Targeting of Cdc42 GTPase in regulatory T cells unleashes antitumor T-cell immunity

Introduction

CD4+ and CD8+ effector T lymphocytes have the potential to inhibit tumor cells. However, tumor cells can evade immune surveillance partly by engaging immune checkpoint proteins (eg, programmed cell death protein-1 (PD-1), cytotoxic T-lymphocytes-associated protein 4 (CTLA-4)) on effector T cells to cause their exhaustion.1 Several immune checkpoint inhibitors (ICIs) (eg, anti-PD-1) have been approved by the Food and Drug Administration (FDA) for the treatment of a number of cancer types.2 However, these ICIs are only efficacious in a small proportion of patients with cancer,3 necessitating improved cancer immunotherapies.

CD4+Foxp3+ regulatory T (Treg) cells are important for maintaining immune tolerance, primarily by inhibiting effector T cells.4 Consequently, defects in Treg cell homeostasis or their suppressive function can lead to excessive effector T-cell responses and autoimmunity.5 6 In the tumor microenvironment (TME), Treg cells contribute to tumor immune evasion.7 8 Depletion of Treg cells may thus benefit patients with cancer.9 However, systemic removal of Treg cells likely causes autoimmune diseases.9 It has recently been suggested that cancer may be treated by induction of Treg cell instability that in general is reflected by loss of stable expression of Foxp3, the signature transcription factor and an important functional marker of Treg cells, effector T-cell reprogramming, and/or impairment of Treg cell function.10–13 The mechanisms underlying Treg cell instability remain poorly defined, understanding of which may provide novel biological targets for cancer immunotherapy.

Cdc42 is a Rho family GTPase that regulates a variety of cellular events including cell proliferation, survival, actin cytoskeletal organization, and migration.14 We have recently reported that Treg cell-specific homozygous gene deletion of Cdc42 decreases Treg cell numbers, induces Treg cell instability, and causes early, fatal inflammatory diseases.15 This suggests that Cdc42 is important for maintenance of Treg cell homeostasis and stability and for controlling autoimmunity. Here we report that Treg cell-specific heterozygous gene deletion of Cdc42 also induces Treg cell instability but does not reduce Treg cell number nor does it cause autoimmunity. Importantly, heterozygous deletion of Cdc42 activates antitumor T-cell immunity. Mechanistically, heterozygous deletion of Cdc42 induces Treg cell instability and unleashes antitumor T-cell immunity through upregulation of carbonic anhydrase I (CAI) that functions to modulate cellular pH by catalyzing the hydration of CO2 to HCO3− and H+.16 A Cdc42 inhibitor, CASIN,17 mimics heterozygous deletion of Cdc42 in inducing Treg cell instability and causes antitumor T-cell immunity, without incurring autoimmune responses. Additionally, CASIN synergizes with anti-PD-1 in triggering antitumor T-cell immunity. Our findings indicate that pharmacological titration of Cdc42 to induce Treg cell instability without altering their homeostasis may be useful for immunotherapy modulations without causing autoimmunity.

MethodsTumor growth studies and treatments

Unless otherwise noted, 8×105 MC38 or KPC tumor cells were injected subcutaneously in 100 µL phosphate-buffered saline (PBS) into one of the flanks of the indicated mice. Two million HCT116 cells were injected subcutaneously in 100 µL PBS into one flank of NSGS mice 15 weeks after the mice were transplanted with CD34+ hematopoietic stem cells.18 Wherever possible, animals were randomized into treatment groups. Tumor volumes were measured every day after the tumor became visible and calculated as V=(length×width2×0.50). All the mice were euthanized when tumor volume of control mice reached about 2 cm3 (unless otherwise noted) and tumors were harvested.

CASIN (Cayman Chemical, 17694) in 60% PBS and 40% β-cyclodextrin (Cayman Chemical, 23387) was injected intraperitoneally (i.p.) at 30 mg/kg two times per day for 7 days and then 40 mg/kg one time per day until the end of the experiments. For prophylactic treatment, CASIN injection was started at the same time as tumor cell injection. For therapeutic treatment, CASIN injection was started at around day 10 when tumors became visible. Carbonic anhydrase (CA) inhibitor acetazolamide (Sigma-Aldrich, A6011) in 90% PBS, 5% Tween-20 and 5% polyethylene glycol was injected i.p. at 40 mg/kg one time per day. Wiskott-Aldrich syndrome protein (WASP) inhibitor wiskostatin (Cayman Chemical, 15047) in 97.5% PBS and 2.5% dimethyl sulfoxide was injected i.p. at 60 mg/kg one time per day. GATA binding protein 3 (GATA3) inhibitor pyrrothiogatain (Santa Cruz, sc-352288A) in 90% PBS, 5% Tween-20 and 5% polyethylene glycol was injected i.p. at 60 mg/kg one time per day. For T-cell depletion experiments, anti-CD4 (150 µg/mouse) (Bio X Cell, BE0003-3, Clone ID: YTS 177, RRID:AB_1107642), anti-CD8 (150 µg/mouse) (Bio X Cell, BE0118, Clone ID: HB-129, RRID: AB_10949065), or rat IgG2a isotype control (150 µg/mouse) (Bio X Cell, BE0090, Clone ID: LTF-2, RRID:AB_1107780) were injected i.p. every other day for four injections and then two times a week until the end of the experiments. Injection of acetazolamide, wiskostatin, pyrrothiogatain, or anti-CD4/CD8 was started at the same time as tumor cell injection. For Treg cell depletion experiment, anti-CD25 (500 µg/mouse) (Bio X Cell, BE0012, Clone ID: PC61.5.3, RRID: AB_1107619) or rat IgG1 isotype control (500 µg/mouse) (Bio X Cell, BE0088, Clone ID: HRPN, RRID: AB_1107775) was injected i.p. 1 day before tumor cell injection.19 For experiments involving ICIs, anti-PD-1 (150 µg/mouse) (Bio X Cell, BE0146, Clone ID: RMP1-14, RRID: AB_10949053) was injected every other day for a total of five injections starting on tumor onset. The order of treatments was not controlled.

Adoptive cell transfer studies

Splenic Treg cells were obtained by magnetic-activated cell sorting of CD4+ T cells (Miltenyi, 130-117-043) followed by fluorescence-activated cell sorting (FACS) of CD4+YFP+ Treg cells to >99% purity. Splenic T cells were isolated from congenic BoyJ mice by a pan T-cell isolation kit (Miltenyi, 130-095-130) followed by Treg cell depletion with CD25 MicroBead Kit (Miltenyi, 130-091-072). The sorted Treg cells (2–3×105 per mouse) with or without congenic T cells (6×105 per mouse) were transferred i.p. into Rag1–/– mice 1 day before or 9 days after MC38 cell injection into the recipient mice.

Flow cytometry

Spleens were mashed with a syringe plunger and passed through 40 µm cell strainer, followed by treatment with red blood cell lysis buffer (BD Biosciences, 555899). Tumors were minced into small fragments and treated with 1.5 mg/mL collagenase IV (Sigma, C5138) for 30 min at 37°C under agitation. The digested tumor tissue was then filtered through a 70 µm cell strainer and centrifuged at 1500 rpm at 4°C for 5 min. The pellets were dissolved in 8 mL of 40% percoll and slowly layered over 5 mL of 80% percoll in a 15 mL falcon tube. The falcon tube was then centrifuged at 2000 rpm at 4°C for 20 min, stopping without brakes. Cells at the interface between 40% percoll and 80% percoll were carefully removed and washed twice with complete Roswell Park Memorial Institute (RPMI) T-cell medium. The cells from spleens and tumors were restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 5 hours in the presence of GolgiPlug for the last 4 hours, and then subjected to antibody staining (see online supplemental materials and methods for the list of antibodies). The stained cells were analyzed by BD LSRII, FACSCanto, or LSRFortessa flow cytometers. Data were analyzed with BD FACSDiva.

In certain experiments, Treg cells isolated by flow cytometry sorting of CD4+YFP+ cells to >99% purity were expanded with Treg cell expansion kit (Miltenyi, 130-095-925) for 3 days, in the presence or absence of 47.6 mM NaHCO3 (to make culture medium of pH 7.60), 3 µM acetazolamide, 50 µM pyrrothiogatain, or the indicated concentrations of wiskostatin or CASIN. The cells were then subjected to the indicated analysis. For cytokine detection, the cells were restimulated with PMA and ionomycin along with GolgiPlug followed by flow cytometry analysis. For lentiviral shRNA-mediated knockdown, scrambled shRNA, CAI shRNA, and GATA3 shRNA lentiviral supernatants were produced by transfection of 293T cells with packaging lentiviral plasmids and either scrambled shRNA (Origene, TR30021), CAI shRNA (Origene, TL510087) or GATA3 shRNA (Origene, TL513036) lentiviral vectors followed by concentrating with Lenti-X concentrator (Takara, 631231). The viral supernatants were transduced into Treg cells that were expanded for 1 day before the transduction, by centrifugation at 1000 g at 32°C for 90 min. The transduced Treg cells were further expanded for another 2 days and subjected to the indicated analysis.

For 5-bromo-29-deoxyuridine (BrdU) incorporation assay, mice were injected i.p. with 500 mg BrdU. Two hours after injection, splenocytes were isolated and immunolabeled with anti-CD4 and anti-Foxp3 antibodies and BrdU incorporation was analyzed by a BrdU Flow kit per the manufacturer’s protocol (BD Pharmingen, 552598).15

For cell apoptosis assay, freshly isolated splenocytes were immunolabeled with anti-CD4 and anti-Foxp3 antibodies and cell apoptosis was analyzed by anti-active caspase 3 antibody (BD Pharmingen, 559341, Clone ID: C92-605, RRID:AB_397234) staining followed by flow cytometry.

Autoantibody detection

Serum was isolated from blood using serum separator tubes. Anti-dsDNA and ANA in serum were detected by ELISA using the corresponding ELISA Kits (Alpha Diagnostic International, 5120–1 and 5210, respectively), according to the manufacturer’s protocol.

Bisulfite pyrosequencing of methylation of Foxp3 enhancer

A total of 200 ng genomic DNA was subjected to sodium bisulfite treatment and purified using the EZ DNA Methylation-Gold Kit (Zymo research, D5007) according to the manufacturer’s specifications. Two rounds of standard polymerase chain reaction (PCR) amplification reaction were performed to amplify targeted gene fragment at an annealing temperature of 50°C before being subjected to pyrosequencing. The generated pyrograms were automatically analyzed using PyroMark analysis software (Qiagen). The pyrosequencing assay was validated using SssI-treated human genomic DNA as a 100% methylation control and human genomic DNA amplified by GenomePlex Complete Whole Genome Amplification kit (Sigma-Aldrich, WGA2-50RXN) as 0% methylation control.15 The primers used for bisulfite pyrosequencing are listed in online supplemental materials and methods.

Histopathological analysis

Tissues were sectioned, fixed in 4% formaldehyde solution, embedded in paraffin, and stained with H&E. The sections were analyzed by light microscopy from Fisher Scientific Moticam at 20× magnification at room temperature. The images were acquired by using the software Motic Images Plus V.2.0.15

Quantitative real-time reverse transcription (RT)-PCR analysis

Total RNA was extracted with the RNeasy mini kit from Qiagen. Isolated RNA was converted to complementary DNA (cDNA) by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Real-time RT-PCR was performed with Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, 11-744-500) and measured on StepOnePlus Real-Time PCR System (Applied Biosystems, 4376600). Data were normalized to 18S rRNA. The primers used are listed in online supplemental materials and methods.

Chromatin immunoprecipitation-quantitative real-time PCR

Treg cells were fixed with 1% formaldehyde at room temperature for 10 min. Formaldehyde was quenched by addition of 0.125 M glycine and incubation for another 5 min. The cells were lysed in 1 mL buffer 1 (50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.25% Triton X-100, and 10% glycerol) supplemented with protease inhibitor cocktail (Roche, 36363600) for 10 min. Nuclei were collected and resuspended in 1 mL buffer 2 (10 mM Tris (pH 8.0), 200 mM NaCl, 0.5 mM EGTA and 1 mM EDTA), and incubated at 4°C for 10 min. Pelleted nuclei were resuspended and sonicated for 10 min in 1 mL sonication buffer (100 mM NaCl, 50 mM Tris (pH 8), 5 mM EDTA pH 8, and 0.5% SDS) in a Covaris sonicator. After removal of an input control (whole cell lysate), chromatin was incubated with anti-GATA3 antibody (Invitrogen, MA1-028, Clone ID: 1A12-1D9, RRID:AB_2536713) or normal mouse IgG (Santa Cruz, sc-2025, RRID:AB_737182) at 4°C overnight, and pre-blocked Dynabeads Protein G for Immunoprecipitation (Thermo Fisher Scientific, 10003D) were added to the samples. After incubation at 4°C for 2 hours, the beads were washed with wash buffer 1 (150 mM NaCl, 20 mM Tris (pH 8), 5 mM EDTA (pH 8), 1% Triton X-100 and 0.2% SDS), Buffer 2 (0.1% deoxycholic acid, 1 mM EDTA (pH 8), 50 mM HEPES (pH 7.5), 1% Triton X-100, 500 mM NaCl), LiCl buffer (10 mM Tris (pH 8), 0.5% deoxycholic acid, 1 mM EDTA (pH 8), 250 mM LiCl, 0.5% NP-40), and Tris-EDTA (TE). DNA was eluted in elution buffer (0.1% SDS) and crosslinking was reversed by incubation at 65°C for 10 hours.20 After RNase A and proteinase K treatment, the precipitated chromatin DNA was purified by using a PCR purification kit (Qiagen, 28104) and then subjected to quantitative real-time PCR with the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, 4304437). Primers targeting the Car1 locus containing GATA3 binding site are: Forward 5’ GTTGTCAGTTGCCTGGCATC 3’; Reverse: 5’ GACAGTGGTAGTGGCTGCAC 3’.

Statistical analysis

Statistical analysis was carried out using Microsoft Excel 2013. Tumor growth and body weight loss in dextran sulfate sodium (DSS)-induced mouse model of colitis were analyzed by two-way analysis of variance. The rest of the statistics were performed with Mann-Whitney test. Data were expressed as mean±SD. P value<0.05 was considered significant.

ResultsHeterozygous deletion of Cdc42 induces Treg cell instability but does not cause autoimmunity

By crossing Cdc42Flox/Flox mice with Foxp3YFP-Cre mice, we generated Cdc42Flox/+Foxp3YFP-Cre mice that harbor heterozygous knockout of Cdc42 specifically in Treg cells. We found that heterozygous knockout of Cdc42 did not affect Treg cell homeostasis (figure 1A), proliferation (figure 1B), and survival (figure 1C). A variety of Treg cell functional markers such as PD-1, CTLA-4, ICOS, GITR, CD39, and CD73 also remained unchanged (figure 1D and online supplemental figure 1A). Cdc42Flox/+Foxp3YFP-Cre mice had no visible inflammatory disorders. H&E staining showed no inflammatory cell infiltration into the colon, liver, lung, and kidney of Cdc42Flox/+Foxp3YFP-Cre mice (figure 1E). However, Cdc42Flox/+Foxp3YFP-Cre Treg cells showed reduced expression of Foxp3 (figure 1F) and increased methylation in conserved non-coding sequence 2 (CNS2) of the Foxp3 locus (figure 1G), a CpG-rich Foxp3 intronic cis-element whose hypomethylation is important for stable Foxp3 expression.13 The increased methylation in CNS2 of the Foxp3 locus was associated with increased expression of DNA methyltransferase 3A (DNMT3a) but not alteration in DNA demethylases TET1/2/3 (online supplemental figure 1B). Cdc42Flox/+Foxp3YFP-Cre Treg cells underwent effector T-cell reprogramming, as evidenced by increased expression of interleukin (IL)-4, a signature cytokine of CD4+ T helper 2 (Th2) cells, and interferon (IFN)-γ, a signature cytokine of CD4+ Th1 cells, but not IL-17, a signature cytokine of CD4+ Th17 cells (figure 1H and online supplemental figure 2A). Although IFN-γ-expressing CD8+ effector T cells were not altered (figure 1I and online supplemental figure 2B), IL-4-expressing and IFN-γ-expressing CD4+ effector T cells (CD4+Foxp3−) were increased in Cdc42Flox/+Foxp3YFP-Cre mice (figure 1J and online supplemental figure 2A). Likewise, heterozygous deletion of Cdc42 in Treg cells did not affect CD62L+CD44– naïve T cells, CD62L+CD44+ central memory T cells and CD62L–CD44+ effector memory T cells in CD8+ T-cell compartment (figure 1K) but increased memory T cells at the expense of naïve T cells in CD4+ T-cell compartment (figure 1L). The increased CD4+ memory T cells may reflect the increased IL-4-producing and IFN-γ-producing CD4+ T cells that would have resulted from a conversion of unstable Treg cells to effector T cells. In support, ex vivo culture of Treg cells found that heterozygous loss of Cdc42 caused more Treg cell conversion to CD4+Foxp3– ex-Treg cells (figure 1M). To determine whether the same occurs in vivo, we took advantage of Foxp3eGFP-Cre-ERT2Rosa26eYFP mice. In Foxp3eGFP-Cre-ERT2Rosa26eYFP mice, treatment with tamoxifen induces the expression of Cre and GFP in Foxp3+ cells. Cre expression causes excision of floxed stop codon in the Rosa26 locus, leading to YFP expression. Thus, Treg cells from Foxp3eGFP-Cre-ERT2Rosa26eYFP mice are marked by both GFP and YFP. When Treg cells convert to CD4+Foxp3– ex-Treg cells, their GFP is lost but YFP is retained, because the Rosa26 locus is ubiquitously expressed.21 We crossed Foxp3eGFP-Cre-ERT2Rosa26eYFP mice with Cdc42Flox/Flox mice and found that the resultant Cdc42Flox/+Foxp3eGFP-Cre-ERT2Rosa26eYFP mice contained more CD4+GFP–YFP+ ex-Treg cells, compared with Cdc42+/+Foxp3eGFP-Cre-ERT2Rosa26eYFP mice (figure 1N). Together, these results indicate that heterozygous loss of Cdc42 primes Treg cell instability without invoking autoimmune responses.

Figure 1Figure 1Figure 1

Heterozygous loss of Cdc42 in Treg cells induces Treg cell instability and increases CD4+ effector T cells but does not result in autoimmunity. (A–M) The spleen from wild type (Cdc42+/+Foxp3YFP-Cre) and Cdc42 heterozygous knockout (Cdc42Flox/+Foxp3YFP-Cre) mice was subjected to flow cytometry analysis of the percentages and number of Treg cells (A), Treg cell proliferation (Brdu incorporation) (B), Treg cell apoptosis (expression of cleaved caspase 3) (C), Treg cell functional markers (D), the expression of Foxp3 (MFI) in Treg cells (F), the expression (percentages and MFI) of IFN-γ, IL-17 and/or IL-4 in Treg cells (H), CD8+ T cells (I) and CD4+ T cells (J), and the percentages of naïve (CD62L+CD44–), central memory (CD62L+CD44+) and effector memory (CD62L–CD44+) T cells in CD8+ (K) and CD4+ (L) T-cell compartments. Of note, MFI of IFN-γ, IL-17 and IL-4 was analyzed in IFN-γ+, IL-17+ and IL-4+ cells, respectively. Splenic Treg cells from the mice were examined for the methylation status of 14 methylation sites of the Foxp3 conserved non-coding sequence 2 by bisulfite pyrosequencing (G) or cultured for 3 days followed by flow cytometry analysis of induction of ex-Treg cells (CD4+Foxp3–) (M). The liver, kidney, colon, and lung from the mice were subjected to H&E staining (E). (N) Cdc42Flox/+Foxp3eGFP-Cre-ERT2Rosa26eYFP and Cdc42+/+Foxp3eGFP-Cre-ERT2Rosa26eYFP mice were treated (intraperitoneally) with 2 mg of tamoxifen daily for five consecutive days. One week and 6 weeks after the last tamoxifen injection, the spleen was analyzed for CD4+GFP–YFP+ ex-Treg cells by flow cytometry. (A–D, F, and H–L) Error bars indicate SD of 5–8 mice. Data are representative of three independent experiments. (N) Error bars indicate SD of five mice. Data are representative of two independent experiments. (G and M) Error bars indicate SD of triplicates. Data are from one experiment with four mice pooled. (G) Data are expressed as percentage of methylation which indicates per cent cells with a particular methylation site methylated. *p<0.05; **p<0.01. BrdU, bromo-29-deoxyuridine; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; Treg, regulatory T cells.

Treg cell-specific heterozygous deletion of Cdc42 inhibits tumor growth

To determine whether Cdc42Flox/+Foxp3YFP-Cre Treg cells can be harnessed for tumor control, we inoculated Cdc42Flox/+Foxp3YFP-Cre and control Cdc42+/+Foxp3YFP-Cre mice with MC38 mouse colon cancer cells. We found that tumor growth (measured by tumor volume) was drastically suppressed in Cdc42Flox/+Foxp3YFP-Cre mice (figure 2A). Although Cdc42Flox/+Foxp3YFP-Cre mice did not show a change in the ratio of tumor-infiltrating effector T cells versus Treg cells (figure 2B), these mice had increased tumor-infiltrating IFN-γ+ Treg cells (figure 2C and online supplemental figure 3A) and effector T cells (figure 2D and E and online supplemental figure 3B, C). A modest increase in IFN-γ-producing Treg cells and CD4+ effector T cells and a marked increase in IFN-γ-producing CD8+ effector T cells were also observed in the spleen of tumor-bearing Cdc42Flox/+Foxp3YFP-Cre mice (online supplemental figure 3D–F), indicating that Cdc42 heterozygosity generates a global immuno-effect in tumor-bearing mice. Nonetheless, IFN-γ+ cells seemed to be less in the spleen than that in tumor (online supplemental figure 3D–F compared with figure 2C–E), suggesting that IFN-γ+ cells unproportionally migrated from tumor/tumor draining lymph nodes to the spleen. Furthermore, heterozygous loss of Cdc42 in Treg cells inhibited tumor growth of KPC pancreatic cancer cells (figure 2F). Together, these results suggest that unstable Cdc42Flox/+Foxp3YFP-Cre Treg cells trigger T-cell immunity against tumor growth.

Figure 2Figure 2Figure 2

Heterozygous loss of Cdc42 in Treg cells inhibits tumor growth by promoting Treg cell instability. (A–F) MC38 colon cancer cells (A–E) or KPC pancreatic cancer cells (F) were inoculated into Cdc42+/+Foxp3YFP-Cre and Cdc42Flox/+Foxp3YFP-Cre mice. Tumor growth (volume) was recorded (A, F). Tumor was dissected and subjected to flow cytometry analysis of the percentages of CD4+Foxp3+ Treg cells and CD4+Foxp3− T cells among total CD4+ T cells, and the percentages of total CD4+Foxp3+ Treg cells and CD8+ T cells from singlet cells. The ratio of CD4+Foxp3− T cells versus CD4+Foxp3+ Treg cells and the ratio of total CD8+ T cells versus total CD4+Foxp3+ Treg cells were calculated (B). IFN-γ+, IL-17+ and/or IL-4+ cells in tumor-infiltrating CD4+Foxp3+ Treg cells (C), CD4+Foxp3− T cells (D) and CD8+ T cells (E) were also analyzed by flow cytometry. (G–J) Cdc42+/+Foxp3YFP-Cre or Cdc42Flox/+Foxp3YFP-Cre Treg cells were adoptively transferred into Rag1−/− mice. One day later, the recipient mice were inoculated with MC38 cells (G). Tumor volume was recorded (H). The percentages of IFN-γ+, IL-17+ and/or IL-4+ cells in tumor-infiltrating Treg cells (CD4+Foxp3+) (I) and CD4+ effector T cells (CD4+Foxp3−) (J) were analyzed by flow cytometry. (K–N) Cdc42+/+Foxp3YFP-Cre or Cdc42Flox/+Foxp3YFP-Cre Treg cells were adoptively co-transferred with congenic T cells into Rag1−/− mice. One day later, the recipient mice were inoculated with MC38 cells (K). Tumor volume was recorded (L). The percentages of IFN-γ+, IL-17+ and/or IL-4+ cells in tumor-infiltrating congenic CD4+ (M) and CD8+ (N) effector T cells were analyzed by flow cytometry. Error bars indicate SD of 5–6 mice. Data are representative of two independent experiments. *p<0.05; **p<0.01. IFN, interferon; IL, interleukin; Treg, regulatory T cells.

The tumor suppression in Cdc42Flox/+Foxp3YFP-Cre mice may be mediated by the increased CD4+ effector T cells that are converted from unstable Treg cells. To test this possibility, we transferred Cdc42Flox/+Foxp3YFP-Cre and Cdc42+/+Foxp3YFP-Cre Treg cells into immunodeficient Rag1–/– mice followed by MC38 tumor cell inoculation (figure 2G). We found that tumor growth was modestly but significantly inhibited in the mice receiving Cdc42Flox/+Foxp3YFP-Cre Treg cells (figure 2H). Tumors in the mice receiving Cdc42Flox/+Foxp3YFP-Cre Treg cells showed an increase in unstable Treg cells (figure 2I) and in CD4+ effector T cells (figure 2J). As only Treg cells were transferred, the increased CD4+ effector T cells were presumably ex-Treg cells converted from unstable Cdc42Flox/+Foxp3YFP-Cre Treg cells, similar to that on ex vivo culture (figure 1M) and in Treg lineage tracing mice (figure 1N). It is conceivable that these ex-Treg cells contributed to the tumor suppression. However, we cannot rule out that effector T-cell activities (eg, IFN-γ production) in Treg cells may have also played a role in the tumor suppression. Taken together, our data demonstrate that the instability of Cdc42Flox/+Foxp3YFP-Cre Treg cells contributes to the tumor suppression in Cdc42Flox/+Foxp3YFP-Cre mice.

The tumor suppression in Cdc42Flox/+Foxp3YFP-Cre mice may also be mediated by dampened suppressive function of unstable Treg cells.11 12 In this context, the increased CD4+ effector T cells in Cdc42Flox/+Foxp3YFP-Cre mice may not only result from unstable Treg cell conversion to effector T cells but also compromised function of Cdc42Flox/+Foxp3YFP-Cre Treg cells, which is supported by the increased CD8+ effector T cells in tumor-bearing Cdc42Flox/+Foxp3YFP-Cre mice. To test this, we co-transferred Cdc42Flox/+Foxp3YFP-Cre or Cdc42+/+Foxp3YFP-Cre Treg cells with congenically marked (CD45.1+) effector T cells into Rag1−/− mice followed by tumor cell inoculation (figure 2K). As expected, tumor growth in the mice receiving Cdc42Flox/+Foxp3YFP-Cre Treg cells and effector T cells was inhibited (figure 2L). The mice contained increased tumor-infiltrating CD45.1+ effector T cells (figure 2M,N), suggesting that Cdc42Flox/+Foxp3YFP-Cre Treg cells were indeed impaired in their suppressive function. In this setting, the co-transfer of Cdc42Flox/+Foxp3YFP-Cre Treg cells with effector T cells appeared to diminish tumor growth to a greater extent than the transfer of Cdc42Flox/+Foxp3YFP-Cre Treg cells only (figure 2L, compared with figure 2H), suggesting that the dampened suppressive function of Cdc42Flox/+Foxp3YFP-Cre Treg cells also contributes to the tumor suppression in Cdc42Flox/+Foxp3YFP-Cre mice.

Of note, unstable Treg cells (online supplemental figure 4A, B) and/or the resultant effector T cells (online supplemental figure 4C,D) in 12–18 months old Cdc42Flox/+Foxp3YFP-Cre mice did not cause spontaneous systemic inflammatory disorders (online supplemental figure 4E), similar to that in 6–8 weeks old Cdc42Flox/+Foxp3YFP-Cre mice (figure 1) but distinct from that in aged CNS2–/– mice that show spontaneous lymphoproliferative disease.22 We speculate that the instability phenotypes of Cdc42Flox/+Foxp3YFP-Cre Treg cells may be milder than that of aged CNS2–/– Treg cells. As that in 6–8 weeks old Cdc42Flox/+Foxp3YFP-Cre mice (figure 2), Cdc42Flox/+Foxp3YFP-Cre Treg cells in 12–18 months old Cdc42Flox/+Foxp3YFP-Cre mice were able to cause tumor suppression attributable to instability of tumor-infiltrating Treg cells (online supplemental figure 4F–I).

Treg cell-specific heterozygous deletion of Cdc42 induces Treg cell instability and elicits antitumor T-cell immunity through a WASP-GATA3-CAI signaling node

To determine the mechanism of heterozygous Cdc42 loss-induced Treg cell instability, we carried out global gene expression profiling of Cdc42Flox/+Foxp3YFP-Cre Treg and Cdc42+/+Foxp3YFP-Cre Treg cells, by RNA sequencing. We found that 319 genes were upregulated (>1.2-fold) and 140 genes were downregulated (>1.2-fold) in Cdc42Flox/+Foxp3YFP-Cre Treg cells (online supplemental table 1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that cytokine–cytokine receptor interaction pathway was upregulated (FDR <0.25) (online supplemental table 2) and ribosome pathway was downregulated (FDR <0.25) (online supplemental Table 3) in Cdc42Flox/+Foxp3YFP-Cre Treg cells. Among the upregulated genes, Car1 that encodes CAI was of particular interest, since it is the second most upregulated gene in Cdc42Flox/+Foxp3YFP-Cre Treg cells (online supplemental table 1 and figure 5A) and the most upregulated gene in Cdc42Flox/FloxFoxp3YFP-Cre Treg cells (online supplemental table 4 and figure 5B). The expression levels of CAI messenger RNA (mRNA) were 369-fold in Cdc42Flox/+Foxp3YFP-Cre Treg cells and 7828-fold in Cdc42Flox/FloxFoxp3YFP-Cre Treg cells more than that in Cdc42+/+Foxp3YFP-Cre Treg cells (online supplemental tables 1 and 4). We confirmed the upregulation of CAI mRNA and CAI protein in Cdc42Flox/+Foxp3YFP-Cre Treg cells (figure 3A and online supplemental figure 6A). CAI functions to modulate cellular pH by catalyzing the hydration of CO2 to HCO3− and H+.16 CAI is expressed in the cytoplasm.16 We hypothesized that overexpression of CAI in Cdc42Flox/+Foxp3YFP-Cre Treg cells generated excessive HCO3– and H+, leading to HCO3– transportation to extracellular space and thus extracellular alkalization that caused Treg cell instability. To test this, we measured the medium pH of Cdc42Flox/+Foxp3YFP-Cre and Cdc42+/+Foxp3YFP-Cre Treg cell culture. We found that while the pH of Cdc42+/+Foxp3YFP-Cre Treg cell culture was maintained at 7.40, the pH of Cdc42Flox/+Foxp3YFP-Cre Treg cell culture was changed to 7.60 (figure 3B). Importantly, Cdc42+/+Foxp3YFP-Cre Treg cells became unstable on incubation with culture medium of pH 7.60 (figure 3C–E) or with conditional medium from Cdc42Flox/+Foxp3YFP-Cre Treg cell culture (figure 3F–H). The extracellular alkalization and instability of Cdc42Flox/+Foxp3YFP-Cre Treg cells were rescued by treatment with a CA inhibitor, acetazolamide (figure 3I–K), and by CAI knockdown (figure 3L–O). Thus, the instability of Cdc42Flox/+Foxp3YFP-Cre Treg cells is likely caused by CAI-mediated extracellular alkalization. Acetazolamide treatment of Cdc42Flox/+Foxp3YFP-Cre mice were able to restore tumor growth (figure 3P), correlating with reduced instability of tumor-infiltrating Treg cells and decreased tumor-infiltrating effector T cells (online supplemental Figure 6B-E). We conclude that heterozygous Cdc42 deletion induces Treg cell instability and revokes antitumor T-cell immunity through upregulation of CAI. Of note, inhibition of CAI in Cdc42+/+Foxp3YFP-Cre Treg cells caused extracellular alkalization and Treg cell instability (figure 3I–K and L–O), similar to that caused by the upregulation of CAI in Cdc42Flox/+Foxp3YFP-Cre Treg cells (figure 3A, I–K and L–O). Furthermore, Cdc42+/+Foxp3YFP-Cre mice treated with acetazolamide showed similar tumor suppression to Cdc42Flox/+Foxp3YFP-Cre mice (figure 3P). These data suggest that physiological levels of CAI are essential for Treg cell stability and Treg cell-mediated tumor growth. Given that only ~4% of Cdc42+/+Foxp3YFP-Cre Treg cells expressed CAI (figure 3A), the drastic inhibition of tumor growth in acetazolamide-treated Cdc42+/+Foxp3YFP-Cre mice suggests that even small proportion of CAI+ Treg cells could be important for maintaining overall Treg cell extracellular pH and their stability and that inhibition of CAI in tumor-infiltrating Treg cells may alter the TME pH, which may not only affect Treg cell stability but also other cells in the TME. It is conceivable that acetazolamide may directly affect effector T cells and/or MC38 tumor cells in tumor-bearing mice. Nonetheless, as effector T cells and MC38 tumor cells from Cdc42Flox/+Foxp3YFP-Cre mice presumably express CAI at comparable levels to that from Cdc42+/+Foxp3YFP-Cre mice, any direct effect of acetazolamide on effector T cells and MC38 tumor cells is expected to affect tumor growth similarly in Cdc42Flox/+Foxp3YFP-Cre and Cdc42+/+Foxp3YFP-Cre mice. Indeed, we did not detect a change in CAI expression in tumor-infiltrating effector T cells from Cdc42Flox/+Foxp3YFP-Cre mice (data not shown). In this context, the restoration of tumor growth in acetazolamide-treated Cdc42Flox/+Foxp3YFP-Cre mice and the inhibition of tumor growth in acetazolamide-treated Cdc42+/+Foxp3YFP-Cre mice unlikely resulted from an off-Treg cell effect of acetazolamide but from differential expression of CAI in Cdc42Flox/+Foxp3YFP-Cre and Cdc42+/+Foxp3YFP-Cre Treg cells.

Figure 3Figure 3Figure 3

Heterozygous loss of Cdc42 induces Treg cell instability and inhibits tumor growth through upregulation of CAI. (A, B) Splenic Treg cells from Cdc42+/+Foxp3YFP-Cre and Cdc42Flox/+Foxp3YFP-Cre mice were examined for the expression of CAI mRNA (left) and CAI protein (right) by quantitative real-time RT-PCR and flow cytometry, respectively (A), or cultured for 3 days followed by measurement of culture medium pH (B). (C–H) Splenic Cdc42+/+Foxp3YFP-Cre Treg cells were cultured for 3 days with medium of pH 7.40 or pH 7.60 (C–E) or with conditional medium from Cdc42+/+Foxp3YFP-Cre or Cdc42Flox/+Foxp3YFP-Cre Treg cell culture (F–H). The expression of Foxp3 (MFI) in Treg cells (C, F) and the percentages of IFN-γ+ cells in Treg cells (D, G) and CD4+ effector T cells (ex-Treg) (E, H) were analyzed by flow cytometry. (I–K) Splenic Cdc42+/+Foxp3YFP-Cre and Cdc42Flox/+Foxp3YFP-Cre Treg cells were cultured for 3 days with AZA or vehicle. The medium pH was measured (I). The percentages of IFN-γ+ cells in Treg cells (J) and CD4+ effector T cells (ex-Treg) (K) were analyzed by flow cytometry. (L–O) Splenic Cdc42+/+Foxp3YFP-Cre and Cdc42Flox/+Foxp3YFP-Cre Treg cells were transduced with CAI shRNA or scramble shRNA during 3 days’ culture. The expression of CAI mRNA (left) and CAI protein (right) was detected by quantitative real-time RT-PCR and flow cytometry, respectively (L). The medium pH was measured (M). The percentages of IFN-γ+ cells in Treg cells (N) and CD4+ effector T cells (ex-Treg) (O) were analyzed by flow cytometry. (P) Cdc42+/+Foxp3YFP-Cre and Cdc42Flox/+Foxp3YFP-Cre mice were inoculated with MC38 cells and treated with or without AZA at 40 mg/kg one time per day, starting at the same time as MC38 cell inoculation. Tumor volume was recorded. (A) Error bars indicate SD of four mice. (B–O) Error bars indicate SD of triplicates. Data are from five m

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