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 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.
Regulatory T cells (Tregs) play an essential role in shaping T cell responses and maintaining immune homeostasis (1). Deficits in Treg function or number allow T cell responses to go unchecked, leading to the development of autoimmunity and chronic inflammatory diseases (2). Dysregulation of the balance between activation and suppression of T cells can also occur when T cells become resistant to Treg-mediated suppression (2). Many autoimmune diseases, including type 1 diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease, feature not only impaired Tregs but also T cells that are resistant to suppression (3). However, the potential mechanisms by which T cells might acquire resistance to Treg-mediated suppression remain unclear. Although several extracellular factors have been linked to inducing resistance in T cells (3), the intracellular signaling mechanisms that can render T cells resistant to Treg suppression are poorly defined. Furthermore, strong activation through the TCR and/or costimulatory receptors can cause T cells to become refractory to Treg suppression (4–8), but the specific pathways allowing this resistance remain elusive. Similarly, whereas resistance to suppression occurs in both CD4+ and CD8+ T cells (3), whether resistance is induced by the same mechanism in both subsets is unknown.
SHP-1 is a cytoplasmic protein tyrosine phosphatase expressed in all hematopoietic cells, which has been implicated in the regulation of TCR-mediated signaling in T cells (9), including the PI3K/Akt pathway (10). We (11) and others (12, 13) have previously shown that SHP-1–deficient T cells are hyperresponsive to TCR stimulation. This was done using the motheaten (me/me) mouse model, in which all hematopoietic cells lack SHP-1 due to a splicing mutation (14), as well as cell lines expressing dominant-negative mutant forms of SHP-1 (12). However, one recent study (15), using conditional T cell deletion of SHP-1 via CD4-Cre, challenged the role of SHP-1 in regulating T cell development, whereas another report using the same mouse model confirmed the role of SHP-1 during T cell development (16). In this study, we generated a conditional knockout mouse model wherein SHP-1 deletion is driven by the distal Lck promoter (17), resulting in abrogation of SHP-1 expression in postselection thymocytes. This model allows largely normal T cell development such that any phenotypic and/or functional alterations observed due to SHP-1 deficiency can be directly ascribed to its role in mature T cells (11, 12, 18). Using this approach, we show that SHP-1 negatively regulates the activation and proliferation of CD4+ and CD8+ T cells in response to TCR stimulation, and that in the absence of SHP-1, T cells become resistant to Treg-mediated suppression. Such resistance is T cell–intrinsic, as SHP-1−/− T cells could not induce “bystander resistance” when cocultured with wild-type T cells. Our data also suggest a role for the PI3K/Akt pathway in mediating both CD4+ and CD8+ T cells to resist suppression. This resistance of CD4+ SHP-1−/− T cells to Treg-mediated suppression was also observed during homeostatic expansion in vivo. Collectively, these data identify a novel function of SHP-1 in regulating the susceptibility of T cells to Treg-mediated suppression in vitro and in vivo, through controlling the strength of signal received via the TCR and attenuating subsequent activation of the downstream PI3K/Akt pathway.
Materials and Methods
SHP-1flox/flox (SHP-1f/f) mice (19) (provided by B. Neel) were crossed to distal Lck-Cre (dLck-Cre) mice (17) purchased from The Jackson Laboratory (Bar Harbor, ME). Genotyping of all mice was done by PCR as described previously for the SHP-1f/f allele (19) and dLck-Cre alelle (17). For all experiments, 6- to 10-wk-old female and male mice were used, and control mice were dLck-Cre− SHP-1f/f or dLck-Cre+ SHP-1+/+ littermates of dLck-Cre+ SHP-1f/f mice. Rag1−/− mice were purchased from The Jackson Laboratory. CD45.1 wild-type C57BL/6 mice were purchased from Charles River Laboratories. All mice were bred and maintained in accordance with the policies of the Institutional Animal Care and Use Committee at the University of Virginia. All experiments involving mice were conducted with the approval of Institutional Animal Care and Use Committee.
Isolation and purification of primary cells
CD4+ T cells were isolated from peripheral lymph nodes (combined inguinal, axillary, brachial, cervical, sacral, and renal nodes) or spleens by negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol. CD8+ T cells were isolated from spleens by negative selection using the CD8α+ T cell isolation kit (Miltenyi Biotec). For naive CD4 or CD8 T cell experiments, CD4+CD44lo or CD8+CD44lo T cells were isolated from spleens by negative selection using a naive CD4 T cell isolation kit or the CD8 T cell isolation kit, respectively (Miltenyi Biotec). For splenic T cell isolation, RBCs were lysed using BD Pharm Lyse buffer (BD Biosciences, San Jose, CA) before T cell isolation. For Treg isolation, CD4+ T cells were subsequently labeled with CD25-PE to separate conventional T (Tcon) cells (defined as CD4+CD25−) and Tregs (CD4+CD25+). Labeled CD4+ cells were run on an autoMACS Pro separator (Miltenyi Biotec) using the posseld2 program to obtain Tregs with >85% purity as assessed by Foxp3+CD25+ staining. CD4+ T cell–depleted splenocytes were irradiated (2000 rad) and used as APCs in culture where indicated.
Cells were stained directly after isolation or harvested after 24, 72, or 96 h of culture as indicated. Cells were surface stained with anti-CD4, anti-CD25 (eBioscience, San Diego, CA), anti-CD8, anti-CD44, anti-CD62L, anti-CD69, anti-CD45.1, and anti-CD45.2 (BD Biosciences) in PBS supplemented with 1% BSA and 0.1% sodium azide. Staining for live cells was done following surface staining and washing, using Live/Dead fixable dye (Life Technologies, Carlsbad, CA). Cells were then fixed with BD Fix/Lyse (BD Biosciences) and washed. For intracellular Foxp3 staining, cells were fixed and permeabilized using a Foxp3 staining buffer set (eBioscience) according to the manufacturer’s protocol and stained with anti-Foxp3 (eBioscience). For pAkt intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s protocol and stained with anti-pAkt T308 (Cell Signaling Technology, Danvers, MA). For caspase-3 staining, cells were stained with the CaspGLOW kit (eBioscience) according to the manufacturer’s protocol for the last 60 min of in vitro culture. Stained cells were collected on a BD FACSCanto I or II, using FACSDiva version 8 software (BD Biosciences), or using Beckman Coulter CytoFLEX and CytExpert software (Beckman Coulter, Brea, CA), and subsequent analyses were done using FlowJo software version 9.9 or version 10.1 (FlowJo, Ashland, OR). Analyses were performed on singlet-gated cells as defined by forward scatter width versus forward scatter area, and live cells as defined by Live/Dead dye negative. Gates were set based on fluorescence minus one controls.
Proliferation and suppression assays
Assessment via CellTrace Violet dilution.
To assess proliferation, isolated T cells (CD4+CD25− [Tcon cells], CD4+CD44lo [naive CD4+ T cells], CD8+, or CD8+CD44lo [naive CD8+ T cells]) were stained with 5 μM CellTrace Violet for 20 min at 37°C followed by quenching with prewarmed complete RPMI 1640 for 5 min at 37°C (Life Technologies). Stained cells were washed, and 2.5 × 104 T cells were plated (in quadruplicate, pooled at time of harvest) in a total volume of 200 μl of RPMI 1640 complete medium (supplemented with 10% FBS, 50 μM 2-ME, 2 mM l-glutamine, 10 mM HEPES, MEM nonessential amino acids, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin) in round-bottom 96-well plates. Irradiated (2000 rad), CD4+ T cell–depleted splenocytes were added at 5 × 104 cells per well along with anti-CD3 Ab (2C11; Cedarlane Laboratories, Burlington, NC) at 10–1000 ng/ml as indicated. For suppression assays, CD4+CD25+ Tregs were plated with responder T cells at indicated ratios. For proliferation assays, cells were cultured for 72 or 96 h, and for suppression assays cells were cultured for 96 h followed by flow cytometric analyses.
Analysis of proliferation assay.
CellTrace Violet dilution was assessed by flow cytometry and subsequently analyzed using FlowJo v9.9 software proliferation Wizard platform (FlowJo). Briefly, after sequentially gating on singlets, live cells, CD4+ cells, and CellTrace Violet+ cells, the percentage of responding (dividing) cells relative to the input was obtained using the provided software algorithm.
Analysis of suppression assay.
To compensate for the increased baseline responsiveness of SHP-1−/− T cells, the percentage of responding cells in the no Treg condition was set to 100% (maximum responsiveness) for each genotype. The percentage of responding cells was calculated as described above for the proliferation analyses for all Treg/T cell ratios and normalized to the maximum responsiveness for their own genotype (no Treg condition). Percentage suppression equals 100 minus the percentage responding cells.
Twenty-four hour T cell activation
CD4+CD25− Tcon cells or naive (CD44lo) CD8+ T cells were isolated from spleens of indicated mice, and 2.5 × 104 cells were cultured per well in a 96-well round-bottom plate with 5 × 104 irradiated (2000 rad) CD4+ T cell–depleted splenocytes and indicated doses of anti-CD3 Ab (2C11; Cedarlane Laboratories). After 24 h, cells were harvested and stained for flow cytometric analysis of CD25 and pAkt T308 expression.
SHP-1 protein level in CD4+ T cells was assessed by lysing 5 × 105 cells in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM sodium chloride, 50 mM Tris, 4 mM sodium pyrophosphate, 5 mM sodium fluoride, 10 μg/ml sodium vanadate, 50 μg/ml antipain, 40 μg/ml PMSF, 1× protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO]) and resolving lysates on an Any KD TGX Tris-glycine-SDS gel (Bio-Rad Laboratories, Hercules, CA). Blots were probed with monoclonal anti–SHP-1 (clone 1SH01; NeoMarkers/Thermo Fisher Scientific, Fremont, CA) and reprobed for β-actin as a loading control (anti–β-actin-HRP, clone AC-15; Sigma-Aldrich). Blots were imaged using the ChemiDoc Touch gel imaging system (Bio-Rad Laboratories). Bands densities were quantified using ImageLab (Bio-Rad Laboratories) software after normalization to loading control.
In vivo T cell transfer
Tcon (CD4+CD25−) cells were isolated by MACS as described above from spleens of CD45.2 SHP-1+/+ or SHP-1−/− mice and CD45.1 wild-type mice. Tcon cells were labeled with 5 μM CellTrace Violet, and Treg (CD4+CD25+) cells were isolated from SHP-1+/+ mice and pooled. Tcon cells were resuspended at a 1:1 ratio of either CD45.2 SHP-1+/+/CD45.1 wild-type Tcon cells or CD45.2 SHP-1−/−/CD45.1 wild-type Tcon cells, and a total of 3 × 106 Tcon cells from either mix were injected i.v. in 200 μl of sterile PBS via the tail vein into Rag1−/− recipients. Additionally, half the recipient mice also received 7.5 × 105 SHP-1+/+ Tregs along with Tcon cells (1:4 Treg/Tcon ratio). After 10 d, spleens were harvested from recipient mice and stained for flow cytometric analysis. Donor and recipient mice were age matched.
T cell proliferation, CD25 upregulation, proliferation index, and suppression assays using CD4+CD25− or total CD8+ T cells were analyzed using a three-way ANOVA with a 95% confidence interval. A Student t test was used to analyze the comparison of percentage and absolute number of CD44hi T cells from SHP-1+/+ or SHP-1−/− mice. A Student t test was 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 comparison posttest was used to analyze cell death and apoptosis data. A one-way ANOVA with a Tukey multiple comparison posttest was performed to analyze the absolute number of lymph node and splenic T cells in dLck-Cre SHP-1f/f mice, the percentage of responding cells in coculture experiments, and the percentage suppression of in vivo T cell transfer experiments. For pAkt T308 flow cytometric data, a one-column t test was applied to the fold change pAkt mean fluorescence intensity (MFI) values of SHP-1−/− T cells compared with the pAkt MFI of SHP-1+/+ T cells for each anti-CD3 dose, with a null hypothesis of 1 (if no change from control, fold change = 1). A p value ≤0.05 was considered significant.
SHP-1 sets threshold for activation and proliferation of T cells in response to TCR stimulation
Although several previous studies have suggested that SHP-1 is a negative regulator of signaling downstream of the TCR, based on in vitro cell culture studies and primary T cells from total body knockout of SHP-1 (reviewed in Ref. 9), a recent study performed using conditional deletion of SHP-1 in T cells has disagreed with this notion (15). One potential reason for this discrepancy might have been the type of Cre line that was used to delete SHP-1 by Johnson et al. (15) as the CD4-Cre used gets expressed from earlier stages of T cell development. To test this possibility, we crossed mice carrying floxed alleles of Ptpn6 (Shp1) (19) with mice that express Cre recombinase under the control of the distal promoter of Lck (17). The dLck promoter drives Cre expression at late stages of T cell development, allowing TCR-dependent selection to occur under conditions of SHP-1 sufficiency (12, 16, 18, 20). We confirmed that SHP-1 was deleted in peripheral CD4+ (Supplemental Fig. 1A) and CD8+ T cells (Supplemental Fig. 1B) from the lymph nodes and spleen of dLck-Cre+ SHP-1f/f (referred to here as SHP-1−/−) mice. Importantly, we observed no changes in the composition of the thymic or peripheral T cell compartments with respect to absolute numbers (Supplemental Fig. 1C) or percentages of CD4+ or CD8+ T cells or Tregs in the lymph nodes or spleens (Supplemental Fig. 1D, 1E). Expression of dLck-Cre alone did not affect the peripheral T cell compartment, consistent with previous reports (21).
To assess the role of SHP-1 during T cell activation, we first compared the proliferation capacities of SHP-1+/+ and SHP-1−/− CD4+CD25− T cells, hereafter referred to as Tcon cells, to differentiate them from CD4+ Tregs. We found that a greater percentage of SHP-1−/− Tcon cells proliferated compared with Tcon cells from SHP-1+/+ mice. Enhanced proliferation in SHP-1−/−Tcon cells was especially apparent at suboptimal concentrations of anti-CD3 stimulation (Fig. 1A). We considered three reasons for SHP-1−/− Tcon cells to display the observed increase in proliferation, which are not mutually exclusive: 1) an increase in the percentage of cells that initially become activated and go on to proliferate, 2) a decreased cell cycle time, and/or 3) an increased survival of cells in the culture. We first determined whether a greater proportion of SHP-1−/− Tcon cells responded to TCR stimulation by using the FlowJo proliferation platform algorithm, which takes into account the number of cells in each round of division relative to the input cells (22), and thereby estimates the fraction of T cells that initially responded to the stimulation. Based on this metric, we found a significant increase in the percentage of responding SHP-1−/− Tcon cells compared with SHP-1+/+ Tcon cells, with the largest difference at the lowest stimulation dose (Fig. 1B). To complement this finding, we assessed the upregulation of CD25 (IL-2Rα) as a measure of early Tcon cell activation and found that a significantly greater percentage of SHP-1−/− Tcon cells were CD25+ after 24 h of stimulation compared with SHP-1+/+ Tcon cells (Fig. 1C). Importantly, we observed no CD25 upregulation on Tcon cells of either genotype in the absence of anti-CD3 stimulation, indicating that any observed T cell activation was TCR/CD3 stimulation-dependent. Interestingly, SHP-1+/+ Tcon cells reached a maximum percentage of CD25+ cells at 150 ng/ml anti-CD3, with no further increase at 1000 ng/ml anti-CD3, whereas the subpopulation of responding SHP-1−/− Tcon cells increased further at 1000 ng/ml anti-CD3 compared with 150 ng/ml. Upregulation of CD69, another marker of activation, followed the same pattern (data not shown). These data suggest that there is a greater percentage of SHP-1−/− Tcon cells that respond and are activated by any given stimulation.
Second, when we calculated the proliferation index of each sample (using the FlowJo proliferation platform that provides the average number of cellular divisions of the cells that divided in culture), SHP-1−/− and SHP-1+/+ Tcon cells underwent comparable rounds of divisions at any given dose of stimulation, with a slight increase in divisions at higher concentrations of stimulation (Fig. 1D). These data indicate that SHP-1 deficiency did not affect cell cycle time. To assess whether SHP-1−/− Tcon cells had an in vitro survival advantage over SHP-1+/+ Tcon cells, we stained cells for activated caspase-3, a marker of apoptosis. After 24 h in culture, we observed very little apoptosis among Tcon cells (<1%) with no significant difference between SHP-1+/+ or SHP-1−/− Tcon cells in terms of apoptosis or cell death (Supplemental Fig. 2A, 2B). By 72 h, we still observed very low levels of apoptosis (≤2%) with no statistically significant differences between SHP-1−/− Tcon cells and SHP-1+/+ Tcon cells (Fig. 1E). Taken together, these data demonstrate that SHP-1 controls the extent of TCR/CD3-driven proliferation by setting the threshold that determines the subpopulation of T cells responding to a given TCR stimulation.
CD4+ T cells lacking SHP-1 resist in vitro Treg suppression
Because our data indicated that SHP-1 lowered the threshold for Tcon cell activation and proliferation, we asked whether SHP-1 also regulated the susceptibility of Tcon cells to Treg-mediated suppression. Using an in vitro suppression assay, Tcon cells from SHP-1−/− and SHP-1+/+ mice were assessed for their susceptibility to wild-type Treg-mediated suppression (Fig. 2A). Strikingly, SHP-1−/− Tcon cells displayed ∼3-fold greater responsiveness compared with SHP-1+/+ Tcon cells, even at the maximally suppressive condition (Fig. 2A). Even after normalization to account for the increased baseline proliferation (no Treg condition in Fig. 2A), SHP-1−/− Tcon cells were significantly less suppressed by Tregs than were SHP-1+/+ Tcon cells (Fig. 2B), indicating that SHP-1 can influence the level of susceptibility to in vitro Treg-mediated suppression.
To determine whether the observed resistance to suppression in Tcon cells could be attributable to an expanded memory T cell population in mice with SHP-1−/− T cells (23, 24), we first assessed whether there were any differences in the memory T cell compartment of dLck-Cre SHP-1f/f mice compared with SHP-1+/+ mice, as has been described for me/me mice (25) and CD4-Cre SHP-1f/f mice (15, 16). However, we did not observe an increase in percentage (Supplemental Fig. 3A, 3B) or absolute number (Supplemental Fig. 3C) of Ag-experienced/memory-like CD44hiCD4+CD25−Foxp3− Tcon cells in the lymph nodes or spleens of dLck-Cre+ SHP-1f/f mice compared with control SHP-1–sufficient mice. As further indication that the composition of the CD4+ T cell compartment in the dLck-Cre+ SHP-1f/f mice was not altered, we detected no differences in the percentage of cells expressing activation markers CD69 or CD25 (data not shown). Furthermore, SHP-1−/− CD4+ T cells depleted of the CD44hi subpopulation (referred to here as naive CD44lo T cells, Supplemental Fig. 3D) retained a greater responsiveness to TCR stimulation (Supplemental Fig. 3E, 3F), without any changes in cell cycle time. These data are consistent with SHP-1 regulating signaling downstream of the TCR in naive T cell subsets. Moreover, SHP-1−/− naive CD4+ T cells were resistant to Treg-mediated suppression in vitro (Fig. 2C, 2D), confirming what we observed in the CD4+CD25− Tcon population. Taken together, these data suggest that SHP-1 regulates the susceptibility of CD4+ T cells to Treg-mediated suppression in vitro.
CD8+ T cells lacking SHP-1 also resist in vitro Treg suppression
Resistance of T cells to Treg-mediated suppression has not only been observed in CD4+ T cells, but also in CD8+ T cells (26–29), which has important clinical implications for cancer immunotherapy and chronic viral infection therapies. Similar to SHP-1−/− CD4+ T cells, SHP-1−/− CD8+ T cells exhibited greater responsiveness to TCR stimulation compared with SHP-1+/+ CD8 T cells (Fig. 3A) without any detectable changes in cell cycle time (data not shown). SHP-1−/− CD8+ T cells also resisted Treg-mediated suppression (Fig. 3A, 3B). However, in contrast to the CD4+ T cell compartment, we did observe a substantial increase in the percentage and number of CD8+CD44hi T cells in the lymph nodes and spleens of SHP-1−/− mice compared with SHP-1+/+ mice (Supplemental Fig. 4). To determine whether SHP-1 deficiency also conferred naive CD8+ T cells with resistance to Treg suppression, we isolated naive CD8+ (CD44lo) T cells and measured their suppression in vitro (Fig. 3C). A greater proportion of SHP-1−/− naive CD8+ T cells responded to TCR stimulation in the absence of Tregs (Fig. 3C). Similar to what we observed for CD4+ T cells, there were no significant differences in cell death or apoptosis between SHP-1+/+ and SHP-1−/− naive CD8+ T cells after 24 h of stimulation across a range of anti-CD3 stimulation (Supplemental Fig. 2C, 2D). There was also no observed survival advantage in SHP-1−/− naive CD8+ T cells after 3 d of stimulation, and in fact at the highest dose of stimulation, SHP-1−/− naive CD8+ T cells displayed enhanced apoptosis compared with SHP-1+/+ cells, possibly due to an increase in activation-induced cell death (Supplemental Fig. 2E). Furthermore, SHP-1−/− naive CD8+ T cells exhibited resistance to suppression, similar to the total CD8+ T cell population (Fig. 3D), indicating that the phenotype was independent of the expanded Ag-experienced/memory-like CD8+ T cell subpopulation. These data demonstrate a role for SHP-1 in regulating susceptibility of not only CD4+ T cells, but also CD8+ T cells, to Treg-mediated suppression, likely by a similar mechanism in both T cell subsets.
SHP-1 regulates TCR signaling and susceptibility to Treg suppression in a cell-intrinsic manner
To further understand how SHP-1 regulates signaling downstream of TCR/CD3 stimulation and susceptibility to Treg suppression, we asked whether these phenotypes occurred in a cell-intrinsic and/or cell-extrinsic manner. To investigate whether SHP-1−/− CD4+CD25− Tcon cells could transfer their enhanced TCR responsiveness to neighboring SHP-1+/+ Tcon cells via a soluble mediator, we set up cocultures (Fig. 4A); we labeled either SHP-1+/+ or SHP-1−/− Tcon cells with CellTrace Violet proliferation dye, mixed them at a 1:1 ratio with SHP-1+/+ or SHP-1−/− Tcon cells, respectively, and assessed the proliferation of labeled cells (Fig. 4B). We found that adding SHP-1−/− Tcon cells to SHP-1+/+ Tcon cells did not enhance the response of the SHP-1+/+ Tcon cells (Fig. 4B), indicating that the enhanced responsiveness to TCR stimulation cannot be transmitted to neighboring T cells. We next asked whether SHP-1−/− Tcon cells could render their local environment resistance promoting for Treg-mediated suppression, perhaps via the production of cytokines or other factors that could directly influence APCs. If this were the case, one would expect that SHP-1−/− Tcon cells would be capable of inducing bystander resistance in SHP-1+/+ Tcon cells exposed to the same environment. Using the same experimental coculture setup as above, but in the presence of wild-type Tregs, the addition of SHP-1−/− Tcon cells to SHP-1+/+ Tcon cells did not induce any resistance to suppression in the SHP-1+/+ Tcon cell population (Fig. 4C). These data suggest that SHP-1−/− Tcon cells resist Treg suppression by a cell-intrinsic mechanism, which does not affect neighboring cells or induce bystander resistance.
SHP-1 deficiency enhances activation of the Akt pathway
Our data suggested that SHP-1 deficiency led to intracellular changes in the signaling pathways downstream of the TCR, which might ultimately mediate T cell resistance to suppression. A number of reports have implicated enhanced activation of PI3K/Akt in conferring T cells with resistance to suppression (24, 27, 29–33). Moreover, it was previously demonstrated that SHP-1 negatively regulates the PI3K/Akt pathway in me/me thymocytes (10, 34). We therefore assessed the phosphorylation of Akt at T308 as a measure of Akt activation (35) in response to TCR/CD3 stimulation. At 24 h poststimulation, SHP-1−/− CD4+ T cells displayed enhanced Akt phosphorylation over a range of TCR stimulation conditions compared with SHP-1+/+ CD4+ T cells (Fig. 5A, 5B). Additionally, there was also a slightly higher baseline activation of Akt in SHP-1−/− CD4+ T cells that received no TCR/CD3 stimulation. We observed the same enhanced Akt activation in SHP-1−/− CD8+ T cells compared with SHP-1+/+ CD8+ T cells, both at baseline as well as following TCR/CD3 stimulation (Fig. 5C, 5D). Taken together, these data suggest that enhanced activation of the PI3K/Akt pathway in SHP-1−/− T cells may provide one component of resistance to Treg-mediated suppression, similar to what has been described for T cells isolated from patients with lupus (32), multiple sclerosis (29), and juvenile idiopathic arthritis (24, 33).
Tcon cells lacking SHP-1 resist Treg-mediated suppression in vivo
To assess whether SHP-1 regulates the susceptibility to Treg mediated suppression in vivo, we used a murine model of Treg-mediated control of homeostatic expansion (36). We i.v. injected SHP-1+/+ or SHP-1−/− CD4+CD25− T cells (CD45.2) at a 1:1 ratio with CD45.1 wild-type CD4+CD25− T cells into Rag1−/− mice, with or without wild-type Tregs. After 10 d, we assessed the expansion of CD4+Foxp3− T cells in the spleens of Rag1−/− recipient mice (Fig. 6A). In the absence of Tregs, we observed no significant differences in the expansion of SHP-1+/+ or SHP-1−/− CD4+ T cells, when compared with the percentages (Fig. 6B) and absolute numbers of coinjected wild-type CD45.1 CD4+ T cells, suggesting that SHP-1 does not regulate homeostatic expansion. In the presence of Tregs, we observed a substantial reduction in absolute number of T cells recovered, indicating Treg-mediated suppression of homeostatic expansion (Fig. 6C). There was no difference in the extent of suppression between SHP-1+/+ T cells and wild-type CD45.1 T cells coinjected with SHP-1+/+ or SHP-1−/− T cells. However, SHP-1−/− T cells exhibited significantly greater homeostatic expansion (∼2.5-fold) in the presence of Tregs compared with SHP-1+/+ T cells or coinjected wild-type CD45.1 T cells, indicating a resistance to Treg-mediated suppression (Fig. 6B, 6C). Taken together, these data strongly suggest that SHP-1 regulates the susceptibility of CD4+ T cells to Treg-mediated suppression in vitro as well as in vivo.
For T cells to mount a productive response against a pathogen, they must be able to transiently overcome constraints imposed by Tregs. Environmental factors as well as strong antigenic signals through the TCR in the presence of costimulation have been shown to allow T cells to become refractory to Treg suppression (4–8). However, the intracellular signaling pathways that result in resistance to suppression are not well defined. In this study, we identify the tyrosine phosphatase SHP-1 as one of the intracellular regulators of Tcon cells that influence their susceptibility to Treg suppression. Both SHP-1−/− CD4+ and CD8+ T cells resisted Treg suppression of proliferation in vitro, and SHP-1−/− CD4+ T cells resisted Treg suppression of homeostatic expansion in vivo. Moreover, SHP-1−/− T cells resisted Treg suppression in a T cell–intrinsic manner, as coculture (Fig. 4) or coinjection (Fig. 6) of SHP-1+/+ (wild-type) and SHP-1−/− CD4+ T cells could not induce SHP-1+/+ (wild-type) CD4+ T cells to become resistant to suppression. SHP-1−/− T cells have been reported to produce increased amounts of IL-4 when stimulated in vitro, and SHP-1 additionally negatively regulates the subsequent downstream phosphorylation of STAT6, suggesting that SHP-1−/− T cells are hyperresponsive to IL-4 signaling (15). Because IL-4 has been shown to induce resistance to Treg suppression in vitro (37), it raised the possibility that IL-4 might play a role in mediating the observed resistance to suppression in SHP-1−/− T cells. However, we found that neither IL-4–neutralizing Abs nor Ab blockade of IL-4Rα–mediated signaling altered the resistance of SHP-1−/− T cells to Treg suppression (data not shown), indicating that the resistance reported here is IL-4–independent. This is consistent with a T cell–intrinsic mechanism and likely mediated by alterations in intracellular signaling events.
Previous studies demonstrated that deficiency of Cbl-b (38) and TNFR-associated factor 6 (31), two other negative regulators of T cell activation, also resulted in T cells that resist Treg suppression. A recent study suggested that SHP-1 regulates the degradation of Cbl-b, such that SHP-1–deficient T cells have decreased levels of Cbl-b protein after TCR stimulation alone (39). Although there are striking similarities between SHP-1−/− and Cbl-b−/− T cells, our proliferation and suppression assays included costimulatory signals from irradiated APCs, which led to Cbl-b degradation (40) in both SHP-1+/+ and SHP-1−/− T cells, and therefore would not account for the observed resistance to Treg suppression. Moreover, we did not detect any differences in Cbl-b protein expression between SHP-1−/− T cells and SHP-1+/+ T cells (data not shown). We did, however, observe enhanced activation of the Akt pathway in SHP-1−/− CD4+ T cells and naive CD8+ T cells, both basally and upon TCR stimulation. The PI3K/Akt pathway is primarily activated downstream of the TCR and CD28 costimulatory signaling, and the resultant signaling cascade allows T cells to proliferate by increasing cell size and glucose metabolism, inactivating cell cycle inhibitors, and enhancing cellular survival (41). An important mechanism of Treg suppression is depriving T cells of costimulatory signals via downregulation of costimulatory molecules CD80/CD86 (B7.1/B7.2) on APCs and upregulation of inhibitory molecules such as CTLA-4 and LAG3 (42). CTLA-4 can outcompete CD28 for binding of B7 molecules on APCs, and LAG3 can prevent maturation of APCs to adequately engage T cells (43). Previous work suggested that SHP-1–deficient T cells have a reduced requirement for costimulation (13). Because SHP-1−/− T cells show enhanced Akt activation upon TCR stimulation, they likely resist Treg suppressive mechanisms that specifically inhibit costimulation, as their need for costimulation is reduced by the enhancement in Akt activation. Interestingly, many of the environmental factors shown to induce suppression-refractory T cells have been linked to enhancing activation of the PI3K/Akt pathway (3).
Our work also helps to clarify recent discrepancies reported on SHP-1 function in negative regulation of TCR signaling due to the use of CD4-Cre–mediated deletion. Using the dLck-Cre line, in which SHP-1 deletion is temporally distinct from early stages of thymic selection, minimized developmental or potential repertoire changes to the T cell compartment. Importantly, dLck-Cre+ SHP-1f/f mice did not display any detectable differences in the composition of the thymic or peripheral T cell compartments compared with SHP-1–sufficient control mice, nor the expansion of CD4+ memory (CD44hi) T cells. However, consistent with data published by others and us (reviewed in Ref. 9), we observed increased responsiveness to TCR stimulation in SHP-1−/− CD4+ and CD8+ T cells, which was directly attributable to loss of SHP-1 within the T cells rather than an expansion of Ag-experienced T cells.
Aside from gaining insight into the molecular mechanisms of Treg resistance, which has been linked to the pathophysiology of autoimmune diseases, our findings might also be applicable toward tumor immunotherapy. Tumors actively recruit and generate Tregs to maintain a suppressive microenvironment (44). Thus, the goal of current adoptive cell transfer and/or chimeric Ag receptor (CAR) T cell therapies is to modify or create CD8+ T cells with enhanced responsiveness toward tumor Ag (45). Many of the signaling components being incorporated into CAR T cells are from costimulatory molecules, which have also been found to induce resistance to Treg suppression. For example, both 4-1BB and OX40 signaling in T cells has been found to induce Treg resistance (46–51), and components of both have been used in second generation CAR T cells (52). Along these lines, adoptive transfer of SHP-1−/− or SHP-1 knockdown (via small interfering RNA) CD8+ T cells improved tumor control in a mouse model of disseminated leukemia (53). However, whether CD8+ T cell resistance to Treg suppression played a role in tumor control was not examined. Our findings suggest that incorporating SHP-1 ablation could be useful in current CAR T cell or adoptive cell transfer therapies to allow CD8+ cytotoxic T lymphocytes to overcome Treg suppression and better control tumor outgrowth. Signaling through many of the costimulatory molecules being used currently in CAR T cell trials also enhance Akt activation. Directly enforced constitutive Akt activation induced human CD8+ T cells to resist Treg suppression and led to enhanced cytotoxicity toward a neuroblastoma cell line (54). Not only are these findings translatable to tumor immunotherapy, but also for treatment of chronic viral infections. It has been shown that chronic viral infection induces Tregs to suppress the function of CD8+ T cells, preventing viral clearance (55). Stimulation of CD8+ T cells with the costimulatory molecule 4-1BB rendered T cells resistant to Treg suppression and able to clear a chronic viral infection in mice (48). Therefore, our data reveal SHP-1 as a possible target to modulate the activation and function of T cells for tumor and chronic viral infection immunotherapies, and they provide more evidence pointing to the critical nature of the PI3K/Akt pathway in regulating the balance between T cells and Tregs.
We thank Dr. Mark Conaway for lending us expertise in statistical analyses, Amber Woods for help with the in vivo suppression experiments, and Dr. Kodi Ravichandran for critical reading of the manuscript.
This work was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant 1F31AI110146 (to E.R.M.), National Institute of General Medical Sciences Grant 5R01GM064709 (to U.M.L.), and by National Heart, Lung, and Blood Institute Grant 1P01HL120840 (to U.M.L.).
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.