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

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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). For naive CD4 or CD8 T cell experiments, Cedarlane Laboratories). After 24 h, cells were harvested and stained for CD4+CD44lo or CD8+CD44lo T cells were isolated from spleens by neg- flow cytometric analysis of CD25 and pAkt T308 expression. ative selection using a naive CD4 T cell isolation kit or the CD8 T cell isolation kit, respectively (Miltenyi Biotec). For splenic T cell isolation, Immunoblotting RBCs were lysed using BD Pharm Lyse buffer (BD Biosciences, San Jose, + 3 5 Downloaded from CA) before T cell isolation. For Treg isolation, CD4+ T cells were sub- SHP-1 protein level in CD4 T cells was assessed by lysing 5 10 cells in sequently labeled with CD25-PE to separate conventional T (Tcon) cells Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM sodium chloride, + 2 + + + m (defined as CD4 CD25 ) and Tregs (CD4 CD25 ). Labeled CD4 cells 50 mM Tris, 4 mM sodium pyrophosphate, 5 mM sodium fluoride, 10 g/ml sodium vanadate, 50 mg/ml antipain, 40 mg/ml PMSF, 13 protease inhibitor were run on an autoMACS Pro separator (Miltenyi Biotec) using the + cocktail [Sigma-Aldrich, St. Louis, MO]) and resolving lysates on an Any posseld2 program to obtain Tregs with .85% purity as assessed by Foxp3 CD25+ staining. CD4+ T cell–depleted splenocytes were irradiated (2000 KD TGX Tris-glycine-SDS gel (Bio-Rad Laboratories, Hercules, CA). Blots rad) and used as APCs in culture where indicated. were probed with monoclonal anti–SHP-1 (clone 1SH01; NeoMarkers/ Thermo Fisher Scientific, Fremont, CA) and reprobed for b-actin as a http://www.jimmunol.org/ Flow cytometry loading control (anti–b-actin-HRP, clone AC-15; Sigma-Aldrich). Blots were imaged using the ChemiDoc Touch gel imaging system (Bio-Rad Cells were stained directly after isolation or harvested after 24, 72, or 96 h of Laboratories). Bands densities were quantified using ImageLab (Bio-Rad culture as indicated. Cells were surface stained with anti-CD4, anti-CD25 Laboratories) software after normalization to loading control. (eBioscience, San Diego, CA), anti-CD8, anti-CD44, anti-CD62L, anti- CD69, anti-CD45.1, and anti-CD45.2 (BD Biosciences) in PBS supple- In vivo T cell transfer mented with 1% BSA and 0.1% sodium azide. Staining for live cells was Tcon (CD4+CD252) cells were isolated by MACS as described above from done following surface staining and washing, using Live/Dead fixable dye spleens of CD45.2 SHP-1+/+ or SHP-12/2 mice and CD45.1 wild-type (Life Technologies, Carlsbad, CA). Cells were then fixed with BD Fix/Lyse mice. Tcon cells were labeled with 5 mM CellTrace Violet, and Treg (BD Biosciences) and washed. For intracellular Foxp3 staining, cells were (CD4+CD25+) cells were isolated from SHP-1+/+ mice and pooled. Tcon fixed and permeabilized using a Foxp3 staining buffer set (eBioscience) cells were resuspended at a 1:1 ratio of either CD45.2 SHP-1+/+/CD45.1 by guest on September 29, 2021 according to the manufacturer’s protocol and stained with anti-Foxp3 wild-type Tcon cells or CD45.2 SHP-12/2/CD45.1 wild-type Tcon cells, (eBioscience). For pAkt intracellular staining, cells were fixed and per- and a total of 3 3 106 Tcon cells from either mix were injected i.v. in 200 ml meabilized using BD Cytofix/Cytoperm (BD Biosciences) according to the of sterile PBS via the tail vein into Rag12/2 recipients. Additionally, half manufacturer’s protocol and stained with anti-pAkt T308 (Cell Signaling the recipient mice also received 7.5 3 105 SHP-1+/+ Tregs along with Tcon Technology, Danvers, MA). For caspase-3 staining, cells were stained with cells (1:4 Treg/Tcon ratio). After 10 d, spleens were harvested from recipient the CaspGLOW kit (eBioscience) according to the manufacturer’s protocol mice and stained for flow cytometric analysis. Donor and recipient mice for the last 60 min of in vitro culture. Stained cells were collected on a BD were age matched. FACSCanto I or II, using FACSDiva version 8 software (BD Biosciences), or using Beckman Coulter CytoFLEX and CytExpert software (Beckman Statistical analysis Coulter, Brea, CA), and subsequent analyses were done using FlowJo software version 9.9 or version 10.1 (FlowJo, Ashland, OR). Analyses T cell proliferation, CD25 upregulation, proliferation index, and suppres- + 2 + were performed on singlet-gated cells as defined by forward scatter width sion assays using CD4 CD25 or total CD8 T cells were analyzed using a versus forward scatter area, and live cells as defined by Live/Dead dye three-way ANOVA with a 95% confidence interval. A Student t test was negative. Gates were set based on fluorescence minus one controls. used to analyze the comparison of percentage and absolute number of CD44hi T cells from SHP-1+/+ or SHP-12/2 mice. A Student t test was Proliferation and suppression assays used to analyze naive CD4+ and naive CD8+ T cell suppression assay data for each Treg/T cell ratio. A two-way ANOVA with a Sidak multiple Assessment via CellTrace Violet dilution. To assess proliferation, isolated comparison posttest was used to analyze cell death and apoptosis data. A T cells (CD4+CD252 [Tcon cells], CD4+CD44lo [naive CD4+ T cells], + + lo + one-way ANOVA with a Tukey multiple comparison posttest was per- CD8 , or CD8 CD44 [naive CD8 T cells]) were stained with 5 mM formed to analyze the absolute number of lymph node and splenic T cells CellTrace Violet for 20 min at 37˚C followed by quenching with pre- in dLck-Cre SHP-1f/f mice, the percentage of responding cells in coculture warmed complete RPMI 1640 for 5 min at 37˚C (Life Technologies). experiments, and the percentage suppression of in vivo T cell transfer 3 4 Stained cells were washed, and 2.5 10 T cells were plated (in qua- experiments. For pAkt T308 flow cytometric data, a one-column t test was druplicate, pooled at time of harvest) in a total volume of 200 ml of RPMI applied to the fold change pAkt mean fluorescence intensity (MFI) values 1640 complete medium (supplemented with 10% FBS, 50 mM 2-ME, of SHP-12/2 T cells compared with the pAkt MFI of SHP-1+/+ T cells for 2mML-glutamine, 10 mM HEPES, MEM nonessential amino acids, each anti-CD3 dose, with a null hypothesis of 1 (if no change from control, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin) in round- fold change = 1). A p value of #0.05 was considered significant. bottom 96-well plates. Irradiated (2000 rad), CD4+ T cell–depleted splenocytes were added at 5 3 104 cells per well along with anti-CD3 Ab (2C11; Cedarlane Laboratories, Burlington, NC) at 10–1000 ng/ml as in- Results dicated. For suppression assays, CD4+CD25+ Tregs were plated with re- SHP-1 sets threshold for activation and proliferation of T cells sponder T cells at indicated ratios. For proliferation assays, cells were in response to TCR stimulation cultured for 72 or 96 h, and for suppression assays cells were cultured for 96 h followed by flow cytometric analyses. Although several previous studies have suggested that SHP-1 is a Analysis of proliferation assay. CellTrace Violet dilution was assessed by negative regulator of signaling downstream of the TCR, based on flow cytometry and subsequently analyzed using FlowJo v9.9 software in vitro cell culture studies and primary T cells from total body proliferation Wizard platform (FlowJo). Briefly, after sequentially gating on knockout of SHP-1 (reviewed in Ref. 9), a recent study performed The Journal of Immunology 3 using conditional deletion of SHP-1 in T cells has disagreed with cell cycle time. To assess whether SHP-12/2 Tcon cells had an this notion (15). One potential reason for this discrepancy might in vitro survival advantage over SHP-1+/+ Tcon cells, we stained have been the type of Cre line that was used to delete SHP-1 by cells for activated caspase-3, a marker of apoptosis. After 24 h in Johnson et al. (15) as the CD4-Cre used gets expressed from culture, we observed very little apoptosis among Tcon cells (,1%) earlier stages of T cell development. To test this possibility, we with no significant difference between SHP-1+/+ or SHP-12/2 Tcon crossed mice carrying floxed alleles of Ptpn6 (Shp1) (19) with cells in terms of apoptosis or cell death (Supplemental Fig. 2A, 2B). mice that express Cre recombinase under the control of the distal By 72 h, we still observed very low levels of apoptosis (#2%) with promoter of Lck (17). The dLck promoter drives Cre expression at no statistically significant differences between SHP-12/2 Tcon cells late stages of T cell development, allowing TCR-dependent se- and SHP-1+/+ Tcon cells (Fig. 1E). Taken together, these data lection to occur under conditions of SHP-1 sufficiency (12, 16, 18, demonstrate that SHP-1 controls the extent of TCR/CD3-driven 20). We confirmed that SHP-1 was deleted in peripheral CD4+ proliferation by setting the threshold that determines the subpopu- (Supplemental Fig. 1A) and CD8+ T cells (Supplemental Fig. 1B) lation of T cells responding to a given TCR stimulation. + f/f from the lymph nodes and spleen of dLck-Cre SHP-1 (referred + to here as SHP-12/2) mice. Importantly, we observed no changes CD4 T cells lacking SHP-1 resist in vitro Treg suppression in the composition of the thymic or peripheral T cell compart- Because our data indicated that SHP-1 lowered the threshold for ments with respect to absolute numbers (Supplemental Fig. 1C) or Tcon cell activation and proliferation, we asked whether SHP-1 percentages of CD4+ or CD8+ T cells or Tregs in the lymph nodes also regulated the susceptibility of Tcon cells to Treg-mediated or spleens (Supplemental Fig. 1D, 1E). Expression of dLck-Cre suppression. Using an in vitro suppression assay, Tcon cells 2 2 alone did not affect the peripheral T cell compartment, consistent from SHP-1 / and SHP-1+/+ mice were assessed for their sus- Downloaded from with previous reports (21). ceptibility to wild-type Treg-mediated suppression (Fig. 2A). To assess the role of SHP-1 during T cell activation, we first Strikingly, SHP-12/2 Tcon cells displayed ∼3-fold greater re- compared the proliferation capacities of SHP-1+/+ and SHP-12/2 sponsiveness compared with SHP-1+/+ Tcon cells, even at the CD4+CD252 T cells, hereafter referred to as Tcon cells, to dif- maximally suppressive condition (Fig. 2A). Even after normali- ferentiate them from CD4+ Tregs. We found that a greater per- zation to account for the increased baseline proliferation (no Treg 2/2 2/2 centage of SHP-1 Tcon cells proliferated compared with Tcon condition in Fig. 2A), SHP-1 Tcon cells were significantly less http://www.jimmunol.org/ cells from SHP-1+/+ mice. Enhanced proliferation in SHP-12/2 suppressed by Tregs than were SHP-1+/+ Tcon cells (Fig. 2B), Tcon cells was especially apparent at suboptimal concentrations indicating that SHP-1 can influence the level of susceptibility to of anti-CD3 stimulation (Fig. 1A). We considered three reasons in vitro Treg-mediated suppression. for SHP-12/2 Tcon cells to display the observed increase in To determine whether the observed resistance to suppression in proliferation, which are not mutually exclusive: 1) an increase in Tcon cells could be attributable to an expanded memory T cell the percentage of cells that initially become activated and go on to population in mice with SHP-12/2 T cells (23, 24), we first proliferate, 2) a decreased cell cycle time, and/or 3) an increased assessed whether there were any differences in the memory T cell survival of cells in the culture. We first determined whether a compartment of dLck-Cre SHP-1f/f mice compared with SHP-1+/+ greater proportion of SHP-12/2 Tcon cells responded to TCR mice, as has been described for me/me mice (25) and CD4-Cre by guest on September 29, 2021 stimulation by using the FlowJo proliferation platform algorithm, SHP-1f/f mice (15, 16). However, we did not observe an increase which takes into account the number of cells in each round of in percentage (Supplemental Fig. 3A, 3B) or absolute number division relative to the input cells (22), and thereby estimates the (Supplemental Fig. 3C) of Ag-experienced/memory-like CD44hi fraction of T cells that initially responded to the stimulation. CD4+CD252Foxp32 Tcon cells in the lymph nodes or spleens of Based on this metric, we found a significant increase in the per- dLck-Cre+ SHP-1f/f mice compared with control SHP-1–sufficient centage of responding SHP-12/2 Tcon cells compared with mice. As further indication that the composition of the CD4+ Tcell SHP-1+/+ Tcon cells, with the largest difference at the lowest compartment in the dLck-Cre+ SHP-1f/f mice was not altered, we stimulation dose (Fig. 1B). To complement this finding, we detected no differences in the percentage of cells expressing acti- assessed the upregulation of CD25 (IL-2Ra) as a measure of early vation markers CD69 or CD25 (data not shown). Furthermore, Tcon cell activation and found that a significantly greater per- SHP-12/2 CD4+ T cells depleted of the CD44hi subpopulation centage of SHP-12/2 Tcon cells were CD25+ after 24 h of stim- (referred to here as naive CD44lo Tcells,SupplementalFig.3D) ulation compared with SHP-1+/+ Tcon cells (Fig. 1C). Importantly, retained a greater responsiveness to TCR stimulation (Supplemental we observed no CD25 upregulation on Tcon cells of either ge- Fig. 3E, 3F), without any changes in cell cycle time. These data are notype in the absence of anti-CD3 stimulation, indicating that any consistent with SHP-1 regulating signaling downstream of the TCR observed T cell activation was TCR/CD3 stimulation-dependent. in naive T cell subsets. Moreover, SHP-12/2 naive CD4+ Tcells Interestingly, SHP-1+/+ Tcon cells reached a maximum percentage were resistant to Treg-mediated suppression in vitro (Fig. 2C, 2D), of CD25+ cells at 150 ng/ml anti-CD3, with no further increase at confirming what we observed in the CD4+CD252 Tcon population. 1000 ng/ml anti-CD3, whereas the subpopulation of responding Taken together, these data suggest that SHP-1 regulates the sus- SHP-12/2 Tcon cells increased further at 1000 ng/ml anti-CD3 ceptibility of CD4+ T cells to Treg-mediated suppression in vitro. compared with 150 ng/ml. Upregulation of CD69, another marker + of activation, followed the same pattern (data not shown). These CD8 T cells lacking SHP-1 also resist in vitro Treg data suggest that there is a greater percentage of SHP-12/2 Tcon suppression cells that respond and are activated by any given stimulation. Resistance of T cells to Treg-mediated suppression has not only Second, when we calculated the proliferation index of each been observed in CD4+ T cells, but also in CD8+ T cells (26–29), sample (using the FlowJo proliferation platform that provides the which has important clinical implications for cancer immuno- average number of cellular divisions of the cells that divided in therapy and chronic viral infection therapies. Similar to SHP-12/2 culture), SHP-12/2 and SHP-1+/+ Tcon cells underwent compa- CD4+ T cells, SHP-12/2 CD8+ T cells exhibited greater respon- rable rounds of divisions at any given dose of stimulation, with a siveness to TCR stimulation compared with SHP-1+/+ CD8 T cells slight increase in divisions at higher concentrations of stimulation (Fig. 3A) without any detectable changes in cell cycle time (data (Fig. 1D). These data indicate that SHP-1 deficiency did not affect not shown). SHP-12/2 CD8+ T cells also resisted Treg-mediated 4 SHP-1–DEFICIENT T CELLS RESIST Treg SUPPRESSION

FIGURE 1. SHP-1 limits the number of T cells responding to TCR stimulation. (A) Seventy-two hour proliferation of splenic CellTrace Violet–labeled CD4+CD252 T cells isolated from SHP-1+/+ or SHP-12/2 mice. Proliferation was measured in response to indicated concentrations of anti-CD3 and irradiated CD4+ T cell–depleted splenocytes as APCs. Histograms shown are representative of three independent experiments; n = 5–7 per genotype. Note 2 2 that the number of cells on the y-axis for histograms is greater for SHP-1 / T cells than SHP-1+/+ T cells. (B) Percentage of CD4+ T cells within each Downloaded from culture initially responding to the indicated stimulation. Data were obtained from the proliferation assays presented in (A). Percentage of T cell responders was calculated using the precursor frequency algorithm of the FlowJo proliferation platform, which takes into account the number of cells in each round of division relative to the input cells, and thereby estimates the fraction of T cells that initially responded to the stimulation. (C) Proliferation assays were set up as described in (A), but cells were harvested after 24 h and assessed for CD25 surface expression. Data represent three independent experiments; n = 5–9 per genotype. (D) Proliferation index, which corresponds to the average rounds of division of T cells, was obtained from the proliferation assays presented in (A) using the FlowJo proliferation platform. (E) Proliferation assays were set up as described in (A). Cells were stained for activated caspase-3 (Casp3) with Fitc-DEVD-FMK for the last hour of culture before harvest and ﬂow cytometric analyses. Data represent percentage of Casp3+ cells within CD4+ T http://www.jimmunol.org/ cell population; n = 3 per genotype. A standard regression ANOVA was performed for (B)–(D), a two-way ANOVA with a Sidak multiple comparison posttest was used for statistical analysis of (E). Error bars indicate 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001. suppression (Fig. 3A, 3B). However, in contrast to the CD4+ T cell (Supplemental Fig. 4). To determine whether SHP-1 deﬁciency compartment, we did observe a substantial increase in the per- also conferred naive CD8+ T cells with resistance to Treg sup- centage and number of CD8+CD44hi T cells in the lymph nodes pression, we isolated naive CD8+ (CD44lo) T cells and measured and spleens of SHP-12/2 mice compared with SHP-1+/+ mice their suppression in vitro (Fig. 3C). A greater proportion of by guest on September 29, 2021

FIGURE 2. SHP-12/2 CD4+ T cells resist Treg-mediated suppression. (A) Splenic CD4+CD252 Tcon cells were isolated from SHP-1+/+ or SHP-12/2 mice, labeled with CellTrace Violet, and cultured either alone or with wild-type Tregs at the indicated ratios in the presence of 150 ng/ml anti-CD3 and irradiated CD4+ T cell–depleted splenocytes as APCs, and proliferation was measured after 4 d. Histograms shown are representative of five independent experiments; n=8–10 mice per genotype. Bold numbers indicate most significant differences observed. (B) Suppression was calculated by normalizing each data point to the corresponding baseline proliferation (no Tregs, maximal response = 100% proliferation), which was then subtracted from 100% proliferation. Note that as described in Fig. 1, proliferation was computed using the FlowJo proliferation platform, which takes into account the number of cells in each round of division relative to the input cells, and thereby estimates the fraction of T cells that initially responded to the stimulation. A three-way ANOVA was performed. (C) Naive CD4+CD44lo T cells were purified from spleens of SHP-1+/+ or SHP-12/2 mice and suppression assays were performed as described in (A) in the presence of 30 ng/ml anti-CD3 and CD4+ T cell–depleted splenocytes as APCs; n = 3 mice per genotype. (D) Suppression was calculated as in (B). Student t tests were performed for each Treg/T cell ratio. Error bars indicate 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001. The Journal of Immunology 5 Downloaded from

FIGURE 3. SHP-12/2 CD8+ T cells resist Treg-mediated suppression. (A) Splenic CD8+ T cells were isolated from SHP-1+/+ or SHP-12/2 mice, labeled http://www.jimmunol.org/ with CellTrace Violet, and cultured either alone or with wild-type Tregs at the indicated ratios in the presence of 10 ng/ml anti-CD3 and CD4+ T cell– depleted splenocytes as APCs, and proliferation was measured after 3 d. Data are representative of two independent experiments; n = 4 mice per genotype. (B) The percentage responding cells was obtained using the FlowJo proliferation platform as described in Figs. 1 and 2. Suppression was calculated by normalizing each data point to the corresponding baseline proliferation (no Tregs, maximal response = 100% proliferation), which was then subtracted from 100% proliferation. A three-way ANOVAwas performed. (C) Naive CD8 T cells (CD8+CD44lo) were isolated, labeled with CellTrace Violet, and cultured with Tregs as described in (A); n = 3 mice per genotype. (D) Percentage suppression was obtained as in (B). Student t tests were performed for each Treg/T cell ratio. Error bars indicate 6 SEM. *p # 0.05, **p # 0.01.

SHP-12/2 naive CD8+ T cells responded to TCR stimulation in (Fig. 4B). We found that adding SHP-12/2 Tcon cells to SHP-1+/+ by guest on September 29, 2021 the absence of Tregs (Fig. 3C). Similar to what we observed for Tcon cells did not enhance the response of the SHP-1+/+ Tcon CD4+ T cells, there were no significant differences in cell death or cells (Fig. 4B), indicating that the enhanced responsiveness to apoptosis between SHP-1+/+ and SHP-12/2 naive CD8+ T cells TCR stimulation cannot be transmitted to neighboring T cells. We after 24 h of stimulation across a range of anti-CD3 stimulation next asked whether SHP-12/2 Tcon cells could render their local (Supplemental Fig. 2C, 2D). There was also no observed survival environment resistance promoting for Treg-mediated suppression, advantage in SHP-12/2 naive CD8+ T cells after 3 d of stimula- perhaps via the production of cytokines or other factors that could tion, and in fact at the highest dose of stimulation, SHP-12/2 directly influence APCs. If this were the case, one would expect naive CD8+ T cells displayed enhanced apoptosis compared with that SHP-12/2 Tcon cells would be capable of inducing bystander SHP-1+/+ cells, possibly due to an increase in activation-induced resistance in SHP-1+/+ Tcon cells exposed to the same environ- cell death (Supplemental Fig. 2E). Furthermore, SHP-12/2 naive ment. Using the same experimental coculture setup as above, but CD8+ T cells exhibited resistance to suppression, similar to the in the presence of wild-type Tregs, the addition of SHP-12/2 Tcon total CD8+ T cell population (Fig. 3D), indicating that the phe- cells to SHP-1+/+ Tcon cells did not induce any resistance to notype was independent of the expanded Ag-experienced/ suppression in the SHP-1+/+ Tcon cell population (Fig. 4C). These memory-like CD8+ T cell subpopulation. These data demon- data suggest that SHP-12/2 Tcon cells resist Treg suppression by strate a role for SHP-1 in regulating susceptibility of not only a cell-intrinsic mechanism, which does not affect neighboring CD4+ T cells, but also CD8+ T cells, to Treg-mediated suppres- cells or induce bystander resistance. sion, likely by a similar mechanism in both T cell subsets. SHP-1 deficiency enhances activation of the Akt pathway SHP-1 regulates TCR signaling and susceptibility to Treg Our data suggested that SHP-1 deficiency led to intracellular suppression in a cell-intrinsic manner changes in the signaling pathways downstream of the TCR, which To further understand how SHP-1 regulates signaling downstream might ultimately mediate T cell resistance to suppression. A of TCR/CD3 stimulation and susceptibility to Treg suppression, we number of reports have implicated enhanced activation of PI3K/ asked whether these phenotypes occurred in a cell-intrinsic and/or Akt in conferring T cells with resistance to suppression (24, 27, cell-extrinsic manner. To investigate whether SHP-12/2 CD4+ 29–33). Moreover, it was previously demonstrated that SHP-1 CD252 Tcon cells could transfer their enhanced TCR respon- negatively regulates the PI3K/Akt pathway in me/me thymocytes siveness to neighboring SHP-1+/+ Tcon cells via a soluble medi- (10, 34). We therefore assessed the phosphorylation of Akt at ator, we set up cocultures (Fig. 4A); we labeled either SHP-1+/+ or T308 as a measure of Akt activation (35) in response to TCR/CD3 SHP-12/2 Tcon cells with CellTrace Violet proliferation dye, stimulation. At 24 h poststimulation, SHP-12/2 CD4+ T cells mixed them at a 1:1 ratio with SHP-1+/+ or SHP-12/2 Tcon cells, displayed enhanced Akt phosphorylation over a range of TCR respectively, and assessed the proliferation of labeled cells stimulation conditions compared with SHP-1+/+ CD4+ T cells 6 SHP-1–DEFICIENT T CELLS RESIST Treg SUPPRESSION

FIGURE 4. SHP-1–mediated T cell phenotypes are cell-intrinsic. (A) Schematic representation of experimental setup. Splenic CD4+CD252 Tcon cells were isolated from SHP-1+/+ or SHP-12/2 mice and labeled with CellTrace dyes. Differently labeled Tcon cells of indicated genotypes were cocultured at a 1:1 ratio in the presence of 30 ng/ml anti-CD3 and irradiated CD4+ T cell–depleted splenocytes as APCs. (B) After 72 h, the proliferation of the CellTrace Violet–labeled cells was measured and assessed using the FlowJo proliferation platform as in Fig. 1. Graph shows percentage responding cells of indicated genotype, compiled from two independent experiments; n = 6 mice per genotype. (C) The same setup as in (B) was used, with the addition of wild-type Tregs at a ratio of 1:4 of Treg/total Tcon cells. After 96 h, proliferation of CellTrace Violet–labeled cells was measured and analyzed as in (B). Graph shows percent suppression (calculated as in Figs. 2 and 3). A one-way ANOVA was performed on data in (B) and (C). Error bars indicate 6 SEM. *p # 0.05.

(Fig. 5A, 5B). Additionally, there was also a slightly higher suppression are not well defined. In this study, we identify the Downloaded from baseline activation of Akt in SHP-12/2 CD4+ T cells that received tyrosine phosphatase SHP-1 as one of the intracellular regulators no TCR/CD3 stimulation. We observed the same enhanced Akt of Tcon cells that influence their susceptibility to Treg suppres- activation in SHP-12/2 CD8+ T cells compared with SHP-1+/+ sion. Both SHP-12/2 CD4+ and CD8+ T cells resisted Treg sup- CD8+ T cells, both at baseline as well as following TCR/CD3 pression of proliferation in vitro, and SHP-12/2 CD4+ T cells resisted stimulation (Fig. 5C, 5D). Taken together, these data suggest Treg suppression of homeostatic expansion in vivo. Moreover, 2/2 2/2 that enhanced activation of the PI3K/Akt pathway in SHP-1 SHP-1 T cells resisted Treg suppression in a T cell–intrinsic http://www.jimmunol.org/ T cells may provide one component of resistance to Treg- manner, as coculture (Fig. 4) or coinjection (Fig. 6) of SHP-1+/+ mediated suppression, similar to what has been described for (wild-type) and SHP-12/2 CD4+ T cells could not induce SHP-1+/+ T cells isolated from patients with lupus (32), multiple sclerosis (wild-type) CD4+ T cells to become resistant to suppression. (29), and juvenile idiopathic arthritis (24, 33). SHP-12/2 T cells have been reported to produce increased amounts of IL-4 when stimulated in vitro, and SHP-1 additionally negatively Tcon cells lacking SHP-1 resist Treg-mediated suppression regulates the subsequent downstream phosphorylation of STAT6, in vivo suggesting that SHP-12/2 T cells are hyperresponsive to IL-4 sig- To assess whether SHP-1 regulates the susceptibility to Treg naling (15). Because IL-4 has been shown to induce resistance to mediated suppression in vivo, we used a murine model of Treg- Treg suppression in vitro (37), it raised the possibility that IL-4 might by guest on September 29, 2021 mediated control of homeostatic expansion (36). We i.v. injected play a role in mediating the observed resistance to suppression in +/+ 2/2 + 2 SHP-1 or SHP-1 CD4 CD25 T cells (CD45.2) at a 1:1 SHP-12/2 T cells. However, we found that neither IL-4–neutralizing + 2 2/2 ratio with CD45.1 wild-type CD4 CD25 T cells into Rag1 Abs nor Ab blockade of IL-4Ra–mediated signaling altered the re- mice, with or without wild-type Tregs. After 10 d, we assessed the sistance of SHP-12/2 T cells to Treg suppression (data not shown), + 2 2/2 expansion of CD4 Foxp3 T cells in the spleens of Rag1 re- indicating that the resistance reported here is IL-4–independent. This cipient mice (Fig. 6A). In the absence of Tregs, we observed no is consistent with a T cell–intrinsic mechanism and likely mediated +/+ 2/2 significant differences in the expansion of SHP-1 or SHP-1 by alterations in intracellular signaling events. + CD4 T cells, when compared with the percentages (Fig. 6B) and Previous studies demonstrated that deficiency of Cbl-b (38) and + absolute numbers of coinjected wild-type CD45.1 CD4 T cells, TNFR-associated factor 6 (31), two other negative regulators of suggesting that SHP-1 does not regulate homeostatic expansion. In T cell activation, also resulted in T cells that resist Treg sup- the presence of Tregs, we observed a substantial reduction in pression. A recent study suggested that SHP-1 regulates the deg- absolute number of T cells recovered, indicating Treg-mediated radation of Cbl-b, such that SHP-1–deficient T cells have suppression of homeostatic expansion (Fig. 6C). There was no decreased levels of Cbl-b protein after TCR stimulation alone +/+ difference in the extent of suppression between SHP-1 T cells (39). Although there are striking similarities between SHP-12/2 +/+ and wild-type CD45.1 T cells coinjected with SHP-1 or and Cbl-b2/2 T cells, our proliferation and suppression assays 2/2 2/2 SHP-1 T cells. However, SHP-1 T cells exhibited signifi- included costimulatory signals from irradiated APCs, which led to cantly greater homeostatic expansion (∼2.5-fold) in the presence Cbl-b degradation (40) in both SHP-1+/+ and SHP-12/2 T cells, +/+ of Tregs compared with SHP-1 T cells or coinjected wild-type and therefore would not account for the observed resistance to CD45.1 T cells, indicating a resistance to Treg-mediated sup- Treg suppression. Moreover, we did not detect any differences in pression (Fig. 6B, 6C). Taken together, these data strongly suggest Cbl-b protein expression between SHP-12/2 T cells and SHP-1+/+ + that SHP-1 regulates the susceptibility of CD4 T cells to Treg- T cells (data not shown). We did, however, observe enhanced mediated suppression in vitro as well as in vivo. activation of the Akt pathway in SHP-12/2 CD4+ T cells and naive CD8+ T cells, both basally and upon TCR stimulation. The Discussion PI3K/Akt pathway is primarily activated downstream of the TCR For T cells to mount a productive response against a pathogen, they and CD28 costimulatory signaling, and the resultant signaling must be able to transiently overcome constraints imposed by Tregs. cascade allows T cells to proliferate by increasing cell size and Environmental factors as well as strong antigenic signals through glucose metabolism, inactivating cell cycle inhibitors, and en- the TCR in the presence of costimulation have been shown to allow hancing cellular survival (41). An important mechanism of Treg T cells to become refractory to Treg suppression (4–8). However, suppression is depriving T cells of costimulatory signals via the intracellular signaling pathways that result in resistance to downregulation of costimulatory molecules CD80/CD86 The Journal of Immunology 7 Downloaded from

FIGURE 5. SHP-12/2 T cells exhibit enhanced activation of Akt. (A)SplenicCD4+CD252 T cells were isolated from SHP-1+/+ or SHP-12/2 mice + and cultured in the presence of indicated concentrations of anti-CD3 and irradiated CD4 T cell–depleted splenocytes as APCs. After 24 h, intracellular http://www.jimmunol.org/ levels of pAkt (T308) were assessed by flow cytometry. Histograms represent pAkt (T308) levels within live CD4+Foxp32 SHP-1+/+ or SHP-12/2 T cells compared with fluorescence minus one (FMO) control. Data represent three independent experiments; n = 6–9 mice per genotype. (B) Bar graph represents compiled relative increase in pAkt MFI compared with baseline (unstimulated SHP-1+/+ CD4+ T cells). (C) Splenic naive (CD44lo)CD8+ T cells were isolated from SHP-1+/+ or SHP-12/2 mice and cultured in the presence of indicated concentrations of anti-CD3 and irradiated CD4+ Tcell– depleted splenocytes as APCs. After 24 h, intracellular levels of pAkt (T308) were assessed by flow cytometry. Histograms represent pAkt (T308) levels within live CD8+ SHP-1+/+ or SHP-12/2 T cells compared with FMO control; n = 3 mice per genotype. (D) Bar graph represents relative increase in pAkt MFI compared with baseline (unstimulated SHP-1+/+ CD8+ T cells). A one-column t test with a null hypothesis of 1 was applied to fold change MFI values in (B)and(D), obtained by comparing MFI of SHP-12/2 cells to the MFI of the SHP-1+/+ cells at each dose. Error bars indicate 6 SEM; *p # 0.05, **p # 0.01, ***p # 0.00.1. by guest on September 29, 2021

(B7.1/B7.2) on APCs and upregulation of inhibitory molecules autoimmune diseases, our findings might also be applicable such as CTLA-4 and LAG3 (42). CTLA-4 can outcompete CD28 toward tumor immunotherapy. Tumors actively recruit and for binding of B7 molecules on APCs, and LAG3 can prevent generate Tregs to maintain a suppressive microenvironment (44). maturation of APCs to adequately engage T cells (43). Previous Thus, the goal of current adoptive cell transfer and/or chimeric work suggested that SHP-1–deficient T cells have a reduced re- Ag receptor (CAR) T cell therapies is to modify or create CD8+ quirement for costimulation (13). Because SHP-12/2 T cells show T cells with enhanced responsiveness toward tumor Ag (45). enhanced Akt activation upon TCR stimulation, they likely resist Many of the signaling components being incorporated into CAR Treg suppressive mechanisms that specifically inhibit costimula- T cells are from costimulatory molecules, which have also been tion, as their need for costimulation is reduced by the enhance- found to induce resistance to Treg suppression. For example, ment in Akt activation. Interestingly, many of the environmental both 4-1BB and OX40 signaling in T cells has been found to factors shown to induce suppression-refractory T cells have been induce Treg resistance (46–51), and components of both have linked to enhancing activation of the PI3K/Akt pathway (3). been used in second generation CAR T cells (52). Along these Our work also helps to clarify recent discrepancies reported on lines, adoptive transfer of SHP-12/2 or SHP-1 knockdown (via SHP-1 function in negative regulation of TCR signaling due to the small interfering RNA) CD8+ T cells improved tumor control in a use of CD4-Cre–mediated deletion. Using the dLck-Cre line, in mouse model of disseminated leukemia (53). However, whether which SHP-1 deletion is temporally distinct from early stages of CD8+ T cell resistance to Treg suppression played a role in tumor thymic selection, minimized developmental or potential repertoire control was not examined. Our findings suggest that incorporating changes to the T cell compartment. Importantly, dLck-Cre+ SHP-1 ablation could be useful in current CAR T cell or adoptive SHP-1f/f mice did not display any detectable differences in the cell transfer therapies to allow CD8+ cytotoxic T lymphocytes to composition of the thymic or peripheral T cell compartments overcome Treg suppression and better control tumor outgrowth. compared with SHP-1–sufficient control mice, nor the expansion Signaling through many of the costimulatory molecules being of CD4+ memory (CD44hi) T cells. However, consistent with data used currently in CAR T cell trials also enhance Akt activation. published by others and us (reviewed in Ref. 9), we observed Directly enforced constitutive Akt activation induced human increased responsiveness to TCR stimulation in SHP-12/2 CD4+ CD8+ T cells to resist Treg suppression and led to enhanced cy- and CD8+ T cells, which was directly attributable to loss of SHP-1 totoxicity toward a neuroblastoma cell line (54). Not only are within the T cells rather than an expansion of Ag-experienced these findings translatable to tumor immunotherapy, but also for T cells. treatment of chronic viral infections. It has been shown that Aside from gaining insight into the molecular mechanisms of chronic viral infection induces Tregs to suppress the function of Treg resistance, which has been linked to the pathophysiology of CD8+ T cells, preventing viral clearance (55). Stimulation of 8 SHP-1–DEFICIENT T CELLS RESIST Treg SUPPRESSION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. SHP-12/2 CD4+ T cells resist Treg suppression in vivo. (A) Schematic representation of experimental setup. Splenic CD4+CD252 Tcon cells were isolated from wild-type CD45.1 mice or SHP-1+/+ or SHP-12/2 CD45.2 mice and labeled with CellTrace Violet. Wild-type Tregs (CD4+CD25+) were isolated from spleens of SHP-1+/+ mice. Tcon cells (3 3 106 total) were injected i.v. via the tail vein into Rag12/2 recipient mice, at a 1:1 ratio of either CD45.2 SHP-1+/+/CD45.1 wild-type Tcon cells [conditions (Ai) and (Aii)] or CD45.2 SHP-12/2/CD45.1 wild-type Tcon cells [conditions (Aiii) and (Aiv)]. Half the recipients received Tcon cells only [conditions (Ai) and (Aiii)], and the other half received Tcon cells along with 7.5 3 105 SHP-1+/+ Tregs (1:4 Treg/Tcon ratio) [conditions (Aii) and (Aiv)]. After 10 d, spleens of recipient mice were harvested and stained for analysis by ﬂow cytometry. (B) Left, Representative ﬂow plots of CD4+CD252 input Tcon cells. Top, Input for conditions (Bi) and (Bii) [as shown in (Ai) and (Aii)]: CD45.2 SHP-1+/+ with CD45.1 wild-type Tcon cells. Bottom, Input for conditions (Biii) and (Biv) [as shown in (Aiii) and (Aiv)]: CD45.2 SHP-12/2 with CD45.1 wild-type Tcon cells. Right, Plots show percentages of splenic CD45.1+ and CD45.2+ CD4+Foxp32 T cells recovered 10 d postinjection; experimental conditions (with or without Tregs) as indicated. (C) Percent suppression was computed by subtracting the percent relative expansion for each indicated genotype from 100%. Percent relative expansion was calculated by dividing the absolute number of CD4+Foxp32 T cells recovered in the presence of coinjected Tregs over the absolute number of CD4+Foxp32 T cells recovered in the absence of Tregs (maximal expansion), multiplied by 100; n = 3–4 recipient mice per donor condition. Error bars indicate 6 SEM. *p # 0.05.

CD8+ T cells with the costimulatory molecule 4-1BB rendered Acknowledgments T cells resistant to Treg suppression and able to clear a chronic viral We thank Dr. Mark Conaway for lending us expertise in statistical analyses, infection in mice (48). Therefore, our data reveal SHP-1 as a pos- Amber Woods for help with the in vivo suppression experiments, and Dr. sible target to modulate the activation and function of T cells for Kodi Ravichandran for critical reading of the manuscript. tumor and chronic viral infection immunotherapies, and they provide more evidence pointing to the critical nature of the PI3K/Akt Disclosures pathway in regulating the balance between T cells and Tregs. The authors have no ﬁnancial conﬂicts of interest. The Journal of Immunology 9

References 28. Nishikawa, H., T. Kato, M. Hirayama, Y. Orito, E. Sato, N. Harada, S. Gnjatic, L. J. Old, and H. Shiku. 2008. Regulatory T cell-resistant CD8+ T cells induced 1. Campbell, D. J., and M. A. Koch. 2011. Phenotypical and functional speciali- by glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res. zation of FOXP3+ regulatory T cells. Nat. Rev. Immunol. 11: 119–130. + + 68: 5948–5954. 2. Buckner, J. H. 2010. Mechanisms of impaired regulation by CD4 CD25 29. Trinschek, B., F. Luessi, J. Haas, B. Wildemann, F. Zipp, H. Wiendl, C. Becker, FOXP3+ regulatory T cells in human autoimmune diseases. Nat. Rev. Immunol. and H. Jonuleit. 2013. Kinetics of IL-6 production defines T effector cell re- 10: 849–859. sponsiveness to regulatory T cells in multiple sclerosis. [Published erratum ap- 3. Mercadante, E. R., and U. M. Lorenz. 2016. Breaking free of control: how con- pears in 2013 PLoS One 8.] PLoS One 8: e77634. ventional T cells overcome regulatory T cell suppression. Front. Immunol. 7: 193. 30. Wohlfert, E. A., and R. B. Clark. 2007. “Vive la re´sistance!”–the PI3K-Akt 4. Thornton, A. M., and E. M. Shevach. 1998. CD4+CD25+ immunoregulatory pathway can determine target sensitivity to regulatory T cell suppression. T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 Trends Immunol. 28: 154–160. production. J. Exp. Med. 188: 287–296. 31. King, C. G., T. Kobayashi, P. J. Cejas, T. Kim, K. Yoon, G. K. Kim, 5. Baecher-Allan, C., V. Viglietta, and D. A. Hafler. 2002. Inhibition of human E. Chiffoleau, S. P. Hickman, P. T. Walsh, L. A. Turka, and Y. Choi. 2006. CD4+CD25+high regulatory T cell function. J. Immunol. 169: 6210–6217. TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of 6. George, T. C., J. Bilsborough, J. L. Viney, and A. M. Norment. 2003. High immune homeostasis. Nat. Med. 12: 1088–1092. antigen dose and activated dendritic cells enable Th cells to escape regulatory 32. Kshirsagar, S., E. Binder, M. Riedl, G. Wechselberger, E. Steichen, and T cell-mediated suppression in vitro. Eur. J. Immunol. 33: 502–511. M. Edelbauer. 2013. Enhanced activity of Akt in Teff cells from children with 7. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, lupus nephritis is associated with reduced induction of tumor necrosis factor and S. Sakaguchi. 1998. Immunologic self-tolerance maintained by CD25+CD4+ receptor-associated factor 6 and increased OX40 expression. Arthritis Rheum. naturally anergic and suppressive T cells : induction of autoimmune disease by 65: 2996–3006. breaking their anergic/suppressive state. Int. Immunol. 10: 1969–1980. 33. Wehrens, E. J., S. J. Vastert, G. Mijnheer, J. Meerding, M. Klein, 8. Sojka, D. K., A. Hughson, T. L. Sukiennicki, and D. J. Fowell. 2005. Early kinetic window of target T cell susceptibility to CD25+ regulatory T cell activity. N. M. Wulffraat, B. J. Prakken, and F. van Wijk. 2013. Anti-tumor necrosis J. Immunol. 175: 7274–7280. factor a targets protein kinase B/c-Akt-induced resistance of effector cells to 9. Lorenz, U. 2009. SHP-1 and SHP-2 in T cells: two phosphatases functioning at suppression in juvenile idiopathic arthritis. Arthritis Rheum. 65: 3279–3284. 34. Cuevas, B. D., Y. Lu, M. Mao, J. Zhang, R. LaPushin, K. Siminovitch, and many levels. Immunol. Rev. 228: 342–359. Downloaded from 10. Cuevas, B., Y. Lu, S. Watt, R. Kumar, J. Zhang, K. A. Siminovitch, and G. B. Mills. 2001. Tyrosine phosphorylation of p85 relieves its inhibitory activity G. B. Mills. 1999. SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase on phosphatidylinositol 3-kinase. J. Biol. Chem. 276: 27455–27461. phosphorylation and activity. J. Biol. Chem. 274: 27583–27589. 35. Liao, Y., and M.-C. Hung. 2010. Physiological regulation of Akt activity and 11. Carter, J. D., B. G. Neel, and U. Lorenz. 1999. The tyrosine phosphatase SHP-1 stability. Am. J. Transl. Res. 2: 19–42. influences thymocyte selection by setting TCR signaling thresholds. Int. 36. Workman, C. J., L. W. Collison, M. Bettini, M. R. Pillai, J. E. Rehg, and D. A. Immunol. 11: 1999–2014. A. Vignali. 2011. In vivo Treg suppression assays. Methods Mol. Biol. 707: 119–156. 37. Pace, L., S. Rizzo, C. Palombi, F. Brombacher, and G. Doria. 2006. Cutting 12. Zhang, J., A. K. Somani, D. Yuen, Y. Yang, P. E. Love, and K. A. Siminovitch. + 2 + + 1999. Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell edge: IL-4-induced protection of CD4 CD25 Th cells from CD4 CD25 reg- selection. J. Immunol. 163: 3012–3021. ulatory T cell-mediated suppression. J. Immunol. 176: 3900–3904. http://www.jimmunol.org/ 38. Wohlfert, E. A., M. K. Callahan, and R. B. Clark. 2004. Resistance to 13. Sathish, J. G., K. G. Johnson, F. G. LeRoy, K. J. Fuller, M. B. Hallett, + + 2/2 P. Brennan, L. K. Borysiewicz, M. J. Sims, and R. J. Matthews. 2001. Re- CD4 CD25 regulatory T cells and TGF-b in Cbl-b mice. J. Immunol. quirement for CD28 co-stimulation is lower in SHP-1-deficient T cells. Eur. J. 173: 1059–1065. Immunol. 31: 3649–3658. 39. Xiao, Y., G. Qiao, J. Tang, R. Tang, H. Guo, S. Warwar, W. Y. Langdon, L. Tao, 14. Shultz, L. D., P. A. Schweitzer, T. V. Rajan, T. Yi, J. N. Ihle, R. J. Matthews, and J. Zhang. 2015. Protein tyrosine phosphatase SHP-1 modulates T cell re- M. L. Thomas, and D. R. Beier. 1993. Mutations at the murine motheaten locus sponses by controlling Cbl-b degradation. J. Immunol. 195: 4218–4227. are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell 40. Paolino, M., and J. M. Penninger. 2010. Cbl-b in T-cell activation. Semin. 73: 1445–1454. Immunopathol. 32: 137–148. 15. Johnson, D. J., L. I. Pao, S. Dhanji, K. Murakami, P. S. Ohashi, and B. G. Neel. 41. Fruman, D. A. 2004. Phosphoinositide 3-kinase and its targets in B-cell and 2013. Shp1 regulates T cell homeostasis by limiting IL-4 signals. J. Exp. Med. T-cell signaling. Curr. Opin. Immunol. 16: 314–320. 210: 1419–1431. 42. Josefowicz, S. Z., L.-F. Lu, and A. Y. Rudensky. 2012. Regulatory T cells: 16. Martinez, R. J., A. B. Morris, D. K. Neeld, and B. D. Evavold. 2016. Targeted mechanisms of differentiation and function. Annu. Rev. Immunol. 30: 531–564. by guest on September 29, 2021 loss of SHP1 in murine thymocytes dampens TCR signaling late in selection. 43. Vignali, D. A. A., L. W. Collison, and C. J. Workman. 2008. How regulatory Eur. J. Immunol. 46: 2103–2110. T cells work. Nat. Rev. Immunol. 8: 523–532. 17. Zhang, D. J., Q. Wang, J. Wei, G. Baimukanova, F. Buchholz, A. F. Stewart, 44. Oleinika, K., R. J. Nibbs, G. J. Graham, and A. R. Fraser. 2013. Suppression, X. Mao, and N. Killeen. 2005. Selective expression of the Cre recombinase in subversion and escape: the role of regulatory T cells in cancer progression. Clin. late-stage thymocytes using the distal promoter of the Lck gene. J. Immunol. 174: Exp. Immunol. 171: 36–45. 6725–6731. 45. Restifo, N. P., M. E. Dudley, and S. A. Rosenberg. 2012. Adoptive immunotherapy 18. Plas, D. R., C. B. Williams, G. J. Kersh, L. S. White, J. M. White, S. Paust, for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12: 269–281. T. Ulyanova, P. M. Allen, and M. L. Thomas. 1999. Cutting edge: the tyrosine 46. Choi, B. K., J. S. Bae, E. M. Choi, W. J. Kang, S. Sakaguchi, D. S. Vinay, and phosphatase SHP-1 regulates thymocyte positive selection. J. Immunol. 162: B. S. Kwon. 2004. 4-1BB-dependent inhibition of immunosuppression by acti- + + 5680–5684. vated CD4 CD25 T cells. J. Leukoc. Biol. 75: 785–791. 19. Pao, L. I., K.-P. Lam, J. M. Henderson, J. L. Kutok, M. Alimzhanov, L. Nitschke, 47.Elpek,K.G.,E.S.Yolcu,D.D.H.Franke,C.Lacelle,R.-H.Schabowsky,and + + + M. L. Thomas, B. G. Neel, and K. Rajewsky. 2007. B cell-specific deletion of H. Shirwan. 2007. Ex vivo expansion of CD4 CD25 FoxP3 T regulatory cells protein-tyrosine phosphatase Shp1 promotes B-1a cell development and causes basedonsynergybetweenIL-2and4-1BBsignaling.J. Immunol. 179: 7295–7304. systemic autoimmunity. Immunity 27: 35–48. 48. Robertson, S. J., R. J. Messer, A. B. Carmody, R. S. Mittler, and K. J. Hasenkrug. 20. Carter, J. D., G. M. Calabrese, M. Naganuma, and U. Lorenz. 2005. Deficiency 2008. CD137-mediated immunotherapy for chronic viral infection. J. Immunol. of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) causes 180: 5267–5274. enrichment of CD4+CD25+ regulatory T cells. J. Immunol. 174: 6627–6638. 49. Takeda, I., S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, 21. Carow, B., Y. Gao, J. Coquet, M. Reilly, and M. E. Rottenberg. 2016. Lck-driven K. Sugamura, and N. Ishii. 2004. Distinct roles for the OX40-OX40 ligand in- Cre expression alters T cell development in the thymus and the frequencies and teraction in regulatory and nonregulatory T cells. J. Immunol. 172: 3580–3589. functions of peripheral T cell subsets. J. Immunol. 197: 2261–2268. 50. Piconese, S., B. Valzasina, and M. P. Colombo. 2008. OX40 triggering blocks 22. Roederer, M. 2011. Interpretation of cellular proliferation data: avoid the pan- suppression by regulatory T cells and facilitates tumor rejection. [Published glossian. Cytometry A 79: 95–101. erratum appears in 2008 J. Exp. Med. 205: 1505.] J. Exp. Med. 205: 825–839. 23. Yang, J., M. O. Brook, M. Carvalho-Gaspar, J. Zhang, H. E. Ramon, 51. Voo, K. S., L. Bover, M. L. Harline, L. T. Vien, V. Facchinetti, K. Arima, M. H. Sayegh, K. J. Wood, L. A. Turka, and N. D. Jones. 2007. Allograft re- L. W. Kwak, and Y. J. Liu. 2013. Antibodies targeting human OX40 expand jection mediated by memory T cells is resistant to regulation. Proc. Natl. Acad. effector T cells and block inducible and natural regulatory T cell function. J. Sci. USA 104: 19954–19959. Immunol. 191: 3641–3650. 24. Wehrens, E. J., G. Mijnheer, C. L. Duurland, M. Klein, J. Meerding, J. van 52. Sadelain, M. 2016. Chimeric antigen receptors: driving immunology towards Loosdregt, W. de Jager, B. Sawitzki, P. J. Coffer, B. Vastert, et al. 2011. Func- synthetic biology. Curr. Opin. Immunol. 41: 68–76. tional human regulatory T cells fail to control autoimmune inflammation due to 53. Stromnes, I. M., C. Fowler, C. C. Casamina, C. M. Georgopolos, M. S. McAfee, PKB/c-akt hyperactivation in effector cells. Blood 118: 3538–3548. T. M. Schmitt, X. Tan, T.-D. Kim, I. Choi, J. N. Blattman, and P. D. Greenberg. 25. Fowler, C. C., L. I. Pao, J. N. Blattman, and P. D. Greenberg. 2010. SHP-1 in 2012. Abrogation of SRC homology region 2 domain-containing phosphatase 1 T cells limits the production of CD8 effector cells without impacting the for- in tumor-specific T cells improves efficacy of adoptive immunotherapy by en- mation of long-lived central memory cells. J. Immunol. 185: 3256–3267. hancing the effector function and accumulation of short-lived effector T cells 26. Petrelli, A., E. J. Wehrens, R. C. Scholman, B. J. Prakken, S. J. Vastert, and in vivo. J. Immunol. 189: 1812–1825. F. van Wijk. 2016. Self-sustained resistance to suppression of CD8+ Teff cells at 54. Sun, J., G. Dotti, L. E. Huye, A. E. Foster, B. Savoldo, M. M. Gramatges, the site of autoimmune inflammation can be reversed by tumor necrosis factor D. M. Spencer, and C. M. Rooney. 2010. T cells expressing constitutively active Akt and interferon-g blockade. Arthritis Rheumatol. 68: 229–236. resist multiple tumor-associated inhibitory mechanisms. Mol. Ther. 18: 2006–2017. 27. Ben Ahmed, M., N. Belhadj Hmida, N. Moes, S. Buyse, M. Abdeladhim, 55. Dittmer, U., H. He, R. J. Messer, S. Schimmer, A. R. M. Olbrich, C. Ohlen, H. Louzir, and N. Cerf-Bensussan. 2009. IL-15 renders conventional lymphocytes P. D. Greenberg, I. M. Stromnes, M. Iwashiro, S. Sakaguchi, et al. 2004. resistant to suppressive functions of regulatory T cells through activation of the Functional impairment of CD8+ T cells by regulatory T cells during persistent phosphatidylinositol 3-kinase pathway. J. Immunol. 182: 6763–6770. retroviral infection. Immunity 20: 293–303. Supplemental Figure 1

A B

LN CD4+ T cells Splenic CD4+ T cells LN CD8+ T cells Splenic CD8+ T cells dLck Cre: - + - + - + - + dLck Cre: - + - + SHP-1: wt/wt wt/wt f/f f/f wt/wt wt/wt f/f f/f SHP-1: f/f f/f f/f f/f

SHP-1 SHP-1

β actin β actin

~91% >95% >95% >95% deletion deletion deletion deletion C D LN Spleen 10 2.0 dLck Cre: - + - + ) 7) 8 8 1.5 x10 x10 SHP-1: wt/wt wt/wt f/f f/f ( 6 ( lls lls 1.0 4.7 78 3.8 80 3.8 82 4.6 77 e e lc

4 lc 0.5 Tota 2 Tota

0 0.0 Thymus dLck Cre: - + - + dLck Cre: - + - + SHP-1: wt/wt wt/wt f/f f/f SHP-1: wt/wt wt/wt f/f f/f 6.7 11 4.5 11 4.1 9.8 5 13 26 1.1 27 0.91 26 1.8 24 1.7

E LN

dLck Cre: - + - + 39 34 42 31 41 31 46 28 0.88 0.34 0.98 0.99 SHP-1: wt/wt wt/wt f/f f/f 11 14 8.8 11 1.9 9.2 1.8 9.2 2 9.7 2.1 10.7 Spleen

LN 70 66 74 72 CD8 18 20 16 15 CD4 85.2 3.7 85.9 3.1 85.1 3.3 83.5 3.7 0.7 6.4 0.4 6.1 0.6 6.4 0.4 7.1

Spleen

89.1 3.8 90.2 3.3 89.2 3.8 88.2 4.3 CD25 FoxP3

Supplemental Figure1: dLck Cre SHP-1f/f mice display normal T cell compartment. (A) CD4+ T cells from spleens and peripheral lymph nodes of mice with indicated genotypes, were lysed in NP40, and run on an Any KD TGX gel (Bio-Rad) and immunoblotted with anti-SHP-1 and β-actin-HRP. SHP-1 levels were normalized to actin and percent SHP-1 deletion was calculated. (B) CD8+ T cells from spleens and peripheral lymph nodes of mice with indicated genotypes, lysed, run on an Any KD TGX gel and immunoblotted as in A. (C) Total numbers of lymph node cells and splenocytes isolated from 6-8 week old mice of indicated genotypes. (D) Representative flow cytometric plot of CD4+ and CD8+ T cells composition in thymus, lymph nodes, and spleens of mice with indicated genotypes. (E) Representative plot of percentage of Treg (CD4+CD25+-Foxp3+) cells in spleens and lymph nodes of mice with indicated genotypes. Data in C were from 8 independent experiments, and a one-way ANOVA was performed.

Mercadante et al Supplemental Figure 2

A B

Apoptotic Tcon cells at 24 hours Dead Tcon cells at 24 hours

2.0 +/+ 80 SHP-1 SHP-1+/+ SHP-1-/- -/- 1.5 4+ 60 SHP-1 CD4+ CD of 1.0 of 40 ead asp3+ 0.5 20 %D %C

0.0 0 0530 150 0530 150 αCD3 ng/mL αCD3 ng/mL

C D Apoptotic naive CD8+ T cells at 24 hours Dead naive CD8+ T cells at 24 hours 0.5 80 +/+ SHP-1 SHP-1+/+ + -/- 0.4 SHP-1 -/- 8+ 60 SHP-1 CD8 CD

of 0.3 of 40 0.2 ead asp3+ 20 0.1 %D %C

0.0 0 0530 150 0530 150 αCD3 ng/mL αCD3 ng/mL E Apoptotic naive CD8+ T cells at 72 hours 30 * SHP-1+/+ + 25 SHP-1-/-

CD8 20 of 15

asp3+ 10

%C 5

0 0530 150 αCD3 ng/mL

Supplemental Figure 2: SHP-1 does not affect in vitro survival of T cells. CD4+CD25- Tcon cells were isolated from spleens of SHP-1+/+ or SHP-1-/- mice and cultured for 24 or 72 hours in the presence of indicated concentrations of anti-CD3 and irradiated CD4+ T cell-depleted splenocytes as APCs. During the last hour of culture, cells were stained for caspase-3 with Fitc-DVED-FMK and then stained for flow cytometric analysis including staining for cell death by a live/dead dye. Data shown represent (A) percent Casp3+ cells and (B) percent dead cells within CD4+ population after 24 hours stimulation. (C-E) Naive (CD44lo) CD8+ T cells were isolated from spleens of SHP-1+/+ or SHP-1-/- mice and cultured as described above. Data shown represent (C) percent Casp3+ cells and (D) percent dead cells within the CD8+ population, and (E) percent Casp3+ cells within the CD8+ population after stimulation for 72 hours. n=3 mice each genotype and dose. Error bars represent ±SEM. A two-way ANOVA with Sidak’s multiple comparison post-test was performed. No statistically significant differences were observed other than where indicated. *p<0.05.

Mercadante et al and spleensofSHP-1 CD4+CD25-Foxp3- Tcon cellsisolatedfromlymphnodes Representative histogram of CD44 expression on expansion ofmemoryCD4+ T cell compartment. (A) CD4+CD25-Foxp3-CD44 n=5-7 mice per genotype.(C) Total number of or SHP-1 Supplemental Figure 3:dLckCre SHP-1 MACS selection of naive (CD44 expression levelsonCD4+ T cellspre-(input)andpost- spleens of SHP-1 tion of percent CD4+CD25-FoxP3-CD44 as APCs. anti-CD3 withirradiated CD4+ T cell-depleted splenocytes cultured in thepresence ofindicated concentrations of ±SEM.* p≤0.05, **p≤0.01,***p≤0.001, ****p≤0.0001 performed on data in B, C, and F; error bars indicate percent responding cells from D. A Student’s t test was CD4+CD44 Mercadante etal C B A CD44hi of CD4+FoxP3-CD25 - 6 %of max (x10 cells) hi CD44 -/- 0 1 2 3 4 LN-derived Tconcells %CD44 of LN n=3 foreachgenotype. mice and labeled with CellTrace Violet and

lo CD4+FoxP3-CD25- -der 20 40 60 80 T cellswereisolatedfromspleensofSHP-1 0 SHP- LN ived Tconcells -der 1 +/ +/+ + ns ived Tconcells orSHP-1 ns +/+ orSHP-1 SHP- hi Tcon cells in lymph nodes and 1 -/ - -/-

mice. %CD44hi of

hi Splenic Tconcells -/- CD44 of CD4+FoxP3-CD25- Splenic Tconcells 20 40 60 80 mice.(B) 0

(F) Quantification of CD4+FoxP3-CD25- Supplemental Figure3 lo 6

) cells. (x10 cells) (D)CD44surface 0 2 4 6 ns f/f SHP- mice show no miceshow hi Splenic Tconcells 1 Tcon cells. +/ Quantifica (E) + SH SH ns Naïve P- P- 1 1 FMO SHP-1 SHP-1 SHP- -/ +/ - +/+ + 1 - -/ - -/- +/+ F E D

(ng/mL) %of max αCD3 CD44 %Respondingcells 150 30 20 40 60 80 5 0 Naive (CD4 p<0.0001

**** # Cells # Cells # Cells 53 1 3 2 2 2 6 4 4 6 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CellTrace Violet Naive (CD44 3 8 7 5 6 1 SHP-1 αCD3 ng/mL . . . 2 6 9 4 p=0.02 lo * ) CD4+Tcel 0 +/+ 6 1 2 3 4 8 . 3 . 6 lo p=0.03 1 2 2 3 4 4 6 5 8 1 CD4+ Tcells Naive (CD44 150 2 4 6 8 Tcells Total inputCD4+ 0 0 0 0 0 0 0 0 0 0 ) CD4+Tcells 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 * 0 0 0 0 0 0 ls 8 8 5 9 4 1 SHP-1 . . . 4 4 6 lo ) selected SH SH P- P- -/- 10 1 1 1 4 4 7 -/ +/ . . - 9 6 + Supplemental Figure 4

A B LN-derived CD8+ T cells Splenic CD8+ T cells LN-derived CD8+ Tcells Splenic-derived CD8 Tcells SHP-1+/+ 80 80 SHP-1-/- SHP-1+/+ FMO -/- 60 60 SHP-1 CD8+

CD8+ p<0.0001 **** of p<0.0001 of hi % of Max of % % of Max of % 40 40 **** hi 4 4 CD4 20 CD4 20 % %

CD44 0 0

C LN-derived CD8+ Tcells Splenic-derived CD8 T cells 4 5

p=0.002 cells) 6 4 ns 3 ** x10

CD8+ 3 +( cells) of

6 2 hi

CD8 2 44 (x10 1 of CD hi 1 44

0 CD 0 SHP-1+/+ SHP-1-/- SHP-1+/+ SHP-1-/-

Supplemental Figure 4: dLck Cre SHP-1f/f mice show increase in CD44hi CD8+ T cells. (A) Representative histogram of CD44 surface expression on CD8+ T cells in lymph nodes and spleens of SHP-1+/+ or SHP-1-/- mice. (B) Quantification of percent CD44hi CD8+ T cells. n=6-10 mice per genotype. (C) Total number of CD44hi CD8+ T cells in lymph nodes and spleens of SHP-1+/+ or SHP-1-/- mice. Note that these mice were 9-10 weeks old, at which time the average absolute number of splenocytes in SHP-1-/- mice was about 1.5 fold lower than in SHP-1+/+ mice (p<0.0001). A Student’s t test was performed on data in (B) and (C); error bars indicate ±SEM. * p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Mercadante et al