METTL3 inhibition reduces N⁶-methyladenosine levels and prevents allogeneic CD4⁺ T-cell responses

Abstract

Alloreactive CD4⁺ T cells play a central role in allograft rejection. However, the post-transcriptional regulation of the effector program in alloreactive CD4⁺ T cells remains unclear. N⁶-methyladenosine (m⁶A) RNA modification is involved in various physiological and pathological processes. Herein, we investigated whether m⁶A methylation plays a role in the allogeneic T-cell effector program. m⁶A levels of CD4⁺ T cells from spleens, draining lymph nodes and skin allografts were determined in a skin transplantation model. The effects of a METTL3 inhibitor (STM2457) on CD4⁺ T-cell characteristics including proliferation, cell cycle, cell apoptosis and effector differentiation were determined after stimulation of polyclonal and alloantigen-specific (TEa; CD4⁺ T cells specific for I-Eα_52–68) CD4⁺ T cells with α-CD3/α-CD28 monoclonal antibodies and cognate CB6F1 alloantigen, respectively. We found that graft-infiltrating CD4⁺ T cells expressed high m⁶A levels. Administration of STM2457 reduced m⁶A levels, inhibited T-cell proliferation and suppressed effector differentiation of polyclonal CD4⁺ T cells. Alloreactive TEa cells challenged with 40 μM STM2457 exhibited deficits in T-cell proliferation and T helper type 1 cell differentiation, a cell cycle arrest in the G0 phase and elevated cell apoptosis. Moreover, these impaired T-cell responses were associated with the diminished expression levels of transcription factors Ki-67, c-Myc and T-bet. Therefore, METTL3 inhibition reduces the expression of several key transcriptional factors for the T-cell effector program and suppresses alloreactive CD4⁺ T-cell effector function and differentiation. Targeting m⁶A-related enzymes and molecular machinery in CD4⁺ T cells represents an attractive therapeutic approach to prevent allograft rejection.

INTRODUCTION

Despite advances in immunosuppression, T-cell-mediated allograft rejection remains a significant hurdle to overcome for long-term transplant survival.^1,2 Following allorecognition of foreign antigens, allogeneic T cells are activated and subsequently proliferate, and differentiate into effector T cells that dominate the rejection process by promoting T-cell cytotoxicity and delayed-type hypersensitivity through proinflammatory cytokines.^3,4 We and others have found previously that transcriptional regulation of T-cell differentiation and function [e.g. interferon regulatory factor 4 (IRF4), signal transducer and activator of transcription 3 (STAT3), basic leucine zipper ATF-like transcription factor (BATF) and BATF3] is critical for allograft rejection.^5–8 However, whether the post-transcriptional modifications play a role in the allogeneic T-cell effector program and allograft rejection remains unclear.

N6-methyladenosine (m⁶A) RNA modification is one of the most prevalent and functional post-transcriptional modifications of messenger RNA in eukaryotes.⁹ Its formation is primarily regulated by m⁶A methyltransferases and demethylases.^9,10 The resultant m⁶A residues are then recognized by different m⁶A reader proteins and impose diverse effects on gene expression through different RNA metabolism pathways.^9,10

m⁶A RNA methylation and the aforementioned m⁶A regulators have been shown to play a role in T-cell-mediated immune responses. Yao et al.¹¹ found previously that METTL3 was required for T follicular helper cell differentiation. Ablation of METTL3 in T cells reduced the stability of Tcf7 transcripts, perturbing T follicular helper cell differentiation and germinal center responses in response to lymphocytic choriomeningitis virus infections.¹¹ Li et al.¹⁰ demonstrated that deletion of METTL3 in T cells disrupted T-cell homeostasis and differentiation, and thus prevented autoimmune disease. It has also been reported that lineage-specific ablation of m⁶A demethylase, such as AlkB homolog 5 (ALKBH5), but not the fat mass and obesity-associated protein (FTO), conferred protection against experimental autoimmune encephalomyelitis.¹² These studies highlight the importance of m⁶A molecular machinery in mediating T-cell immunity; however, its role in allogeneic T-cell response still awaits investigation.

METTL3 is the catalytic component of the m⁶A-methylase complex.^9–11 A potent catalytic inhibitor, STM2457 can directly bind to its SAM-binding site and inhibit its enzyme activity to deposit m⁶A RNA modification.¹³ A recent study has shown that STM2457 treatment can substantially decrease m⁶A levels, restricting the growth of acute myeloid leukemia in vitro and in vivo.¹³ Herein, we investigated the effects of the METTL3 inhibitor STM2457 in allogeneic T-cell response. We found that m⁶A RNA methylation levels increased in graft-infiltrating CD4⁺ T cells. Further, pharmacological inhibition of METTL3 in alloreactive CD4⁺ T cells specific for I-Eα_52–68 (TEa) reduced m⁶A RNA methylation levels, impaired cell proliferation, induced cell cycle arrest in the G0 phase, promoted cell apoptosis and inhibited effector differentiation by repressing c-Myc and T-bet expression. Hence, we propose that pharmacological inhibition of METTL3 reduces m⁶A levels in T cells and represents an attractive therapeutic approach to prevent alloreactive T-cell responses.

RESULTS

Allograft-infiltrating CD4⁺ T cells display a high level of m⁶A RNA methylation

CD4⁺, but not CD8⁺, T cells play a central role in mediating the rejection of transplanted organs.^6,14 To investigate the role of m⁶A RNA methylation in allogeneic T-cell response, we first determined its expression levels in CD4⁺ T cells after skin transplantation. Tail skin allografts from BALB/c donors were transplanted onto wild-type C57BL/6 (B6) recipients on day 0. We then determined m⁶A RNA methylation levels of CD4⁺ T cells from spleens, draining lymph nodes and skin allografts by flow cytometry on day 8 after skin grafting (Figure 1a). Figure 1b shows the gating strategy to detect live CD4⁺ T cells. Significantly higher levels of m⁶A RNA methylation were seen in allograft-infiltrating CD4⁺ T cells present in the skin grafts than in those in the spleens and draining lymph nodes (Figure 1c, d). Further, as T-bet is an essential transcriptional factor that regulates effector differentiation and function in T cells, we examined the levels of T-bet present in CD4⁺ T cells.^3,15 The higher levels of m⁶A RNA methylation were associated with a higher level of T-bet expression of CD4⁺ T cells in the skin allografts (Figure 1e, f). Hence, graft-infiltrating CD4⁺ T cells exhibited a high level of m⁶A RNA methylation, indicating that m⁶A RNA methylation may play a role in allogeneic T-cell differentiation and function.

Figure 1.

Allograft-infiltrating CD4⁺ T cells display a high level of m⁶A RNA methylation. BALB/c skin allografts were transplanted onto B6 recipients on day 0. The levels of m⁶A RNA methylation and T-bet expression in CD4⁺ T cells were determined by flow cytometric analysis on day 8 after skin transplantation. All plots were gated on live CD4⁺ T cells. (a) Schematic of the experimental design. (b) Gating strategy to detect live CD4⁺ T cells. (c, d) Representative plots display the m⁶A RNA methylation of CD4⁺ T cells from spleens, draining lymph nodes and skin allografts. (e, f) Representative plots display the T-bet expression of CD4⁺ T cells. Data represent the mean standard deviation (n = 4) and are from the same batch of biological replicates. **P < 0.01, ****P < 0.0001 (unpaired Student’s t-test). FSC-A, forward scatter-area; m⁶A, N⁶-methyladenosine; MFI, mean fluorescence intensity; SSC-A, side scatter-area; Tx, transplantation; WT, wild type.

STM2457 treatment reduces m⁶A levels and inhibits polyclonal CD4⁺ T-cell proliferation following α-CD3/α-CD28 monoclonal antibody stimulation

STM2457 is a selective METTL3 inhibitor that can reduce m⁶A levels.¹³ To determine the effects of STM2457 on T-cell response, naïve CD4⁺ cells were labeled with CellTrace Violet (CTV) and stimulated with α-CD3/α-CD28 monoclonal antibodies (mAbs) in the presence of 0, 1, 10, 20, 40 and 80 μM STM2457. Cell viability, activation and proliferation markers were identified by flow cytometry (Figure 2a).

Figure 2.

STM2457 treatment reduces m⁶A levels and inhibits T-cell proliferation of polyclonal CD4⁺ T cells following α-CD3/α-CD28 mAb stimulation. CTV-labeled naïve CD4⁺ T cells were stimulated with α-CD3/α-CD28 mAbs for 3 days followed by flow cytometric analysis. (a) Schematic of the experimental design. (b) Cell viability was determined by flow cytometry. (c) Representative plots display division peaks and CTV fluorescence. (d–f) Naïve CD4⁺ T cells were stimulated with α-CD3/α-CD28 mAbs in the presence of 40 μM STM2457 or DMSO for 2 days. (d) Representative plots, (e) bar graphs and (f) m⁶A RNA dot blot display the levels of m⁶A RNA methylation. Data represent the mean standard deviation (n = 3 or 4) and are from the same batch of biological replicates. **P < 0.01, ***P < 0.001 (unpaired Student’s t-test). CTV, CellTrace Violet; DMSO, dimethyl sulfoxide; m⁶A, N⁶-methyladenosine; mAb, monoclonal antibody; ns, no significance.

Supplementary figure 1a shows the gating strategy to detect CD4⁺ T cells in this assay. Treatment with 1, 10, 20 and 40 μM STM2457 barely reduced the mean cell viability of CD4⁺ T cells (Figure 2b). However, the viability of CD4⁺ T cells was significantly decreased in the presence of the 80 μM STM2457 challenge (Figure 2b). Further, treatment of 1, 10, 20 and 40 μM STM2457 failed to largely inhibit the expression levels of CD4⁺ T-cell activation markers CD25 and CD69 (Supplementary figure 1b).

We then investigated whether STM2457 treatment inhibits T-cell proliferation. Results indicated that naïve CD4⁺ T cells did not proliferate and showed only one division peak with the highest CTV intensity (Figure 2c). By contrast, polyclonal CD4⁺ T cells in the dimethyl sulfoxide (DMSO) control group proliferated robustly and showed six division peaks on after 3 post α-CD3/α-CD28 mAb stimulation. Further, STM2457 treatment reduced the division peaks of CD4⁺ T cells in a dose-dependent manner (Figure 2c). Of note, CD4⁺ T cells treated with 40 μM STM2457 displayed significantly lower m⁶A levels than those in the DMSO control group after α-CD3/α-CD28 mAb stimulation (Figure 2d–f). As 40 μM STM2457 treatment effectively reduced m⁶A levels but barely decreased the viability of CD4⁺ T cells, we selected this STM2457 dose for the follow-up experiments.

Taken together, STM2457 treatment reduced m⁶A RNA methylation levels and inhibited polyclonal CD4⁺ T-cell proliferation in response to α-CD3/α-CD28 mAb stimulation.

STM2457 treatment impairs the effector differentiation of polyclonal CD4⁺ T cells

We next investigated whether STM2457 treatment affects T-cell effector differentiation. Naïve CD4⁺ T cells were stimulated with α-CD3/α-CD28 and treated with DMSO or 40 μM STM2457 in the presence of polarizing cytokines for 3 days. The cultured CD4⁺ T cells were restimulated with phorbol 12-myristate 13-acetate, ionomycin and GolgiStop for 4 h, and this was followed by flow cytometric analysis to determine the expression levels of lineage-specific cytokines (Figure 3a). We found that STM2457 treatment significantly inhibited the polarization of T helper type 1 (Th1), Th2 and induced regulatory T cells. In the Th1-, Th2- and induced regulatory T-cell-skewed conditions, the percentages of interferon (IFN)c⁺, interleukin (IL)-13⁺ and Foxp3⁺ cells in the STM2457 treatment group were significantly lower than those in the DMSO control group, respectively (Figure 3b–g). Therefore, m⁶A inhibition impaired the effector differentiation of polyclonal CD4⁺ T cells following α-CD3/α-CD28 mAb stimulation.

Figure 3.

STM2457 treatment inhibits the effector differentiation of polyclonal CD4⁺ T cells following α-CD3/α-CD28 mAb stimulation. CD4⁺ naïve T cells were stimulated with α-CD3/α-CD28 mAbs in the presence of Th1-, Th2- and iTreg-skewed cytokines for 3 days. All plots were gated on live CD4⁺ T cells. (a) Schematic of the experimental design. (b, c) Representative plots and bar graphs exhibit percentage of IFNγ⁺ cells among polyclonal CD4⁺ T cells in the Th1-skewed condition. (d, e) Representative plots and bar graphs exhibit percentage of IL-13⁺ cells among polyclonal CD4⁺ T cells in the Th2-skewed condition. (f, g) Representative plots and bar graphs exhibit percentage of Foxp3⁺ cells among polyclonal CD4⁺ T cells in the iTreg-skewed condition. Data represent the mean standard deviation (n = 4) and are from the same batch of biological replicates. **P < 0.01, ***P < 0.001 (unpaired Student’s t-test). DMSO, dimethyl sulfoxide; IFN, interferon; IL, interleukin; iTreg, induced regulatory T-cell; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate; Th1, T helper type 1; Th2, T helper type 2.

STM2457 treatment represses c-Myc expression and inhibits cell proliferation of alloreactive T cells

TEa cells are T-cell receptor (TCR)-transgenic CD4⁺ T cells that can recognize the IEα_52–68 peptide on CB6F1 stimulators.¹⁶ To further determine the role of STM2457 in the allogeneic T-cell response, we performed a mixed leukocyte reaction assay in which naïve TEa cells were labeled with or without CTV, incubated with 10 ng mL⁻¹ murine IL-2 and stimulated with either allogeneic CB6F1 or B6 syngeneic stimulators in the presence of DMSO or 40 μM STM2457 (Figure 4a).

Figure 4.

STM2457 treatment represses c-Myc expression and inhibits cell proliferation of alloreactive CD4⁺ cells. Naïve TEa cells were labeled with/without CTV and then stimulated with either allogeneic CB6F1 or B6 syngeneic stimulators in the presence of DMSO or 40 μM STM2457. All plots were gated on live Vb6⁺ TEa cells. (a) Schematic of the experimental design. (b) Representative plots display division peaks and CTV fluorescence. (c, d) Representative plots and bar graphs display Ki-67 expression on day 2 after stimulation. (e, f) Representative plots and bar graphs display c-Myc expression on day 2 after stimulation. Data represent the mean standard deviation (n = 3) and are from the same batch of biological replicates. *P < 0.05, **P < 0.01 (unpaired Student’s t-test). APC, antigen-presenting cell; CTV, CellTrace Violet; DMSO, dimethyl sulfoxide; IL, interleukin; MFI, mean fluorescence intensity; TEa, CD4⁺ T cells specific for I-Eα_52–68.

Supplementary figure 2a shows the gating strategy for detecting live TEa cells. Results indicate that TEa cells did not proliferate in response to syngeneic B6 stimulators. By contrast, allogenic CB6F1 stimulators, which contain abundant I-E-α alloantigen that can be recognized by TEa cells, caused a robust proliferation in most TEa cells, as indicated by multiple cell divisions and the loss of CTV fluorescence on days 2 and 3 after stimulation (Figure 4b). By contrast, STM2457 treatment reduced TEa cell proliferation, which was revealed by lower cell divisions and higher CTV fluorescence than those in the DMSO control group (Figure 4b). Ki-67 and c-Myc are critical transcription factors that facilitate cell proliferation. We found that TEa cells stimulated with allogeneic CB6F1 stimulators, but not syngeneic B6 stimulators, expressed high levels of Ki-67 and c-Myc. Further, STM2457 treatment significantly reduced Ki-67 and c-Myc expression of alloreactive TEa cells upon allogeneic CB6F1 stimulation (Figure 4c–f). Hence, STM2457 treatment repressed c-Myc/Ki-67 expression and inhibited cell proliferation of alloreactive TEa cells.

STM2457 treatment induces G₀ phase cell cycle arrest and promotes alloreactive T-cell apoptosis

Impaired cell differentiation is associated with cell cycle arrest and cell apoptosis.¹⁷ To determine the influence of STM2457 treatment on the cell cycle distribution and cell apoptosis rates, we stimulated naïve TEa cells with allogeneic CB6F1 or syngeneic B6 stimulators in the presence of DMSO or 40 μM STM2457 for 3 days. The cell cycle stages and cell apoptosis rates were determined by flow cytometric analysis.

Based on different expressions of Ki-67 and propidium iodide, the cell cycle stages in T cells can be roughly segregated into G₀, G₁ and S–G₂/M phases.¹⁸ Without allogenic CB6F1 stimulation, most TEa cells remained quiescent and did not enter the cell cycle. Following allogeneic CB6F1 stimulation, most TEa cells entered the cell cycle, and 35% of them entered the S–G₂/M phases. By contrast, over 30% of TEa cells in the STM2457 treatment group did not enter the cell cycle and remained in the G₀ phase. Accordingly, the percentage of TEa cells in S–G₂/M phases in the STM2457 group was significantly lower than those of the DMSO control group (Figure 5a, b).

Figure 5.

STM2457 treatment induces G₀ phase cell cycle arrest and promotes cell apoptosis of alloreactive CD4⁺ cells. Naïve TEa cells were stimulated with either allogeneic CB6F1 or B6 syngeneic stimulators in the presence of DMSO or 40 μM STM2457 for 3 days, followed by flow cytometric analysis. (a, b) Representative plots and bar graphs display the percentage of TEa cells in G₀, G₁ and S–G₂/M phases. (c, d) Representative plots and bar graphs display the percentage of Annexin V⁺7-AAD⁻ cells among TEa cells. Data represent the mean standard deviation (n = 3) and are from the same batch of biological replicates. **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired Student’s t-test). APC, antigen-presenting cell; 7-AAD, 7-aminoactinomycin D; DMSO, dimethyl sulfoxide; PI, propidium iodide; TEa, CD4⁺ T cells specific for I-Eα_52–68.

We next determined the cell apoptosis rates of the alloreactive TEa cells following allogeneic stimulation and polyclonal CD4⁺ T cells following α-CD3/α-CD28 mAb stimulation. Over 90% of the alloreactive TEa cells remained Annexin V⁻7-AAD⁻ live cells, and only 2% of the TEa cells were Annexin V⁺7-AAD⁻ preapoptotic cells in the DMSO control group. By contrast, treatment of STM2457 significantly increased the percentage of Annexin V⁺7-AAD⁻ preapoptotic cells to 12% (Figure 5c, d). STM2457 treatment also promoted the cell apoptosis of polyclonal CD4⁺ T cells following α-CD3/α-CD28 mAb stimulation (Supplementary figure 3). Taken together, METTL3 inhibition with STM2457 induced G₀ phase cell cycle arrest in alloreactive TEa cells and promoted cell apoptosis.

STM2457 treatment represses T-bet expression and inhibits Th1 differentiation of alloreactive T cells

Th1 differentiation of alloreactive T cells mediates allograft rejection and hinders transplantation tolerance.³ Herein, we developed an assay to determine the role of STM2457 in the effector differentiation of alloreactive T cells. In this assay, TEa cells from TEa transgenic mice were used as responders, whereas T-cell-depleted splenocytes from CB6F1 mice were used as allogeneic stimulators. Responder TEa cells and allogeneic stimulators were mixed in a 1:2 ratio, incubated with Th1 polarizing cytokines and treated with DMSO or 40 μM STM2457. On day 3 after stimulation, the production of IFNγ by TEa cells was determined by flow cytometric analysis (Figure 6a). Results indicated that TEa cells in the DMSO group expressed a higher level of IFNγ than those in the STM2457 group in response to CB6F1 stimulators in the Th1-skewed condition (Figure 6b, c). We next determined the expression levels of transcription factors IRF4 and T-bet of alloreactive TEa cells. Specifically, we found that treatment of STM2457 did not inhibit the expression level of IRF4 in TEa cells. Nevertheless, TEa cells in the DMSO group expressed a significantly higher level of T-bet (>10-folds) than those in the STM2457 group (Figure 6d, e). Hence, METTL3 inhibition with STM2457 treatment repressed T-bet expression and inhibited Th1 differentiation of alloreactive CD4⁺ T cells.

Figure 6.

STM2457 treatment represses T-bet expression and inhibits Th1 cell differentiation. Naïve TEa cells were stimulated with allogeneic CB6F1 APC or syngeneic B6 APC and subsequently treated with DMSO or 40 μM STM2457 in the Th1-skewed condition for 3 days, followed by flow cytometric analysis. All plots were gated on the live TEa cells. (a) Schematic of the experimental design. (b, c) Percentage of IFNγ⁺ cells among TEa cells in the Th1-skewed condition. (d, e) Percentage of T-bet⁺IRF4⁺ cells among TEa cells. Data represent the mean standard deviation (n = 3) and are from the same batch of biological replicates. *P < 0.05; ****P < 0.0001 (unpaired Student’s t-test). APC, antigen-presenting cell; DMSO, dimethyl sulfoxide; IFN, interferon; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; Th1, T helper type 1; TEa, CD4⁺ T cells specific for I-Eα_52–68.

DISCUSSION

Little is known about the post-transcriptional modification of allogeneic T-cell responses. In this study, we found that STM2457 treatment reduced the levels of m⁶A RNA methylation, inhibited cell proliferation, induced G0 phase cell cycle arrest, promoted cell apoptosis and impaired effector differentiation of alloreactive TEa cells in response to alloantigen stimulation. The attenuated allogeneic T-cell responses were associated with diminished expression levels of key transcription factors (e.g. T-bet, c-Myc) that control the T-cell effector program. Therefore, METTL3 inhibition reduces the levels of m⁶A RNA methylation and inhibits allogeneic CD4⁺ T-cell responses.

m⁶A RNA modification plays a critical role in immune cell differentiation and function. Deletion of the m⁶A methyltransferase METTL14 in tumor-associated macrophages reduces m⁶A levels and increases their production of Ebi3, an IL-27 subunit. Ebi3 in turn promotes CD8⁺ T-cell dysfunction, leading to impaired antitumor function.¹⁹ By contrast, deletion of an m⁶A reader protein (YTHDF1) in dendritic cells enhances their cross-presentation of tumor antigens and promotes the function of antitumor CD8⁺ T cells.²⁰ m⁶A RNA modification also regulates intrinsic T-cell function. Deletion of METTL3 or an m⁶A demethylase (ALKBH5) in T cells disrupts T-cell effector function and differentiation, and thus prevents autoimmune diseases.^10,12 Herein, demonstrated in the context of allogeneic response, we found that METTL3 inhibition impaired the differentiation and effector function of alloreactive CD4⁺ T cells.

Cumulative data have suggested that METTL3 promotes c-Myc protein expression by increasing its m⁶A RNA methylation.^21,22 Yankova et al.¹³ found that STM2457, an METTL3 inhibitor, reduces c-Myc protein expression and inhibits tumor growth in vivo. Therefore, we speculate that the drug STM2457 inhibits c-Myc expression by reducing the m⁶A level on the c-Myc mRNA transcript. In our study, we found that in allogeneic T-cell response, METTL3 inhibition decreases the expression of the c-Myc protein, which plausibly results in impaired alloreactive CD4⁺ T-cell proliferation, elevated cell cycle arrest in the G₀ phase and increased cell apoptosis.

T-bet is a key lineage-defining transcription factor that is required for Th1 cell differentiation.¹⁵ Deletion of METTL3 in T cells has been shown to impair T-bet expression and inhibit Th1 cell differentiation in an acute infection model.¹¹ Further, Li et al.¹⁰ found that deletion of METTL3 in T cells inhibited Th1/Th17 cell differentiation and promoted Th2 cell differentiation in vitro. By contrast, deletion of another “writer,” methyltransferase METTL14, in T cells has been shown to induce spontaneous colitis and display higher levels of Th1- and Th17-type cytokines as a result of the impaired Treg function.²³ Herein, we found that METTL3 inhibition suppressed T-bet expression and impaired Th1 cell differentiation in allogeneic T-cell response.

There are several limitations in our study that required further examination. First, although we found that METTL3 inhibition by STM2457 reduced the m⁶A levels and decreased the expression of Ki-67, c-Myc and T-bet in allogeneic T-cell response, whether STM2457 affected these transcription factors by directly regulating their m⁶A RNA methylation remains unclear. Further studies will be required to elucidate the underpinning mechanism by which STM2457 affected the expression levels of Ki-67, c-Myc and T-bet. Second, we currently did not include in vivo data about how STM2457 suppressed alloreactive CD4⁺ T-cell effector function. These limitations should be carefully addressed in future studies.

In summary, the results from this study show that METTL3 inhibition reduced m⁶A levels and disrupted cell proliferation and Th1 differentiation of alloreactive TEa cells. Together, these results suggest that targeting the RNA-modifying enzyme METTL3 represents a promising therapeutic strategy to eliminate undesirable allogeneic T-cell responses.

METHODS

Mice

C57BL/6 (B6), BALB/c and TEa TCR transgenic mice (B6 background) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Male B6 mice were crossed with female BALB/c mice to create CB6F1 mice. Mice were housed in a specific pathogen-free facility at Houston Methodist Research Institute in Houston, Texas. Male mice (aged 8 to 12 weeks) were randomly assigned and used in experiments. All animal-related experiments in this study were approved by the Houston Methodist Animal Care Committee under the institutional animal care and use guidelines.

Skin transplantation

Murine skin transplantation was performed as previously described.¹⁶ In brief, about 1.0 × 1.0 cm² tail skin grafts from BALB/c donors were transplanted onto the backs of wild-type B6 recipients using 5–0 sutures. The skin grafts were then covered with gauze and a secure doubled-up bandage for 7 days. On day 7 after skin grafting, the sutures were removed by sterile scissors.

Isolation of graft-infiltrating immune cells

On day 8 after skin transplantation, skin grafts were harvested, minced into small pieces and transferred to a 2-mL microcentrifuge tube with 800 lL Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, Waltham, MA, USA). Samples were incubated with 450 U mL⁻¹ collagenase I (Thermo Fisher Scientific) and 60 U mL⁻¹ DNase-I (Thermo Fisher Scientific) at 37°C on a rocking shaker at 100 rpm for 45 min. The digested samples were filtered by a 40-μm cell strainer, washed with 2% fetal bovine serum (Thermo Fisher Scientific) in Hank’s balanced salt solution (Thermo Fisher Scientific) and centrifuged at 500 g for 5 min. The supernatant was discarded, and the samples were further purified using Percoll (Cytiva, Marlborough, MA, USA).

Cell preparation

The Dynabeads untouched mouse CD4 cells kit (Thermo Fisher Scientific) was used to purify both CD4⁺ T cells from the spleens of wild-type B6 mice and TEa cells from the spleens of TEa TCR transgenic mice. Anti-CD44 mAb (clone IM7; BioLegend, San Diego, CA, USA) and the Depletion Dynabeads were used to obtain CD44^Low naïve CD4⁺ T cells or TEa cells from the purified CD4⁺ T cells or TEa cells.

As previously described,⁸ CB6F1 (allogeneic stimulators) or B6 (syngeneic stimulators) splenocytes were obtained by depleting T cells using an anti-CD3 mAb (clone 17A2; BioLegend) and the Depletion Dynabeads.

T-cell anti-CD3/anti-CD28 mAb stimulation

Ninety-six-well flat-bottomed tissue culture plates were coated with 5 lg mL⁻¹ anti-CD3 mAb (clone 2C11; BioLegend) overnight at 4°C. Polyclonal naïve CD4⁺ T cells isolated from B6 mice were plated in anti-CD3 mAb-coated 96-well flat-bottomed tissue culture plates (1 × 10⁵ cells/well) and treated with either STM2457 (catalog number S9870; Selleckchem, Houston, TX, USA) or DMSO (BioLegend) in the presence of 1 lg mL⁻¹ anti-CD28 mAb (clone 37.51; Bio X Cell, Lebanon, NH, USA). Stimulated CD4⁺ T cells were analyzed by an LSR II or Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Cell viability

Polyclonal CD4⁺ T cells were incubated with the Zombie Aqua Fixable Viability Kit (BioLegend) for 15 min at room temperature in the dark, washed with phosphate-buffered saline (Thermo Fisher Scientific) twice and analyzed on an LSR II or Fortessa flow cytometer. Cell viability was defined as the percentage of Zombie Aqua⁻ CD4⁺ T cells among total CD4⁺ T cells.

m⁶A dot blot assay

Total RNA was isolated using TRIzol (Thermo Fisher Scientific) and the concentration of the isolated RNA was determined by NanoDrop. Serial dilutions of RNA samples were spotted on the Amersham Hybond-N⁺ membrane (GE Healthcare, Chicago, IL, USA). The membrane was ultraviolet-crosslinked twice by a UVP crosslinker, blocked with 3% nonfat dry milk, immunoblotted with the anti-m⁶A antibody (catalog number 202 003; Synaptic Systems, Go€ttingen, Lower Saxony, Germany) overnight at 4°C and mouse anti-rabbit IgG-horseradish peroxidase (catalog number sc-2357; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. The blot was developed with Clarity Western ECL Substrate (catalog number 1 705 061; Bio-Rad, Hercules, CA, USA). The same membrane was stained with 0.02% methylene blue (catalog number AC414240250; Acros Organics, Geel, Flanders, Belgium) in 0.3 m sodium acetate (pH 5.2) as a loading control.

Mixed leukocyte reaction

Naïve TEa cells isolated from TEa TCR transgenic mice were mixed with CB6F1 (allogeneic stimulators) or B6 (syngeneic stimulators) splenocytes in a 1:2 ratio, plated into 96-well round bottomed plates (a total of 3 × 10⁵ cells/well) and incubated with 10 ng mL⁻¹ murine IL-2 (PeproTech, Rocky Hill, NJ, USA) for 72 h. The stimulated TEa cells were analyzed by an LSR II or Fortessa flow cytometer.

CellTrace Violet T-cell proliferation assay

Naïve CD4⁺ T cells or TEa cells were labeled with the CTV Cell Proliferation Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. CTV-labeled polyclonal naïve CD4⁺ T cells and alloantigen-specific naïve TEa cells were stimulated with anti-CD3/anti-CD28 mAbs and allogeneic/syngeneic stimulators, respectively. These stimulated CD4⁺ T cells were analyzed by an LSR II or Fortessa flow cytometer.

T-cell polarization assay

Naïve CD4⁺ T cells or TEa cells were activated in the presence of four distinct polarizing cytokines for 3 days. In brief, IL-12 (10 ng mL⁻¹) and IL-2 (10 ng mL⁻¹) were used to induce Th1 cells. IL-4 (10 ng mL⁻¹) and IL-2 (10 ng mL⁻¹) were used to induce Th2 cells. Transforming growth factor-b1 (3 ng mL⁻¹) and IL-6 (30 ng mL⁻¹) were used to induce Th17 cells. Transforming growth factor-b1 (3 ng mL⁻¹) and IL-2 (10 ng mL⁻¹) were used to induce induced regulatory T cells. All cytokines were purchased from PeproTech.

Intracellular cytokine production

Cultured CD4⁺ T cells or TEa cells were restimulated for 4 h with 50 ng mL⁻¹ phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA) and 500 ng mL⁻¹ ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences). Intracellular expression of cytokines (IFNγ, IL-17A, IL-4 and IL-13) was determined by Cytofix/Cytoperm solution (BD Biosciences), as previously described.⁸

Flow cytometric analysis

Fluorochrome-conjugated antibodies specific for mouse CD3 (clone 17A2), CD4 (GK1.5), CD69 (H1.2F3), IFNγ (XMG1.2), IL-17A (TC11–18H10.1), IL-4 (11B11), T-bet (4B10) and Ki-67 (16A8), as well as 7-aminoactinomycin D (7-AAD; catalog number 420 404), Annexin V (catalog number 640 920) and propidium iodide (catalog number 421 301) were purchased from BioLegend. Goat anti-rabbit IgG (catalog number A-21244), fluorochrome-conjugated antibodies specific for CD25 (PC61.5), IL-13 (eBio13A) and Foxp3 (FJK-16 s) were purchased from Thermo Fisher. Goat anti-mouse IgG (catalog number 115–225-166) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). A purified antibody specific for m⁶A (catalog number 202 003) was purchased from Synaptic Systems. A purified antibody specific for IRF4 (D9P5H) was purchased from Cell Signaling Technology (Danvers, MA, USA). A purified antibody specific for c-Myc (9E10) was purchased from Santa Cruz Biotechnology.

In brief, polyclonal CD4⁺ T cells or TEa cells were stained with the aforesaid antibodies and analyzed on an LSR II or Fortessa flow cytometer using a previously described method.⁸ Dead cells were excluded from the analysis using the Zombie Aqua Fixable Viability Kit (BioLegend). For staining of surface antigens, polyclonal CD4⁺ T cells or TEa cells were incubated with the Zombie Aqua Fixable Viability Kit following the manufacturer’s instructions, and fluorochrome-conjugated antibodies against the surface antigens for 20 min in the dark at 4°C. For intracellular staining of transcription factors, polyclonal CD4⁺ T cells or TEa cells were fixed, permeabilized and stained with fluorochrome-conjugated antibodies against transcription factors using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) as previously described.⁶ Fixed and permeabilized CD4⁺ T cells were stained with anti-IRF4, anti-c-Myc or anti-m⁶A primary antibodies and then stained with goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies. For cell cycle analysis, TEa cells were stained with Ki-67 and propidium iodide as previously described.²⁴ For cell apoptosis analysis, polyclonal CD4⁺ T cells or TEa cells were resuspended in Annexin V binding buffer (catalog number 422 201; BioLegend), incubated with Annexin V and 7-AAD for 15 min at room temperature in the dark and analyzed by an LSR II or Fortessa flow cytometer. Data were processed using the FlowJo version 10 software (Tree Star, Inc, Ashland, OR, USA).

Statistical analysis

Data were represented as mean standard deviation and analyzed with Prism version 8 (GraphPad Software, La Jolla, CA, USA). Differences were calculated by unpaired Student’s t-test. P < 0.05 was considered statistically significant.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.