermatology Foundation.
Author information
Authors and Affiliations
Contributions
M.D. and N.S.J. conceptualized the research and wrote the paper. M.D., N.I.H., I.W., K.C., S.V., J.H., E.F., J.L.L., J.B., A.T. and J.S. performed the research. M.D., N.I.H., A.V., D.K., C.C. and S.K. analysed data. N.I.H. collected biopsies for human scRNA-seq analysis. W.E.D. and J.S.L. provided histology data from human samples. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature thanks Laura Mackay, Evan Newell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.
Additional information
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 Induction of Ag expression in EpCAM+CD45- skin cells coupled to CPIs leads to quantifiable cutaneous pathology.
A) NINJA allele and Dox/4-OH-Tam-driven recombinations required for induction of NINJA Ag expression. B) Average ± s.d. of frequencies of Ag (GFP)-expressing skin cell populations identified by EpCAM and CD45 expression. n = 3 (Ag ON, Ag ON/CPI, Ag ON/CPI + αCD8) or 6 (Ag OFF), representative of > 3 experimental repeats. C) Representative pictures of cutaneous pathology by range of pathological scores. D) Frequency of pathological scores by experimental condition. n = 6 (Ag ON) or 7 (Ag OFF, Ag ON/CPI), representative of > 3 experimental repeats.
Extended Data Fig. 2 Ag-specific CD8 T cells infiltrate skin upon local Ag expression with and without CPIs.
A) Average ± s.d. of normalized counts of skin-infiltrating CD4 and CD8 T cells from mice in the indicated conditions. * P = 0.0495 (Ag ON vs. Ag OFF), * P = 0.0437 (Ag ON/CPI vs. Ag OFF), and ns = not significant by two-tailed t test. n = 4 (Ag OFF, Ag OFF/CPI) or 5 (Ag ON, Ag ON/CPI), representative of 3 experimental repeats. B) Gating strategy and average ± s.d. of frequencies of gated populations from skin in the indicated experimental conditions. n = 5 (Ag OFF, Ag OFF/CPI) or 6 (Ag ON, Ag ON/CPI), representative of 3 experimental repeats. C) Average ± s.d. of normalized counts of endogenous GP33-specific CD8 T cells from skin of mice in the indicated experimental conditions. * P = 0.0196, ** P = 0.004 and ns = not significant by two-tailed t test. n = 5 (Ag ON); 6 (Ag OFF/CPI); or 7 (Ag OFF, Ag ON/CPI). Representative of 3 experimental repeats.
Extended Data Fig. 3 PD-1 allows Ag-specific CD8 T cells to co-exist with Ag-expressing skin cells without local pathology.
A) Experimental schedule. B) IVIS imaging of fLuc-expressing P14 CD8 T cells in representative mice from the indicated conditions. n = 4 (Ag OFF) or 6 (Ag ON), representative of 2 experimental repeats. Numbers indicate total counts of luminescence. C) Average ± s.d. of frequencies of Ag (GFP)-expressing skin cells (top) and Thy1.1+ P14 CD8 T cells (bottom) in 4-OH-Tam-treated skin from mice in the indicated conditions. n = 4 (Ag OFF) or 6 (Ag ON), representative of 2 experimental repeats. D) Representative pictures of the back of mice from the indicated conditions in C. E) Pathological scores assigned to mice in the indicated conditions in C. ns = not significant by two-tailed t test. F) Confocal microscopy analysis of skin from mice in C. Dotted line = epidermis (top)/dermis (bottom) interface. G) Experimental schedule for comparison of concomitant vs. delayed administration of αPD-1 antibodies in Dox/4-OH-Tam-treated N/C mice. H) Pathological scores of mice from the indicated conditions. Black and red dotted lines indicate median pathological scores of Ag OFF and Ag ON conditions, respectively, from Fig. 1e. ns = not significant by two-tailed t test. n = 5 (day 0) or 6 (day 5), representative of 2 experimental repeats.
Extended Data Fig. 4 Skin-specific Ag expression leads to changes in local cell populations identified by scRNAseq analysis.
A) UMAP projection of total skin cells sequenced across experimental samples and annotated by cell type. n = 21,178 total cells sequenced. B) Dot plot of genes defining populations of skin cells shown in A. C) UMAP projections of total skin cells from A by experimental condition. D) Frequencies of skin cell types from A by experimental condition. E) PHATE map of total skin cells sequenced across experimental samples and annotated by cell type. F) PHATE maps of total skin cells from E by experimental condition. G) UMAP projection of Cd3e-expressing cells from A sequenced across experimental samples and annotated by T cell type. H) Dot plot of genes defining populations of CD3e-expressing cells in skin in G. I) UMAP projections of Cd3e-expressing cells in G by experimental condition. J) PHATE map of Cd3e-expressing cells from E annotated by T cell type. K) PHATE maps of Cd3e-expressing cells in J by experimental condition.
Extended Data Fig. 5 Transcriptomic analysis of myeloid cell populations from the skin of Ag OFF, Ag ON and Ag ON/CPI mice.
A) Dot plot of genes defining populations of skin-infiltrating myeloid cells from Fig. 4a. B) PHATE map of myeloid cell populations sequenced across experimental samples. C) PHATE maps of myeloid cell populations in B by experimental condition. D) Gating strategy and representative histograms for skin-infiltrating CD11b+CD11c+CD16+ cells in the indicated conditions. Numbers represent average ± s.d. of frequencies of the gated populations (dot plots) or average ± s.d. of MFIs of the indicated markers (histograms). n = 3, representative of 3 experimental repeats. E) PHATE map of Ifng expression by total skin cells from Extended Data Fig. 4e. F) PHATE map of Ifng expression by CD3e+ skin cells from Extended Data Fig. 4j. G) PHATE maps of expression level of the indicated genes by skin-infiltrating myeloid cells sequenced across samples shown in B.
Extended Data Fig. 6 Multi-layer interactions between skin-infiltrating myeloid cells and T cells are revealed by scRNAseq analysis.
A,B) CellPhoneDB algorithm and Pearson correlation analysis of the gene expression levels of the indicated ligand/receptor pairs in skin-infiltrating myeloid cells and T cells (A) and T cells and myeloid cells (B), respectively, sequenced from mice in the indicated experimental conditions. One-sided P values are shown.
Extended Data Fig. 7 Transcriptomic analysis of P14 CD8 T cells from the skin of Ag ON and Ag ON/CPI mice.
A) PHATE maps of skin-infiltrating P14 CD8 T cells sequenced in the indicated experimental conditions. B) PHATE maps of expression level of the indicated genes by skin-infiltrating P14 CD8 T cells sequenced across samples. C) PHATE maps of the likelihood of skin-infiltrating P14 CD8 T cells calculated by MELD. D) PHATE maps of the pseudotime analysis of skin-infiltrating P14 CD8 T cells sequenced across samples. E) Skin-infiltrating P14 CD8 T cell number distribution along the pseudotime shown by experimental condition. F) Z-score-normalized expression levels of the indicated genes over pseudotime in skin-infiltrating P14 CD8 T cells in B.
Extended Data Fig. 8 Integrated transcriptomic analysis supports a linear differentiation trajectory for skin Ag-specific CD8 T cells.
A) PHATE map of skin-infiltrating P14 CD8 T cells from Ag ON and Ag ON/CPI mice and GP33-specific CD8 T cells from an LCMV-Clone13-infected B6 mouse (28 days post infection) and GP33-specific CD8 T cells from an LCMV-Armstrong-infected B6 mouse (28 days post infection). B) Average ± s.d. of frequencies of gated populations of skin-infiltrating P14 CD8 T cells from Ag ON and Ag ON/CPI mice, and GP33-specific CD8 T cells from the spleen of LCMV-Clone13-infected B6 mice (28 days post infection) or LCMV-Armstrong-infected B6 mice (8 or 28 days post infection). Naïve CD44- CD8 T cells from the spleen of one B6 mouse are shown as negative controls. n = 3 (LCMV-infected mice); 4 (Ag ON); or 5 (Ag ON/CPI). Representative of 2 experimental repeats. C) PHATE maps of expression level of the indicated genes by skin-infiltrating P14 CD8 T cells and GP33-specific CD8 T cells in A.
Extended Data Fig. 9 Transcriptomic analysis by scRNAseq of human skin from healthy donors and patients with cutaneous lichenoid IRAEs.
A) UMAP projection of scRNAseq data from total cells harvested from matched lesional and non-lesional skin of two patients with lichenoid IRAEs and from three healthy donors. n = 19,851, 11,955, and 4,209 cells from lesional, non-lesional and healthy skin samples, respectively. B) UMAP projection of the dataset in A color-coded by cell population. C) Dot plot of genes defining the cell populations identified in B. D) Frequencies of the skin cell populations in B.
Extended Data Fig. 10 CD8 T cells with a cytotoxic gene expression profile infiltrate healthy and diseased skin in patients with cutaneous lichenoid IRAEs.
A) UMAP projection of CD3e-expressing skin cells from the dataset shown in Extended Data Fig. 9. B) UMAP projection of CD3e-expressing skin cells in A color-coded by Cluster. C) Dot plot of genes defining individual Clusters in B. D) Frequencies of the T cell populations identified in B in lesional vs. non-lesional skin. E-H) Dot plot of genes characterizing activation, cytotoxicity, exhaustion and interferon-response signatures50 in T cell Clusters from B,C. I) Frequency of cells with and without associated TRAV/TRBV sequences from Clusters 1–5 defined in B,C. J) Number and size of all T cell clones from Clusters 2–5 sequenced in lesional and non-lesional skin.
Supplementary information
Supplementary Figures
This file contains Supplementary Figs. 1–7
Supplementary Table 1
Raw gene expression in cell populations from human lesional, non-lesional, and healthy skin.
Supplementary Table 2
Raw gene expression in cells from clusters 1–7 in human lesional, non-lesional, and healthy skin.
Supplementary Table 3
Gene expression in cells from cluster 1 in human lesional and non-lesional skin.
Supplementary Table 4
Clonal analysis of T cells in clusters 1–5 in human lesional, non-lesional, and healthy skin.
Source data
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Damo, M., Hornick, N.I., Venkat, A. et al. PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens. Nature 619, 151–159 (2023). https://doi.org/10.1038/s41586-023-06217-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-023-06217-y
This article is cited by
-
Clinical and translational attributes of immune-related adverse events
Nature Cancer (2024)
-
Functional T cell tolerance by peripheral tissue-based checkpoint control
Nature Immunology (2023)
-
γδ T cells control murine skin inflammation and subcutaneous adipose wasting during chronic Trypanosoma brucei infection
Nature Communications (2023)
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.
