To identify orphan GPCR engaged in the generation of cAMP in mouse T cells, we analyzed available RNA sequencing data (27) and found a subset-specific expression of several orphan GPCRs ( Figure 1A ; Figures S1 , S2 ). The orphan Gs-coupled GPR52 that has previously been shown to alter intracellular cAMP levels in transfected HEK cells and primary neurons (28, 29) was also expressed in T cells and differentially higher in Tregs compared to Teffs. Since GPR52 has not been studied in the context of T cell function, we selected this GPCR for further functional investigations. We verified the differential expression of GPR52 in mouse T cell subsets using multiplexed gene expression analysis and RT-qPCR ( Figures 1B, C ; Figure S3 ). Next, we tested the specificity of 4-(3-(3-fluoro-5-(trifluoromethyl)benzyl)-5-methyl-1H-1,2,4-triazol-1-yl)-2-methyl-benzamide (FTBMT), a selective GPR52 agonist, in HEK293A cells. As GPR52 is not endogenously expressed in HEK293A cells, we transfected them with the cytosolic FRET-based cAMP sensor Epac1-camps together with Gpr52-GFP or GFP-control. We observed a significant increase in cAMP levels upon FTBMT treatment of Gpr52-transfected HEK293A cells in comparison to control-transfected cells ( Figure 1D ; Figures S4A, B ). Having validated FTBMT specificity, we detected, in line with expression data, higher cAMP production in Tregs compared to Teffs from CAG-Epac1-camps transgenic mice (30) after FTBMT pre-treatment ( Figure 1E ; Figures S4C, D ). According to the calibration curve of our cAMP biosensor (31) in its relevant range, the difference of approx. 15% response corresponds to 1.5–2 µM higher intracellular cAMP in Treg. By contrast, pre-treatment of T cell subsets with E7, an antagonist of GPR52 (28), decreased intracellular cAMP levels in both Teffs and Tregs compared to FTBMT treatment alone and resulted in a comparable FRET signal in Teffs and Tregs ( Figure 1E ; Figures S4E, F ). Together, this indicates that GPR52 activation on T cells increases their intracellular cAMP levels with Tregs mounting a stronger response to the selective GPR52 agonist than Teffs.

As we have shown that pharmacological manipulation of GPR52 alters intracellular cAMP levels, we hypothesized that T cell function is also affected by Gpr52 deletion. To test this, we used primary T cells isolated from Gpr52-deficient mice with a verified knockout of Gpr52 as shown by RT-qPCR ( Figure S5 ). However, Gpr52 deficiency did not result in an altered T cell activation by antibody-mediated CD3 and CD28 stimulation ( Figures 2A, B ). Moreover, Gpr52 deletion did not affect the immunosuppressive capacity of Tregs ( Figure 2C ; Figure S6 ). Differentiation of naïve CD4+ T cells into T helper subsets (Th1, Th2, Th17, and Treg) was also not different in T cells derived from Gpr52-deficient mice compared to wildtype littermate controls ( Figures 2D, E ). Thus, GPR52 is dispensable for T cell activation, differentiation and Treg-mediated immunosuppression in in vitro assays.

As Gpr52 deficiency had no apparent impact on T cell function, we next determined whether treatment of T cells with the selective GPR52 antagonist E7 (28) or agonist FTBMT (32) results in altered T cell function. We first tested the effect of E7 and FTBMT treatment on wildtype T cells derived from C57BL/6. Treatment with 100 nM E7 did not alter the differentiation of naïve CD4+ T cells to Th1, Th2, Th17 or Tregs ( Figure 3A ; Figure S7A ). Similarly, FTBMT agonist treatment did not alter differentiation from naïve CD4+ T cell into T cell subsets ( Figure 3B ; Figure S7B ). FTBMT did neither affect T cell proliferation ( Figures 3C, D ) nor did FTBMT or E7 alter T cell activation ( Figures 3E, F ) of CD4+ T cells. Upon higher concentration of E7, as suggested in literature (28, 29), increased cell death was observed ( Figure S7C, D ). Toxic effect of E7 was observed on both, Gpr52-deficient and wildtype T cells ( Figure S7E ). In line with that, decrease of T cell activity upon higher concentration of E7 was observed in both, Gpr52-proficient and Gpr52-deficient T cells, independent of GPR52 abundance ( Figure S7F, G ). Together, the GPR52 agonist FTBMT did not alter T cell function while the observed effects after E7 antagonist treatment at high concentrations were GPR52-independent.

As cAMP is crucial for Teff functions and the immunosuppressive capacity of Tregs, we finally tested whether GPR52 exerts its function in an autoimmune response. To do so, we induced three independent EAE in Gpr52-deficient mice and age- and sex-matched wildtype littermate controls. However, neither clinical score ( Figure 4A ), nor weight loss differed between the experimental groups ( Figure 4B ). Also the EAE incidence ( Figure 4C ), disease onset ( Figure 4D ), defined as day when first symptoms were recorded, cumulative clinical score ( Figure 4E ) and maximal clinical score ( Figure 4F ) did not differ between Gpr52-deficient animals and wildtype littermate controls. Thus, we compiled evidence that GPR52 is dispensable for encephalitogenic T cell generation and CNS auto-aggression.

Seven- to 12-week-old C57BL/6 wildtype, CAG-Epac1-camps and Gpr52-deficient mice (Gpr52^tm1Kohi) were used in this study (30, 46, 48). Gpr52-deficient mice were kindly provided by Takeda Pharmaceutical Company Limited. Mice were kept under specific pathogen-free conditions in the central animal facility of the University Medical Center Hamburg-Eppendorf (UKE) and were handled in accordance to international and national animal welfare guidelines with the organ isolation procedures approved by the Behörde für Justiz und Verbraucherschutz Hamburg (ORG1010, ORG946 and ORG1075). Induction of experimental autoimmune encephalomyelitis and tissue sample collection was licensed under No. 45/17 and 83/19.

HEK293A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich) at 37°C and 5% CO₂. HEK293A were plated on 25 mm glass coverslips. To ensure proper cell attachment, cells were cultured overnight and transfected with a mixture consisting of plasmid DNA (0.5 μg/well), non-supplemented DMEM, and Lipofectamine 2000 (Thermo Fisher Scientific, following the manufacturer’s transfection protocol) in a total volume of 50 μl/well. Cells were transfected with cytosolic FRET-based sensor, Epac1-camps, and pcDNA3 (empty vector) or GPR52-encoding plasmid labeled with GFP. 40–48 hours post-transfection, cells with proper expression of the plasmid DNA were used to validate GPR52 agonist drug (FTBMT) specificity via real-time FRET measurements described below.

For live-cell imaging via FRET, spleens from eight- to 12-week-old CAG-Epac1-camps transgenic mice (30) were collected on ice-cold PBS. CD4+ T cells were obtained from single-cell suspension by negative selection following the manufacturer’s protocol of murine CD4+ T cell Isolation Kit (Miltenyi Biotec). Separation of CD4+CD25- and CD4+CD25+ T cells were processed by further labeling with CD25-biotin antibody (Miltenyi Biotec). T cell activation was achieved upon overnight culture of T cell subsets in presence of Dynabeads Mouse T-Cell Activator CD3/CD28 (Thermo Fisher) at 37°C and 5% CO₂. Real-time measurements were performed on T cells plated on 25 mm glass coverslips coated with poly-D-Lysine (Sigma Aldrich). For glass coverslips, an Autofluor cell chamber was used, as well as FRET buffer (144 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES; pH 7.3), to dilute chemical compounds tested. Non-adherent cells were washed before the start of the measurement. To monitor FRET response Leica DMI 3000 B was used, an inverted fluorescent microscope equipped with an oil-immersion 63x/1.40 objective and a MicroManager 1.4. software. As the fluorescent light source CoolLED was used, to excite cyan fluorescent protein (CFP) at 440 nm. Beam-splitter, DV2 Dual View (Photometrics; Cube 05-EM, 505 dcxr, D480/30, D535/40), was used to split the emission light into CFP and yellow fluorescent protein (YFP) channel, simultaneously monitored on a CMOS (OptiMOS, QImaging) camera chip. Images were taken every 5- or 10-seconds. Data were analyzed by ImageJ (RRID: SCR:003070) and Microsoft Excel for offline corrections with a spectral bleed-through correction factor as described (31).

Lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from C57BL/6 wildtype mice were collected in ice-cold PBS. CD4+ T cells were enriched by negative selection using the MojoSort CD4+ T cell isolation kit (BioLegend). CD4+ T cells were stained for T cell surface marker for 30 min at 4°C. Antibodies that were used are listed in Supplementary Table 1 . Dead cells were excluded by adding Alexa Fluor 750 NHS Ester (Succinimidyl Ester) (Thermo Fisher). Nonspecific Fc receptor-mediated antibody binding was minimized by blocking with TruStain FCX anti-mouse CD16/32 (clone 93, BioLegend). Stained cells were FACS-purified using a FACS Aria III cell sorter (BD Bioscience). RNA of sorted samples was isolated using RNeasy Mini Kit (Qiagen). The yield of total RNA was determined by using the Qubit RNA high sensitivity, broad range, and extended range Assay Kit (Thermo Fisher Scientific). mRNA expression was measured with the NanoString nCounter FLEX Analysis System (RRID: SCR_021712, NanoString Technologies) using 30–35 ng of total RNA. The custom-made CodeSet for 36 genes including 6 housekeeping genes (Actb, Gapdh, Gusb, Hprt, Rpl19, Tbp) was hybridized to total RNA for 16 hours at 67°C. The expression data were analyzed utilizing the NanoString nSolver Analysis Software 4.0. Raw data were analyzed and normalized to the housekeeping genes together with quality control performed using nSolver 4.0 User Manual in addition to default settings and algorithm within the nSolver Analysis software.

For T cell activation assay, lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from C57BL/6 wild-type mice or Gpr52 ^–/– and respective littermate controls were collected in ice-cold PBS. CD4+ T cells were isolated from single-cell suspension using the MojoSort CD4+ T cell isolation kit (BioLegend) according to the manufacturer’s protocol. CD4+ T cells were seeded in an anti-CD3 (1 µg/ml; clone 145-2CL11, BioLegend)-coated 96-well plate. Cells were supplemented with compounds (E7 and FTBMT) and soluble anti-CD28 (2 µg/ml; clone 37.51, BioLegend). Samples were incubated for 6 hours at 37°C and 5% CO₂. After that cells were stained for T cell surface markers for 30 minutes at 4°C. Dead cells were excluded by adding Alexa Fluor 750 NHS Ester (Succinimidyl Ester) (Thermo Fisher). Nonspecific Fc receptor-mediated antibody binding was minimized by blocking with TruStain FCX anti-mouse CD16/32 (clone 93, BioLegend). After fixation and permeabilization at RT with eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher), FOXP3 was stained intranuclearly for 30 min at RT. Cells were stained with antibodies listed in Supplementary Table 1 . Samples were acquired on the BD FACS LSR II analyzer (BD Bioscience) or FACSymphony A3 (BD Bioscience). Data were analyzed using FlowJo (version 10, BD Bioscience, RRID: SCR_008520).

For T cell proliferation assay lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from C57BL/6 wildtype mice were collected in ice-cold PBS. Single-cell suspension was generated and splenocytes were seeded in an anti-CD3 (clone 145-2CL11, BioLegend)-coated 96-well plate. Cells were supplemented with soluble anti-CD28, IL2 (Peprotech) and FTBMT (100 nM and 1 µM; MedChemExpress). Samples were incubated for 72 hours at 37°C and 5% CO₂. During the last 16 hours cells were pulsed with 1 µg/ml bromodeoxyuridine (BrdU, BioLegend). Cells were stained for surface markers, fixated and permeabilized as described above. Next, cells were incubated in PBS supplemented with Ca²⁺ and Mg²⁺ supplemented with 40 KU/ml DNase I (Merck) for 1 hour at 37°C and 5% CO₂. After DNA digestion, Foxp3 and incorporated BrdU was stained for 30 min at RT. Used antibodies are listed in Supplementary Table 1 . Samples were acquired and data were analyzed as indicated above.

For T cell differentiation assays lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from C57BL/6 wildtype mice or Gpr52 ^–/– and littermate controls were collected in ice-cold PBS. Naïve CD4+ T cells were isolated from single-cell suspension using the MojoSort naïve CD4+ T cell isolation kit (BioLegend) according to manufacturer’s protocol. Naïve CD4+ T cells were seeded in an anti-CD3 (clone 145-2CL11, BioLegend)-coated 96-well plate and supplemented with soluble anti-CD28, compounds (100 nM E7, 1 µM E7 or 1 µM FTBMT), as well as respective cytokines and antibodies required for induction of each T cell subset (see Supplementary Table 2 ). Cells were incubated for 72 hours at 37°C and 5% CO₂. During the last 5 hours of incubation, cells were stimulated with 1 µg/ml ionomycin (Sigma Aldrich), 20 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich), and brefeldin A (BioLegend) to induce cytokine production and accumulation. Cells were stained for surface markers, fixed and permeabilized as well as stained for intracellular marker as indicated above. Antibodies used are listed in Supplementary Table 1 . Samples were acquired and data were analyzed as described above.

For Treg suppression assay, lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from Gpr52 ^–/– and littermate controls were collected in ice-cold PBS. Teffs and Tregs were isolated from single-cell suspension using CD4+ CD25+ Regulatory T cell isolation kit (Miltenyi Biotec) according to manufacturer’s protocol. Staining of wildtype Teffs was performed with CellTrace™ CFSE Cell Proliferation Kit (Thermo Fisher). Briefly, Teffs were stained for 15 minutes at 37°C with reconstituted CFSE in PBS containing 0.1% BSA (Miltenyi Biotec). Tregs and labeled wildtype Teffs were seeded in 96-well plate together with Dynabeads™ mouse T-activator CD3/CD28 (ratio Teffs:beads 1:1; Thermo Fisher) and IL-2 (10 IU/ml, Peprotech). Cells were incubated for 72 hours at 37°C and 5% CO₂. Cells were stained for surface markers, fixed, permeabilized, and stained for intranuclear FOXP3 as indicated above. Antibodies used are listed in Supplementary Table 1 . Samples were acquired and data were analyzed as indicated above.

T cells were isolated from lymph nodes (superficial cervical, axillary, brachial, inguinal) and spleen from C57BL/6 wild-type mice from single-cell suspension using the MojoSort CD4+ T cell isolation kit (BioLegend) according to the manufacturer’s protocol. After, CD4+ T cells were supplemented with compounds (E7 and FTBMT) at increasing doses and incubated for 72 hours at 37°C and 5% CO2. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) on a Spark 10M multimode microplate reader (Tecan).

Teffs and Tregs of seven- to ten-week-old Gpr52-deficient mice and littermate controls were isolated from peripheral lymphatic organs (axillary, brachial, superficial, cervical, inguinal lymph nodes, and spleen). T cells were isolated using the MojoSort CD4+ T cell isolation kit (BioLegend) according to the manufacturer’s instructions. Cells were stained with antibodies as indicated in Supplementary Table 1 . Teffs and Tregs were sorted with FACS Aria III cell sorter (BD Bioscience). Samples were further analyzed using quantitative real-time PCR analysis as described below.

For induction of EAE, eight- to 12-week-old male and female Gpr52–/– and littermate controls were immunized subcutaneously with 100 µg MOG_35-55 (peptides and elephants) in complete Freund’s adjuvants (BD Difco) containing 4 mg/ml Mycobacterium tuberculosis (BD Difco). 250–300 ng pertussis toxin (Merck Millipore) was injected intraperitoneally in 100 µl PBS on the day of immunization and 48 hours later. Weight and clinical signs of disease were scored daily from day 7 to day 30. Mice were scored blinded for clinical signs by the following system: 0: no clinical deficits; 1: tail weakness; 2: hind limb paresis; 3: partial hind limb paralysis; 3.5: full hind limb paralysis; 4: full hind and forelimb paresis. Animals that reached a score of 4, or 3.5 for more than 3 days, or lost weight of ≥ 25% of starting weight were euthanized according to the regulations of the local Animal Welfare Act. Graphs show pooled data from 3 independent experiments.

Total RNA was isolated from MACS-purified Treg and Teff with RNeasy Plus Micro Kit according to manufacturer′s protocol (Qiagen). iScript cDNA Synthesis Kit (Bio-Rad) was used as the manufacturer′s instructions indicated. Primer pairs were synthesized by Eurofins, and the following primer pairs were used: mouse Gpr52 (forward: 5′-TTGTCTTGCTGACATTTCTGATCA-3′ and reverse: 5′-GGAGCACAGTGAAAGACAAAGATG-3′), and mouse Tbp as housekeeping gene (forward: 5′-GTAGCGGTGGCGGGTATC-3′ and reverse: 5′-CATGAAATAGTGATGCTGGGA-3′). To perform quantitive real-time PCR SyberGreen (Perfecta, QuantaBio) was utilized. Samples were analyzed with Rotor Gene-Q cycler and corresponding program for analysis (Qiagen). To obtain relative gene expression level for Gpr52 ΔCt quantification was employed.

Data shown in bar graphs are shown as mean ± standard error of the mean (SEM). For EAE and functional T cell assays, n represents the number of mice. For FRET measurements, number of mice (n) and number of single measured cells (n) are indicated below the graphs. Number of mice was used for statistical analysis. For heatmap of analysis of available RNA sequencing data (27), differences in gene expression between Teffs and Tregs were tested using limma (49) and genes with a Benjamini-Hochberg corrected P value < 0.05 were considered differentially expressed. Statistical analysis of other data was performed using GraphPad software (RRID: SCR_002798). Normal distribution was tested using Kolmogorov-Smirnov or Shapiro-Wilk test. Differences were tested using one-way ANOVA (followed by Dunett’s or Sidak multiple comparison tests), Wilcoxon matched pairs signed rank test, multiple T tests and Mann-Whitney U test (corrected by Benjamini-Hochberg Correction), Area-under-Curve, Fisher’s exact test, Student’s t-test, or Mann-Whitney U test, as appropriate and indicated in the figure legends. Significant differences are indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.