Abstract
The discovery of the highly efficient site-specific nuclease system CRISPR–Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy1,2. The immunogenicity of Cas9 nuclease has been demonstrated in mice3,4. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues3,4,5,6. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease7,8. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell–mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.
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Data availability
The underlying data are available from the corresponding author upon reasonable request. TRB sequencing data have been deposited in the immuneACCESS database at https://doi.org/10.21417/B7ZP86 (http://clients.adaptivebiotech.com/pub/wagner-2018-naturemedicine).
Change history
05 November 2018
An outdated version of the Supplementary Information was originally published with this article. In the correct version, the sizing and layout of the figures were adjusted, and the language in the legends was updated for style such that the supplementary legends and the main-text figure legends were stylistically consistent. However, no data were changed or added. The correct version is now available online.
References
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Cox, D. B. T., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Chew, W. L. et al. A multifunctional AAV–CRISPR–Cas9 and its host response. Nat. Methods 13, 868–874 (2016).
Chew, W. L. Immunity to CRISPR Cas9 and Cas12a therapeutics. Wiley Interdiscip. Rev. Syst. Biol. Med. 10, e1408 (2018).
Lehrman, S. Virus treatment questioned after gene therapy death. Nature 401, 517–518 (1999).
Nayak, S. & Herzog, R. W. Progress and prospects: immune responses to viral vectors. Gene Ther. 17, 295–304 (2010).
Carapetis, J. R., Steer, A. C., Mulholland, E. K. & Weber, M. The global burden of group A streptococcal diseases. Lancet. Infect. Dis. 5, 685–694 (2005).
Charlesworth, C. T. et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. Preprint at https://www.biorxiv.org/content/early/2018/01/05/243345 (2018).
Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med 24, 1216–1224 (2018).
Shaikh, N., Leonard, E. & Martin, J. M. Prevalence of streptococcal pharyngitis and streptococcal carriage in children: a meta-analysis. Pediatrics 126, e557–e564 (2010).
Frentsch, M. et al. Direct access to CD4+ T cells specific for defined antigens according to CD154 expression. Nat. Med. 11, 1118–1124 (2005).
Wolfl, M. et al. Activation-induced expression of CD137 permits detection, isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen without requiring knowledge of epitope specificities. Blood 110, 201–210 (2007).
Schmueck-Henneresse, M. et al. Peripheral blood-derived virus-specific memory stem T cells mature to functional effector memory subsets with self-renewal potency. J. Immunol. 194, 5559–5567 (2015).
Lathrop, S. K. et al. Peripheral education of the immune system by colonic commensal microbiota. Nature 478, 250–254 (2011).
Bacher, P. et al. Regulatory T Cell specificity directs tolerance versus allergy against aeroantigens in humans. Cell 167, 1067–1078.e16 (2016).
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. & Toda, M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155, 1151–1164 (1995).
Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T Cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).
Wing, K. et al. CTLA-4 contrÿol over Foxp3+ regulatory T cell function. Science 322, 271–275 (2008).
Schoenbrunn, A. et al. A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg. J. Immunol. 189, 5985–5994 (2012).
Polansky, J. K. et al. DNA methylation controls Foxp3 gene expression. Eur. J. Immunol. 38, 1654–1663 (2008).
Bacher, P. et al. Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol. 7, 916–928 (2014).
Shmakov, S. et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).
Harrison, O. J. & Powrie, F. M. Regulatory T cells and immune tolerance in the intestine. Cold Spring Harb. Perspect. Biol. 5, a018341 (2013).
Wakelin, S. J. et al. “Dirty little secrets”: endotoxin contamination of recombinant proteins. Immunol. Lett. 106, 1–7 (2006).
Hamano, R., Huang, J., Yoshimura, T., Oppenheim, J. J. & Chen, X. TNF optimally activatives regulatory T cells by inducing TNF receptor superfamily members TNFR2, 4-1BB and OX40. Eur. J. Immunol. 41, 2010–2020 (2011).
Lei, H., Schmidt-Bleek, K., Dienelt, A., Reinke, P. & Volk, H.-D. Regulatory T cell-mediated anti-inflammatory effects promote successful tissue repair in both indirect and direct manners. Front. Pharmacol. 6, 184 (2015).
Chandran, S. et al. Polyclonal regulatory T Cell therapy for control of inflammation in kidney transplants. Am. J. Transplant. 17, 2945–2954 (2017).
Bennett, C. L. et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27, 20–21 (2001).
Guilherme, L., Kalil, J. & Cunningham, M. Molecular mimicry in the autoimmune pathogenesis of rheumatic heart disease. Autoimmunity 39, 31–39 (2006).
Simhadri, V. L. et al. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. Mol. Ther. Methods Clin. Dev 10, 105–112 (2018).
Arruda, V. R., Favaro, P. & Finn, J. D. Strategies to modulate immune responses: a new frontier for gene therapy. Mol. Ther. 17, 1492–1503 (2009).
Robins, H. S. et al. Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells. Blood 114, 4099–4107 (2009).
Sherwood, A. M. et al. Deep sequencing of the human TCRγ and TCRβ repertoires suggests that TCRβ rearranges after αβ and γδ T cell commitment. Sci. Transl. Med. 3, 90ra61 (2011).
Yousfi Monod, M. Y., Giudicelli, V., Chaume, D. & Lefranc, M.-P. IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V-J and V-D-J JUNCTIONs. Bioinformatics 20, i379–i385 (2004).
Johnson, D. R., Kurlan, R., Leckman, J. & Kaplan, E. L. The human immune response to streptococcal extracellular antigens: clinical, diagnostic, and potential pathogenetic implications. Clin. Infect. Dis. 50, 481–490 (2010).
Sen, E. S. & Ramanan, A. V. How to use antistreptolysin O titre. Arch. Dis. Child. Educ. Pract. Ed. 99, 231–238 (2014).
Heslop, H. E. et al. Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat. Med. 2, 551–555 (1996).
Moosmann, A. et al. B cells immortalized by a mini-Epstein–Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells. Blood 100, 1755–1764 (2002).
Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008).
Hammoud, B. et al. HCMV-specific T-cell therapy: do not forget supply of help. J. Immunother. 36, 93–101 (2013).
Acknowledgements
The study was generously supported in parts by the Deutsche Forschungsgemeinschaft (German Research Foundation, SFB-TR36-project A3 grant to H.-D.V., P.R., M.S.H.), the German Federal Ministry of Education and Research (BCRT grant, all authors), a kick-box grant for young scientists by the Einstein Center for Regenerative Therapies (D.L.W.), and the Berlin Institute of Health medical doctoral research stipend (D.L.W.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge the assistance of the BCRT Flow & Mass Cytometry Lab, D. Kunkel and J. Hartwig; G. Grütz and the team of the Core Unit Biomarker—Immunological Study Lab, BCRT, Charité—Universitätsmedizin Berlin; M. Streitz for flow cytometry assistance and CMV lysates; K. Vogt for technical assistance with the TSDR analyses; A. Jurisch, K. Grzeschik, and R. Noster for technical assistance; and A. Floriane Hennig and U. Kornak (Institute for Medical Genetics and Human Genetics, Charité – Universitätsmedizin Berlin) for the PX458_T2mali DNA plasmid. We thank the groups of M. Seifert and A. Thiel (both BCRT, Charité—Universitätsmedizin Berlin) for supplying the reagents, and T. Roch and M. Frentsch for critical discussions.
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D.L.W. led the project, designed the research, performed the experiments, analyzed and interpreted the data, and wrote the manuscript. L. Amini and D.J.W. established the methods, performed the experiments, analyzed the data, and revised the manuscript. L.-M.B. performed the experiments. L. Akyüz designed the experimental approaches, and performed the Meso Scale Diagnostics and Ella systems measurements. P.R. wrote the manuscript and supplied the reagents. H.-D.V. designed the research, interpreted the data, and wrote the manuscript. M.S.-H. led the project, designed the research, analyzed and interpreted the data, and wrote the manuscript.
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D.L.W., L. Amini, D.J.W., M.S.-H., P.R., and H.-D.V. have a patent pending on CRISPR Associated Protein Reactive T Cell Immunity (European patent application EP18163491.6, 2018). L.-M.B. and L. Akyüz have no financial competing interests.
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Wagner, D.L., Amini, L., Wendering, D.J. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med 25, 242–248 (2019). https://doi.org/10.1038/s41591-018-0204-6
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DOI: https://doi.org/10.1038/s41591-018-0204-6
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