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T Cells Deficient in the Tyrosine Phosphatase SHP-1 Resist Suppression by Regulatory T Cells

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T Cells Deficient in the Tyrosine Phosphatase SHP-1 Resist Suppression by Regulatory T Cells T Cells Deficient in the Tyrosine Phosphatase SHP-1 Resist Suppression by Regulatory T Cells This information is current as Emily R. Mercadante and Ulrike M. Lorenz of September 29, 2021. J Immunol published online 26 May 2017 http://www.jimmunol.org/content/early/2017/05/26/jimmun ol.1602171 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/05/26/jimmunol.160217 Material 1.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 26, 2017, doi:10.4049/jimmunol.1602171 The Journal of Immunology T Cells Deficient in the Tyrosine Phosphatase SHP-1 Resist Suppression by Regulatory T Cells Emily R. Mercadante and Ulrike M. Lorenz The balance between activation of T cells and their suppression by regulatory T cells (Tregs) is dysregulated in autoimmune diseases and cancer. Autoimmune diseases feature T cells that are resistant to suppression by Tregs, whereas in cancer, T cells are unable to mount antitumor responses due to the Treg-enriched suppressive microenvironment. In this study, we observed that loss of the tyrosine phosphatase SHP-1, a negative regulator of TCR signaling, renders naive CD4+ and CD8+ T cells resistant to Treg- mediated suppression in a T cell–intrinsic manner. At the intracellular level, SHP-1 controlled the extent of Akt activation, which has been linked to the induction of T cell resistance to Treg suppression. Finally, under conditions of homeostatic expansion, SHP-1–deficient CD4+ T cells resisted Treg suppression in vivo. Collectively, these data establish SHP-1 as a critical player in setting the threshold downstream of TCR signaling and identify a novel function of SHP-1 as a regulator of T cell susceptibility to Downloaded from Treg-mediated suppression in vitro and in vivo. Thus, SHP-1 could represent a potential novel immunotherapeutic target to modulate susceptibility of T cells to Treg suppression. The Journal of Immunology, 2017, 199: 000–000. egulatory T cells (Tregs) play an essential role in shaping SHP-1 is a cytoplasmic protein tyrosine phosphatase expressed T cell responses and maintaining immune homeostasis in all hematopoietic cells, which has been implicated in the reg- (1). Deficits in Treg function or number allow T cell ulation of TCR-mediated signaling in T cells (9), including the R http://www.jimmunol.org/ responses to go unchecked, leading to the development of auto- PI3K/Akt pathway (10). We (11) and others (12, 13) have previ- immunity and chronic inflammatory diseases (2). Dysregulation of ously shown that SHP-1–deficient T cells are hyperresponsive to the balance between activation and suppression of T cells can also TCR stimulation. This was done using the motheaten (me/me) occur when T cells become resistant to Treg-mediated suppression mouse model, in which all hematopoietic cells lack SHP-1 due to (2). Many autoimmune diseases, including type 1 diabetes, mul- a splicing mutation (14), as well as cell lines expressing dominant- tiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, negative mutant forms of SHP-1 (12). However, one recent study and inflammatory bowel disease, feature not only impaired Tregs (15), using conditional T cell deletion of SHP-1 via CD4-Cre, but also T cells that are resistant to suppression (3). However, the challenged the role of SHP-1 in regulating T cell development, potential mechanisms by which T cells might acquire resistance to whereas another report using the same mouse model confirmed the by guest on September 29, 2021 Treg-mediated suppression remain unclear. Although several ex- role of SHP-1 during T cell development (16). In this study, we tracellular factors have been linked to inducing resistance in generated a conditional knockout mouse model wherein SHP-1 T cells (3), the intracellular signaling mechanisms that can render deletion is driven by the distal Lck promoter (17), resulting in T cells resistant to Treg suppression are poorly defined. Further- abrogation of SHP-1 expression in postselection thymocytes. This more, strong activation through the TCR and/or costimulatory model allows largely normal T cell development such that any receptors can cause T cells to become refractory to Treg sup- phenotypic and/or functional alterations observed due to SHP-1 pression (4–8), but the specific pathways allowing this resistance deficiency can be directly ascribed to its role in mature T cells (11, remain elusive. Similarly, whereas resistance to suppression oc- 12, 18). Using this approach, we show that SHP-1 negatively curs in both CD4+ and CD8+ T cells (3), whether resistance is regulates the activation and proliferation of CD4+ and CD8+ induced by the same mechanism in both subsets is unknown. T cells in response to TCR stimulation, and that in the absence of SHP-1, T cells become resistant to Treg-mediated suppression. 2/2 Carter Immunology Center, University of Virginia, Charlottesville, VA 22908; and Such resistance is T cell–intrinsic, as SHP-1 T cells could not Department of Microbiology, Immunology, and Cancer Biology, University of induce “bystander resistance” when cocultured with wild-type Virginia, Charlottesville, VA 22908 T cells. Our data also suggest a role for the PI3K/Akt pathway ORCID: 0000-0002-7933-6855 (U.M.L.). in mediating both CD4+ and CD8+ T cells to resist suppression. Received for publication December 27, 2016. Accepted for publication April 24, This resistance of CD4+ SHP-12/2 T cells to Treg-mediated 2017. suppression was also observed during homeostatic expansion This work was supported by National Institutes of Health/National Institute of Al- in vivo. Collectively, these data identify a novel function of SHP-1 lergy and Infectious Diseases Grant 1F31AI110146 (to E.R.M.), National Institute of General Medical Sciences Grant 5R01GM064709 (to U.M.L.), and by National in regulating the susceptibility of T cells to Treg-mediated sup- Heart, Lung, and Blood Institute Grant 1P01HL120840 (to U.M.L.). pression in vitro and in vivo, through controlling the strength of Address correspondence and reprint requests to Dr. Ulrike M. Lorenz, Univer- signal received via the TCR and attenuating subsequent activation sity of Virginia, Carter Immunology Center, Carter-Harrison Research Build- of the downstream PI3K/Akt pathway. ing, 345 Crispell Drive, Charlottesville, VA 22908. E-mail address: ulorenz@ virginia.edu The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: CAR, chimeric Ag receptor; dLck, distal Lck; f/f, Mice flox/flox; MFI, mean fluorescence intensity; Tcon, conventional T; Treg, regulatory flox/flox f/f T cell. SHP-1 (SHP-1 ) mice (19) (provided by B. Neel) were crossed to distal Lck-Cre (dLck-Cre) mice (17) purchased from The Jackson Labo- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 ratory (Bar Harbor, ME). Genotyping of all mice was done by PCR as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602171 2 SHP-1–DEFICIENT T CELLS RESIST Treg SUPPRESSION described previously for the SHP-1f/f allele (19) and dLck-Cre alelle (17). singlets, live cells, CD4+ cells, and CellTrace Violet+ cells, the percentage For all experiments, 6- to 10-wk-old female and male mice were used, and of responding (dividing) cells relative to the input was obtained using the control mice were dLck-Cre2 SHP-1f/f or dLck-Cre+ SHP-1+/+ littermates provided software algorithm. + f/f 2/2 of dLck-Cre SHP-1 mice. Rag1 mice were purchased from The Analysis of suppression assay. To compensate for the increased baseline Jackson Laboratory. CD45.1 wild-type C57BL/6 mice were purchased responsiveness of SHP-12/2 T cells, the percentage of responding cells in from Charles River Laboratories. All mice were bred and maintained in the no Treg condition was set to 100% (maximum responsiveness) for each accordance with the policies of the Institutional Animal Care and Use genotype. The percentage of responding cells was calculated as described Committee at the University of Virginia. All experiments involving mice above for the proliferation analyses for all Treg/T cell ratios and nor- were conducted with the approval of Institutional Animal Care and Use malized to the maximum responsiveness for their own genotype (no Treg Committee. condition). Percentage suppression equals 100 minus the percentage responding cells. Isolation and purification of primary cells Twenty-four hour T cell activation CD4+ T cells were isolated from peripheral lymph nodes (combined in- guinal, axillary, brachial, cervical, sacral, and renal nodes) or spleens by CD4+CD252 Tcon cells or naive (CD44lo) CD8+ T cells were isolated negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec, from spleens of indicated mice, and 2.5 3 104 cells were cultured per well Auburn, CA) according to the manufacturer’s protocol. CD8+ T cells were in a 96-well round-bottom plate with 5 3 104 irradiated (2000 rad) CD4+ isolated from spleens by negative selection using the CD8a+ T cell iso- T cell–depleted splenocytes and indicated doses of anti-CD3 Ab (2C11; lation kit (Miltenyi Biotec).
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