Trpm2 ^-/- mice (Trpm2 ^tm1Yamo) (29), Rag1 ^-/- mice (B6.129S7−Rag1 ^tm1Mom/J) (36), OT-1 mice [Tg(TcraTcrb)1100Mjb] (37), and CD90.1 congenic mice (B6.PL-Thy1^a /CyJ) were on the C57BL/6 background. All other mice used in this study were derived from intercrosses of these mouse strains. Mice were housed under specific pathogen-free conditions at the University Medical Center Hamburg-Eppendorf. The housing was done under standard conditions with food and water ad libitum in individually ventilated cages. Mice were monitored on a daily basis. Animal experiments were approved by the local committee for animal experiments of the City of Hamburg (registration numbers: N033/2018, N067/2020). Age- and sex-matched mice were used.

Mice were i.p. infected with 10⁴ CFU of a Listeria monocytogenes strain recombinant for ovalbumin (LmOVA) (38). Inocula were controlled by plating on TSB agar plates. From day 2 on, mice were treated with 2 mg/ml ampicillin in the drinking water. Endogenous T-cell responses were analyzed on day 8 postinfection. For T-cell co-transfer studies, CD90.1 congenic mice or Rag1 ^-/- mice were infected with LmOVA. On the same day, infected mice received a mix of WT and Trpm2 ^-/- OT-1 CD8⁺ T cells. Spleen cells from WT CD90.1⁺CD90.2⁺ OT-1 cells and Trpm2 ^-/- CD90.1^-CD90.2⁺ OT-1 cells were purified and mixed to reach a 1:1 ratio of CD8⁺ T cells. Recipient mice intravenously received a total of approx. 10,000 CD8⁺ T cells. Responses in CD90.1 congenic and Rag1 ^-/- recipients were analyzed after 5 days and after 8 weeks, respectively. For analysis of endogenous T-cell response and for 8-week transfer experiments, 2 µg per mouse anti-CD45 mAb (30-F11, AF700) mAb was injected i.v., 3 min before sacrificing to label intravascular cells.

Cells from spleen were isolated by pressing the organ successive through 70- and 40-µm cell strainers. Cells from the kidney, lung, and liver were digested for 40 min at 37°C with 10 U/ml DNase I (Sigma-Aldrich, St. Louis, MO) and 400 µg/ml Collagenase D (Roche, Mannheim, Germany). Leukocytes were enriched by density gradient centrifugation (37.5% Easycoll, Merck Millipore, Darmstadt, Germany) and then filtered through a 30-µm strainer. Erythrocytes were depleted with lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 10 µM EDTA, pH 7.2). After removal of Peyer’s patches, the small intestine was opened longitudinally and washed in PBS 1% FCS. Then, the small intestine was cut into small pieces of approx. 0.5-cm length and incubated in the presence of 5 mM EDTA in complete medium at 37°C for 30 min while shaking. Intraepithelial lymphocytes (IEL) in the supernatant were collected and enriched by centrifugation. For isolation of lamina propria lymphocytes (LPL), the remaining tissue was digested with collagenase IV (100 U/ml, Roche Diagnostics GmbH, Mannheim) and DNase I (10 U/ml Sigma-Aldrich) in complete medium at 37°C for 45 min while shaking. The digested intestinal tissue was further homogenized by passing through a metal strainer, and pooled IEL and LPL fractions were enriched by a 40%/67% Percoll gradient centrifugation. Lymphocytes were collected from the interphase.

For induction of cytokines, cells were incubated for 4 h in IMDM medium supplemented with fetal calf serum, glutamine, gentamicin, and 2-mercaptoethanol. Cells were simulated for 4 h, with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma-Aldrich) and ionomycin (1 µM, Sigma Aldrich) or with the ovalbumin_257-264 peptide (10^-6 M, SIINFEKL) (JPT, Berlin, Germany). Brefeldin A (10 µg/ml, Sigma Aldrich) was added to the cultures to prevent cytokine secretion. In controls, medium only contained brefeldin A.

For induction of CD69, IRF4, and NUR77, spleen cells were cultured for 4, 24, or 48 h in 96W plates coated with anti-CD3ϵ mAb (2 µg/ml, clone: 145-2C11). Anti-CD28 mAb (1 µg/ml, clone: 37.51) was added to the culture. In some of the assays, cells were CFSE labeled prior to stimulation. A division index was calculated with the FlowJo software (Tree Star, Ashland, OR, USA). For long-term culture, cells were stimulated with anti-CD3 mAb, antiCD28, mAb and IL-2 (100 U/ml IL-2, Proleukin S, Novartis, Nürnberg, Germany). After 3 days, cells were washed and further cultured with IL-7 (10 ng/ml mIL-7, PeproTech, Hamburg, Germany).

Lymphocytes were isolated from spleen and lymph nodes of WT and Trpm2 ^-/- mice. Naive CD4⁺ CD25^- CD44^- T cells were enriched by depletion of CD25⁺ and CD44⁺ cells followed by enrichment of CD4⁺ T cells using MACS according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of CD4⁺ T cells obtained was about 80% as determined by flow cytometry. For each differentiation condition, the cells were cultured in a 96-well plate at 2 × 10⁵ cells per well in 200 µl of full Click’s medium (Irvine Scientific, Santa Ana, USA) supplemented with cytokines and antibodies. For differentiation of Th1 cells, naive CD4⁺ T cells were cultured in the presence of 100 U/ml mIL-2, 10 ng/ml mIL-12, 10 µg/ml anti-IL-4 mAb (clone: 11B11), and 2 µg/ml anti-CD28 mAb (clone: 37.51) in plates coated with 10 µg/ml anti-CD3ϵ mAb (clone: 145 2C1). For the differentiation of Th17 cells, naive CD4⁺ T cells were cultured in the presence of 10 ng/ml mIL-6 and 0.25 ng/ml hTGF-β1, 10 µg/ml anti-IL-4 mAb, 10 µg/ml anti-IFN−γ mAb (clone: XMG1.2), and 2 µg/ml anti-CD28 mAb in plates coated with 10 µg/ml anti-CD3ϵ mAb. For differentiation of Treg cells, naive CD4⁺ T cells were cultured in the presence of 50 U/ml mIL-2 and 2 ng/ml hTGF-β1 and 2 µg/ml anti-CD28 mAb in plates coated with 2 µg/ml anti-CD3ϵ mAb. For differentiation of Tr1 cells, naive CD4⁺ T cells were cultured in the presence of 30 ng/ml mIL-27 and 0.25 ng/ml hTGF-β1 and 2 µg/ml anti-CD28 mAb in plates coated with 10 µg/ml anti-CD3 mAb. Cytokines and antibodies were purchased from BioLegend (San Diego, CA) and Miltenyi Biotec. After 3 days, T cells were restimulated with PMA (50 ng/ml, Sigma-Aldrich) and ionomycin (1 µM, Sigma-Aldrich) for 4 h in the presence of monensin (2 µM, BioLegend) and expression of cytokines and Foxp3 was determined by intracellular antibody staining.

After isolation from tissue or after cell culture, cells were incubated in PBS with 1% rat serum and 10 µg/ml anti-Fc-receptor mAb (clone 2.4G2, Bio X Cell, West Lebanon, NH). For extracellular staining, fluorochrome-conjugated antibodies and a fixable dead cell stain (AF750 life/dead staining or Pacific Orange succinimidyl ester, Life Technologies, Carlsbad, CA) were added. Cells were incubated for 15 min on ice. Intracellular antibody staining was conducted with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Carlsbad, CA) according to the manufacturer’s protocol. Cells were washed with PBS 1% FCS and incubated with antibodies for intracellular staining in PBS 1% FCS for 20 min at RT.

Fluorochrome-conjugated antibodies against murine CD3 (clone 17A2, BV421), CD4 (clone RM4-5, FITC/AF700/BV605/BV785), CD8α (clone 53-6.7, PerCP/BV650), CD38 (clone 90, PE-Cy7), CD40L (clone MR1, PerCP-Cy5.5), CD44 (clone IM7, APC/BV785), CD45 (clone 30-F11, APC-Cy7/AF700/BV510/BV785), CD62L (clone MEL-14, APC/PerCP), CD69 (clone H1.2F3, PE-Cy7/V450/BV785), CD90.1 (clone HIS51, FITC/eFlour 450), CD90.2 (clone 53-2.1, PerCP), CD127 (clone A7R34, BV421), CX3CR1 (clone SA011F11, PE), Ly6C (clone AL-21, FITC), Ly6G (clone 1A8, PerCP-Cy5.5), CTLA4 (clone UC10-4F10-11, PE), PD1 (clone 29F.1A12, BV421), LAG3 (clone C9B7W, APC), IFN-γ (clone XMG1.2, APC-Cy7/BV785), IL-10 (clone JES5-16E3/APC/PE), PE), IL-17A (clone TC11-18H10.1, BV785/AF488/APC), TNF-α (clone MP6-XT22; V450), Foxp3 (clones NRRF-30, PE and FJK-16s, APC), IRF4 (clone 3E4, PE-Cy7), KI-67 (clone SolA15, PE), KLRG1 (clone 2F1, BV605), and NUR77 (clone 12.14, PE) were obtained from BioLegend, eBioscience, Thermo Fisher (Darmstadt, Germany) or BD Bioscience (Heidelberg, Germany). The anti-mouse TRPM2 mAb (clone A128) was developed by DNA immunization of rats with a plasmid expressing the full-length cDNA of mouse TRPM2. For analysis of TRPM2 expression, spleen cells were stained intracellularly with unconjugated anti-TRPM2 mAb and then stained with anti-rat-IgG antibodies (R-PE, polyclonal, Dianova, Hamburg, Germany). APC-conjugated H-2K^b/SIINFEKL dextramers were obtained from Immudex (Copenhagen, Denmark).

Cells were analyzed using Canto II, Celesta or Fortessa flow cytometers (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star, Ashland, OR, USA).

Mice were i.p. injected with 15 µg anti-CD3 mAb (clone: 145 2C11) on days 0, 2, and 4. Mice were analyzed 4 h after the last injection. For the T-cell transfer approach, cells were isolated from spleen and peripheral lymph nodes of WT and Trpm2 ^-/- mice. Total CD4⁺ cells were enriched with MACS according to the manufacturer’s instructions. Recipient Rag1 ^-/- mice received 2 × 10⁶ CD4⁺ cells i.v. After 4 weeks, recipients were treated i.p. with 2 µg anti-CD3 mAb on days 0, 2, and 4 and were analyzed 4 h after the last injection.

CD4⁺ and CD8⁺ T cells were isolated from spleens of WT and Trpm2 ^-/- mice and purified by MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA from pooled CD4⁺ and CD8⁺ T cells was isolated using TRIzol^® reagent (Invitrogen) according to the manufacturer’s protocol. RNA was reverse transcribed with the high-capacity cDNA synthesis Kit (Thermo Fisher, Darmstadt, Germany). cDNA concentrations for Ryr1, Tpcn1, Tpcn2, Orai1, and the control Hprt were determined with TaqMan PCR using primer and probes from Thermo Fisher: Hprt Mm03024075_m1, Ryr1 Mm01175211_m1, Tpcn1 Mm00455326_m1, Tpcn2 Mm00628260_m1, and Orai1 Mm00774349_m1.

Statistical analyses were performed with Prism software (GraphPad Software Inc., La Jolla, CA). Results were analyzed with the tests indicated in the figure legends. A p-value of <0.05 was considered significant (p < 0.05) and is indicated with *.

Ca²⁺ signaling is central for T-cell activation and differentiation, and Ca²⁺ influx facilitated by TRPM2 could enhance or modulate these processes. In a first set of experiments, the expression of TRPM2 was determined in CD8⁺ T cells ( Figure 1A and Supplementary Figure 2A ). Compared to Ly6C⁺ and Ly6G⁺ myeloid cells (inflammatory monocytes and neutrophils), we detected only a low level of TRPM2 expression in CD8⁺ T cells from WT mice which was markedly reduced in CD8⁺ T cells from Trpm2 ^-/- mice. Spleen cells from WT and Trpm2 ^-/- mice were stimulated with anti-CD3ϵ and anti-CD28 mAb, and the expression of CD69 and of the transcription factors IRF4 and NUR77 (NR4A1) was determined by flow cytometry. Expression of these proteins is induced within a few hours of TCR stimulation and can be used to determine the quality of the TCR signal. After 4 h, we observed strong upregulation of all three proteins and expression of IRF4 was further increased after 24 and 48 h of culture ( Figures 1B, C ). CD8⁺ T cells from WT and Trpm2 ^-/- mice showed similar upregulation of the proteins. Then, spleen cells were labeled with CFSE and proliferation was determined by loss of the CFSE label. Cells were stimulated with anti-CD3ϵ and anti-CD28 mAb, and after 1, 2, 3, and 4 days, CFSE staining was determined ( Figures 1D, E and Supplementary Figure 2B ). T-cell activation resulted in cumulative loss of CFSE staining in CD8⁺ T cells; however, there was no difference in proliferation between WT and Trpm2 ^-/- cells. TRPM2 could also modulate the T-cell response at later time points. Therefore, CD8⁺ T cells from WT and Trpm2 ^-/- mice were mixed and activated with anti-CD3ϵ and anti-CD28 mAb. After 3 days, activated cells were cultured in medium containing IL-7 ( Figure 1F ). After 21 days, CD8⁺ T cells expressed only low levels of CD69 and of the proliferation marker Ki-67 but had upregulated CD38. Importantly, the ratio of WT to Trpm2^-/- CD8⁺ T cells was similar to the ratio at the start of the culture, indicating that the lack of TRPM2 did not alter the T-cell response.

Similar to WT CD8⁺ T cells, WT CD4⁺ T cells expressed only low levels of TRPM2 and staining was reduced in Trpm2 ^-/- CD4⁺ T cells ( Figure 2A ). The early response following TCR stimulation was also determined in CD4⁺ T cells ( Figures 2B, C ). CD4⁺ T cells showed rapid upregulation of CD69, NUR77, and IRF4, but there was no difference between WT and Trpm2 ^-/- CD4⁺ T cells. We also determined the induction of the inhibitory receptors CTLA4, PD1, and LAG3 ( Supplementary Figure 3 ). We observed an equal expression of these receptors on WT and Trpm2 ^-/- CD4⁺ T cells. Differentiation of CD4⁺ Th cells is regulated by cytokine signals from the environment but also by the strength of the TCR stimulus and the quality of the TCR-induced Ca²⁺ signal. Thus, modulation of the Ca²⁺ signal by TRPM2 could influence the fate of Th-cell differentiation. Purified CD4⁺ T cells from WT and Trpm2 ^-/- mice were stimulated under defined conditions to induce IFN-γ⁺ Th1 cells, IL-17A⁺ Th17 cells, Foxp3⁺ regulatory T cells (Treg cells), and Foxp3^- IL-10⁺ regulatory 1 T cells (Tr1 cells). After 3 days, T cells were activated with PMA and ionomycin, and expression of Foxp3 and cytokines was determined by intracellular staining and flow cytometry ( Figure 2D ). Consistent with the conditions of differentiation, we detected upregulation of IFN-γ, IL-17A, IL-10, and Foxp3; however, WT and Trpm2^-/- CD4⁺ T cells did not significantly differ in the generation of IFN-γ⁺ Th1 cells, IL-17A⁺ Th17 cells, and Foxp3⁺ Treg cells. In the experiment shown, there was a small reduction of Trpm2 ^-/- Tr1 cells; however, this reduction was not consistent in other experiments.

Absence of TRPM2 could be compensated by higher expression of other Ca²⁺ channels. Therefore, mRNA was isolated from WT and Trpm2 ^-/- T cells and the expression of Ryr1, Tpcn1, Tpcn2, and Orai1 coding for Ca²⁺ channels that might compensate for the absence in T cells was measured by RT-PCR ( Supplementary Figure 4 ). We detected similar mRNA levels for all analyzed Ca²⁺ channels in WT and Trpm2 ^-/- T cells.

In conclusion, our data so far provide no evidence for a substantial role of TRPM2 in the activation and differentiation of CD4⁺ and CD8⁺ T cells.

In order to analyze the role TRPM2 in CD8⁺ T cells in vivo, we used the Listeria monocytogenes infection model. WT and Trpm2 ^-/- mice were infected with an ovalbumin-recombinant strain of L. monocytogenes (LmOVA) which induce a strong CD8⁺ T cell response against the OVA_257-264 peptide (38). Since Trpm2 ^-/- mice are more susceptible to L. monocytogenes (30, 31), mice were treated after 2 days with ampicillin in the drinking water which results in the rapid elimination of Listeria but does only marginally effect the T-cell response (39). Eight days postinfection, frequencies and total numbers of ovalbumin-specific CD8⁺ T cells were determined using OVA_257-264H-2K^b dextramers ( Figures 3A–C ). WT and Trpm2 ^-/- mice showed similar numbers of dextramers⁺ CD8⁺ T cells in spleen and liver, the main sites of listeria replication. Dextramer⁺ CD8⁺ T cells in both mouse strains were CD44^hiCD62L^lo and similar frequencies expressed CX3CR1 and KLRG1, indicative for highly activated effector T cells ( Figures 3D–F ). Dextramer⁺ CD8⁺ T cells did also not differ with regard to the upregulation of CD38 ( Figure 3G ). Spleen and liver cells were also incubated with OVA_257-264 peptide, and the induction of TNF-α and IFN-γ was determined by intracellular cytokine staining ( Figure 3H ). Again, we observed similar frequencies of TNF-α⁺IFN-γ⁺ CD8⁺ T cells. There was also no difference in the production of IFN-γ and TNF-α by CD4⁺ T cells in response to polyclonal restimulation ( Figure 3I ).

More excessive initial inflammation and altered function of TRPM2-deficient innate immune cells could mask a defect of CD8⁺ T cells in Trpm2 ^-/- mice. Therefore, we used a competitive T-cell transfer assay to characterize the function of TRPM2 in CD8⁺ T cells. Trpm2 ^-/- mice were crossed with OT-1 mice which are transgenic for an MHC class I-restricted OVA_257-264-specific TCR (37). CD8⁺ T cells from WT and Trpm2 ^-/- mice were mixed roughly at a 1:1 ratio, and 1 × 10⁴ CD8⁺ T cells were transferred into recipient mice infected with LmOVA. Donor and recipient cells differed in the expression of CD90.1 and CD90.2, which allowed identification of the different cell populations. Five days post transfer and infection, CD8⁺ T cells derived from both donors could be detected in spleen and liver ( Figures 4A, B ). However, the ratio of WT to Trpm2 ^-/- cells in both tissues was similar to that of the transferred CD8⁺ T-cells and both populations were similar in their expression profiles of CD44, CD62L, and KLRG1 ( Figures 4C–E ). In addition, after stimulation with the OVA_257-264 peptide, WT and Trpm2 ^-/- OT-1 T-cells showed similar induction of IFN-γ and TNF-α and of NURF77 ( Figures 4F, G ). Thus, TRPM2 deficiency restricted to CD8⁺ T cells did not significantly impair their response during acute infection.

The competitive transfer assay was also used to determine the role of TRPM2 in CD8⁺ memory T-cell formation. To exclude rejection of donor cells, Rag1 ^-/- mice were used as recipients. Eight weeks post transfer and LmOVA infection, donor cells in the spleen, liver, lung, kidney, and bone marrow were analyzed. In the spleen, liver, and bone marrow, we observed ratios of WT to Trpm2 ^-/- cells similar to the ratio of the transferred cell population ( Figure 5A ). Interestingly, WT cells were slightly more prominent in lung and kidney, indicating a disadvantage of Trpm2 ^-/- CD8⁺ T cells in migration into or survival within these tissues. Phenotypical characterization revealed similar expression profiles for CD44 and CD62L cells with high frequencies of CD44^hiCD62L^lo effector/effector memory T cells in the liver, lung, and kidney, and somewhat lower frequencies of these cells in the spleen and bone marrow ( Figure 5B ). Upon peptide restimulation of CD8⁺ T cells from the spleen of recipients, we observed similar frequencies of TNF-α⁺IFN-γ⁺ and NUR77⁺ T cells in both CD8⁺ T-cell populations ( Figures 5C, D ).

In order to test the response of Trpm2 ^-/- CD4⁺ T cells in vivo, we used the model of anti-CD3 mAb-induced intestinal inflammation. In this model, repeated injection of anti-CD3 mAb causes systemic T-cell activation. A main hallmark of the model is the activation and accumulation of Th1 and Th17 cells in the small intestine resulting in inflammation of the intestinal mucosa, and diarrhea and weight loss as disease manifestations. As a consequence of the inflammation, Th17 cells differentiate to IL-10-secreting Tr1 cells and enhanced frequencies of both Tr1 cells and conventional Foxp3⁺ Treg cells are found in the small intestinal mucosa. Thus, the anti-CD3 application model allows the analyses of Th1, Th17, and Treg cell responses as well as the formation of Tr1 cells (40–43). Four days after anti-CD3 mAb treatment, mice had lost about 15% of their weight; however, weight loss was similarly extensive in WT and Trpm2 ^-/- mice ( Figure 6A ). Characterization of T cells from the small intestinal mucosa revealed similar frequencies and numbers of CD4⁺ Th1, Th17, Treg, and Tr1 cells in WT and Trpm2 ^-/- mice ( Figure 6B ). Thus, deficiency of TRPM2 did not affect the CD4⁺ T-cell response in this model.

As discussed in the context of the infection model, TRPM2 deficiency in cells other than T cells could mask an altered CD4⁺ T-cells response. Therefore, Rag1 ^-/- mice were reconstituted with CD4⁺ T cells from either WT or Trpm2 ^-/- mice and then treated with anti-CD3 mAb. Under these conditions, weight loss was less pronounced but similar in mice reconstituted with WT and Trpm2 ^-/- cells ( Figure 7A ). Recipients did not differ in the frequencies and numbers of intestinal IFN-γ⁺ Th1 cells, IL-17A⁺ Th17 cells, and IFN-γ⁺IL-17A⁺ CD4⁺ T cells as well as in frequencies and numbers of Foxp3⁺ cells ( Figure 7B ).

In conclusion, we could not demonstrate a substantial role of TRPM2 in the in vivo response of CD4⁺ T cells.