Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
18 08 2021
18 08 2021
Historique:
received:
20
01
2021
accepted:
05
08
2021
entrez:
19
8
2021
pubmed:
20
8
2021
medline:
12
11
2021
Statut:
epublish
Résumé
Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.
Identifiants
pubmed: 34408239
doi: 10.1038/s41598-021-96184-z
pii: 10.1038/s41598-021-96184-z
pmc: PMC8373891
doi:
Substances chimiques
Bone Morphogenetic Proteins
0
GDF7 protein, human
0
Growth Differentiation Factors
0
IFNA7 protein, human
0
Immunologic Factors
0
Interferon-alpha
0
Pancreatitis-Associated Proteins
0
REG3A protein, human
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
16767Informations de copyright
© 2021. The Author(s).
Références
Adeegbe, D. O. & Nishikawa, H. Natural and induced T regulatory cells in cancer. Front. Immunol. 4, 190 (2013).
pubmed: 23874336
pmcid: 3708155
doi: 10.3389/fimmu.2013.00190
Rudensky, A. Y. Regulatory T cells and Foxp3. Immunol. Rev. 241, 260–268 (2011).
pubmed: 21488902
pmcid: 3077798
doi: 10.1111/j.1600-065X.2011.01018.x
Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune tolerance. Cell 133, 775–787 (2008).
pubmed: 18510923
doi: 10.1016/j.cell.2008.05.009
Grant, C. R., Liberal, R., Mieli-Vergani, G., Vergani, D. & Longhi, M. S. Regulatory T-cells in autoimmune diseases: Challenges, controversies and—yet—unanswered questions. Autoimmun. Rev. 14, 105–116 (2015).
pubmed: 25449680
doi: 10.1016/j.autrev.2014.10.012
Kanamori, M., Nakatsukasa, H., Okada, M., Lu, Q. & Yoshimura, A. Induced regulatory T cells: Their development, stability, and applications. Trends Immunol. 37, 803–811 (2016).
pubmed: 27623114
doi: 10.1016/j.it.2016.08.012
Wing, K. et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271–275 (2008).
pubmed: 18845758
doi: 10.1126/science.1160062
Shevach, E. M. Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 30, 636–645 (2009).
pubmed: 19464986
doi: 10.1016/j.immuni.2009.04.010
Tang, Q. & Bluestone, J. A. The Foxp3+ regulatory T cell: A jack of all trades, master of regulation. Nat. Immunol. 9, 239–244 (2008).
pubmed: 18285775
pmcid: 3075612
doi: 10.1038/ni1572
Vignali, D. A., Collison, L. W. & Workman, C. J. How regulatory T cells work. Nat. Rev. Immunol. 8, 523–532 (2008).
pubmed: 18566595
pmcid: 2665249
doi: 10.1038/nri2343
Cretney, E., Kallies, A. & Nutt, S. L. Differentiation and function of Foxp3(+) effector regulatory T cells. Trends Immunol. 34, 74–80 (2013).
pubmed: 23219401
doi: 10.1016/j.it.2012.11.002
Fan, X. et al. CD49b defines functionally mature Treg cells that survey skin and vascular tissues. J. Exp. Med. 215, 2796–2814 (2018).
pubmed: 30355617
pmcid: 6219731
doi: 10.1084/jem.20181442
Gangaplara, A. et al. Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment. PLoS Pathog 14, e1006985 (2018).
pubmed: 29672594
pmcid: 5929570
doi: 10.1371/journal.ppat.1006985
Mohr, A., Malhotra, R., Mayer, G., Gorochov, G. & Miyara, M. Human FOXP3(+) T regulatory cell heterogeneity. Clin. Transl. Immunol. 7, e1005 (2018).
doi: 10.1002/cti2.1005
Ronchetti, S. et al. Glucocorticoid-induced tumour necrosis factor receptor-related protein: A key marker of functional regulatory T cells. J. Immunol. Res. 2015, 171520 (2015).
pubmed: 25961057
pmcid: 4413981
doi: 10.1155/2015/171520
Chen, W. Tregs in immunotherapy: Opportunities and challenges. Immunotherapy 3, 911–914 (2011).
pubmed: 21843075
doi: 10.2217/imt.11.79
Zheng, S. G. et al. CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft-versus-host disease with a lupus-like syndrome. J. Immunol. 172, 1531–1539 (2004).
pubmed: 14734731
doi: 10.4049/jimmunol.172.3.1531
Davidson, T. S., DiPaolo, R. J., Andersson, J. & Shevach, E. M. Cutting edge: IL-2 is essential for TGF-beta-mediated induction of Foxp3+ T regulatory cells. J. Immunol. 178, 4022–4026 (2007).
pubmed: 17371955
doi: 10.4049/jimmunol.178.7.4022
Murai, M. et al. Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis. Nat. Immunol. 10, 1178–1184 (2009).
pubmed: 19783988
pmcid: 2898179
doi: 10.1038/ni.1791
Srivastava, S., Koch, L. K. & Campbell, D. J. IFNalphaR signaling in effector but not regulatory T cells is required for immune dysregulation during type I IFN-dependent inflammatory disease. J. Immunol. 193, 2733–2742 (2014).
pubmed: 25092894
doi: 10.4049/jimmunol.1401039
Uhlén, M. et al. The human secretome. Sci. Signal. 12, eaaz0274 (2019).
pubmed: 31772123
doi: 10.1126/scisignal.aaz0274
Ding, M. et al. A phenotypic screening approach using human Treg cells identified regulators of Forkhead Box p3 expression. ACS Chem. Biol. 14, 543–553 (2019).
pubmed: 30807094
doi: 10.1021/acschembio.9b00075
Jennbacken, K. et al. Phenotypic screen with the human secretome identifies FGF16 as inducing proliferation of iPSC-derived cardiac progenitor cells. Int. J. Mol. Sci. 20, 1–16 (2019).
doi: 10.3390/ijms20236037
Kanje, S. et al. Improvements of a high-throughput protein purification process using a calcium-dependent setup. Protein Expr. Purif. 175, 105698 (2020).
pubmed: 32681960
doi: 10.1016/j.pep.2020.105698
Tegel, H. et al. High throughput generation of a resource of the human secretome in mammalian cells. New Biotechnol. 58, 45–54 (2020).
doi: 10.1016/j.nbt.2020.05.002
Ding, M. et al. Secretome-based screening in target discovery. SLAS Discov. Adv. Life Sci. R&D 25, 535–551 (2020).
doi: 10.1177/2472555220917113
Lin, H. et al. Discovery of a cytokine and its receptor by functional screening of the extracellular proteome. Science 320, 807–811 (2008).
pubmed: 18467591
doi: 10.1126/science.1154370
Gonzalez, R. et al. Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proc. Natl. Acad. Sci. USA 107, 3552–3557 (2010).
pubmed: 20133595
pmcid: 2840467
doi: 10.1073/pnas.0914019107
Sakaguchi, S., Vignali, D. A., Rudensky, A. Y., Niec, R. E. & Waldmann, H. The plasticity and stability of regulatory T cells. Nat. Rev. Immunol. 13, 461–467 (2013).
pubmed: 23681097
doi: 10.1038/nri3464
Chang, J. H. et al. Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells. Nat. Immunol. 13, 481–490 (2012).
pubmed: 22484734
pmcid: 3361639
doi: 10.1038/ni.2267
van Loosdregt, J. et al. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity 39, 259–271 (2013).
pubmed: 23973222
pmcid: 4133784
doi: 10.1016/j.immuni.2013.05.018
Samstein, R. M. et al. Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 151, 153–166 (2012).
pubmed: 23021222
pmcid: 3493256
doi: 10.1016/j.cell.2012.06.053
Yegutkin, G. G. et al. Consequences of the lack of CD73 and prostatic acid phosphatase in the lymphoid organs. Mediat. Inflamm. 2014, 485743 (2014).
doi: 10.1155/2014/485743
Ortlund, E., LaCount, M. W. & Lebioda, L. Crystal structures of human prostatic acid phosphatase in complex with a phosphate ion and alpha-benzylaminobenzylphosphonic acid update the mechanistic picture and offer new insights into inhibitor design. Biochemistry 42, 383–389 (2003).
pubmed: 12525165
doi: 10.1021/bi0265067
Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003).
pubmed: 12612578
doi: 10.1038/ni904
Hori, S. Developmental plasticity of Foxp3+ regulatory T cells. Curr. Opin. Immunol. 22, 575–582 (2010).
pubmed: 20829012
doi: 10.1016/j.coi.2010.08.004
Chen, Q., Kim, Y. C., Laurence, A., Punkosdy, G. A. & Shevach, E. M. IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ T cells in vivo. J. Immunol. 186, 6329–6337 (2011).
pubmed: 21525380
doi: 10.4049/jimmunol.1100061
Curiel, T. J. Regulatory T cells and treatment of cancer. Curr. Opin. Immunol. 20, 241–246 (2008).
pubmed: 18508251
pmcid: 3319305
doi: 10.1016/j.coi.2008.04.008
Huber, S. & Schramm, C. TGF-beta and CD4+CD25+ regulatory T cells. Front. Biosci. 11, 1014–1023 (2006).
pubmed: 16146793
doi: 10.2741/1859
Mazerbourg, S. et al. Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses. J. Biol. Chem. 280, 32122–32132 (2005).
pubmed: 16049014
doi: 10.1074/jbc.M504629200
Velonas, V. M., Woo, H. H., dos Remedios, C. G. & Assinder, S. J. Current status of biomarkers for prostate cancer. Int. J. Mol. Sci. 14, 11034–11060 (2013).
pubmed: 23708103
pmcid: 3709717
doi: 10.3390/ijms140611034
Quintero, I. B. et al. Prostatic acid phosphatase is not a prostate specific target. Can. Res. 67, 6549–6554 (2007).
doi: 10.1158/0008-5472.CAN-07-1651
Deaglio, S. et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J. Exp. Med. 204, 1257–1265 (2007).
pubmed: 17502665
pmcid: 2118603
doi: 10.1084/jem.20062512
Rissiek, A. et al. The expression of CD39 on regulatory T cells is genetically driven and further upregulated at sites of inflammation. J. Autoimmun. 58, 12–20 (2015).
pubmed: 25640206
doi: 10.1016/j.jaut.2014.12.007
Schuler, P. J. et al. Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients. Eur. J. Immunol. 42, 1876–1885 (2012).
pubmed: 22585562
pmcid: 3689271
doi: 10.1002/eji.201142347
Vijayan, D., Young, A., Teng, M. W. L. & Smyth, M. J. Targeting immunosuppressive adenosine in cancer. Nat. Rev. Cancer 17, 709–724 (2017).
pubmed: 29059149
doi: 10.1038/nrc.2017.86
Theofilopoulos, A. N., Baccala, R., Beutler, B. & Kono, D. H. Type I interferons (alpha/beta) in immunity and autoimmunity. Annu. Rev. Immunol. 23, 307–336 (2005).
pubmed: 15771573
doi: 10.1146/annurev.immunol.23.021704.115843
Chen, K., Liu, J. & Cao, X. Regulation of type I interferon signaling in immunity and inflammation: A comprehensive review. J. Autoimmun. 83, 1–11 (2017).
pubmed: 28330758
doi: 10.1016/j.jaut.2017.03.008
Piconese, S., Pacella, I., Timperi, E. & Barnaba, V. Divergent effects of type-I interferons on regulatory T cells. Cytokine Growth Factor Rev. 26, 133–141 (2015).
pubmed: 25466634
doi: 10.1016/j.cytogfr.2014.10.012
Lee, S. E. et al. Type I interferons maintain Foxp3 expression and T-regulatory cell functions under inflammatory conditions in mice. Gastroenterology 143, 145–154 (2012).
pubmed: 22475534
doi: 10.1053/j.gastro.2012.03.042
Srivastava, S., Koch, M. A., Pepper, M. & Campbell, D. J. Type I interferons directly inhibit regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection. J. Exp. Med. 211, 961–974 (2014).
pubmed: 24711580
pmcid: 4010906
doi: 10.1084/jem.20131556
Loebbermann, J. et al. Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection. Mucosal Immunol. 5, 161–172 (2012).
pubmed: 22236998
pmcid: 3282434
doi: 10.1038/mi.2011.62
Gondek, D. C., Lu, L. F., Quezada, S. A., Sakaguchi, S. & Noelle, R. J. Cutting edge: Contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J. Immunol. 174, 1783–1786 (2005).
pubmed: 15699103
doi: 10.4049/jimmunol.174.4.1783
Schreiber, G. The molecular basis for differential type I interferon signaling. J. Biol. Chem. 292, 7285–7294 (2017).
pubmed: 28289098
pmcid: 5418031
doi: 10.1074/jbc.R116.774562
Matikainen, S. et al. Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells. Blood 93, 1980–1991 (1999).
pubmed: 10068671
doi: 10.1182/blood.V93.6.1980.406k20_1980_1991
Chen, C., Rowell, E. A., Thomas, R. M., Hancock, W. W. & Wells, A. D. Transcriptional regulation by Foxp3 is associated with direct promoter occupancy and modulation of histone acetylation. J. Biol. Chem. 281, 36828–36834 (2006).
pubmed: 17028180
doi: 10.1074/jbc.M608848200
Goldstein, J. D. et al. Inhibition of the JAK/STAT signaling pathway in regulatory T cells reveals a very dynamic regulation of Foxp3 expression. PLoS ONE 11, e0153682 (2016).
pubmed: 27077371
pmcid: 4831811
doi: 10.1371/journal.pone.0153682
Jansson, A. M. et al. The interleukin-like epithelial–mesenchymal transition inducer ILEI exhibits a non-interleukin-like fold and is active as a domain-swapped dimer. J. Biol. Chem. 292, 15501–15511 (2017).
pubmed: 28751379
pmcid: 5602407
doi: 10.1074/jbc.M117.782904
Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).
pubmed: 27043002
doi: 10.1038/nbt.3519
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
pubmed: 25516281
pmcid: 4302049
doi: 10.1186/s13059-014-0550-8
Varemo, L., Nielsen, J. & Nookaew, I. Enriching the gene set analysis of genome-wide data by incorporating directionality of gene expression and combining statistical hypotheses and methods. Nucleic Acids Res. 41, 4378–4391 (2013).
pubmed: 23444143
pmcid: 3632109
doi: 10.1093/nar/gkt111
Liberzon, A. et al. The molecular signatures database (MSigDB) hallmark gene set collection. Cell Syst. 1, 417–425 (2015).
pubmed: 26771021
pmcid: 4707969
doi: 10.1016/j.cels.2015.12.004
Hanzelmann, S., Castelo, R. & Guinney, J. GSVA: Gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics 14, 7 (2013).
pubmed: 23323831
pmcid: 3618321
doi: 10.1186/1471-2105-14-7
Birmingham, A. et al. Statistical methods for analysis of high-throughput RNA interference screens. Nat. Methods 6, 569–575 (2009).
pubmed: 19644458
pmcid: 2789971
doi: 10.1038/nmeth.1351