BALB/c and C57BL/6 mice (male, 7-8 weeks-old) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong Province, China). Mice were housed and maintained under a specific pathogen-free (SPF) condition. The animal protocols performed in this study were approved by the Animal Ethics Committee of Guangdong Provincial Academy of Chinese Medical Sciences (Approval No. 2020091).

ESAT-6 was purchased from China Peptides Co. Ltd. Briefly, ESAT-6 gene was obtained by gene synthesis and cloned into plasmid pET32a containing c-myc tag, which then was transfected into E.coli. Subsequently, ESAT-6 protein was expressed, purified and verified through SDS-PAGE (purity>95%), followed by endotoxin removal using the Protein Endotoxin Removal Kits (Beyotime, China, endotoxin <0.1 EU/ml).

Most recipient mice were randomly divided into four groups, including control (vehicle for ESAT-6), rapamycin (Rapa, MCE, USA), ESAT-6 (China Peptides Co. Ltd., China) and ESAT-6 plus Rapa groups. In some experiments, the culture filtrate protein (CFP-10, China Peptides Co. Ltd.) was also used as another control group. Rapa (1 mg/kg) was administered i.p. daily for three weeks or until allograft rejection, whichever came earlier, while ESAT-6 or CFP-10 (50μg/kg) was injected i.p. on days 0, 2, 4, 6 and 8 following allotransplantation. Both ESAT-6/CFP-10 and rapamycin were dissolved in saline. To deplete Tregs, recipient mice were administered with anti-CD25 mAb (Clone PC61.5, Bio X Cell, USA) at 0.1 mg on days 0, 3 and 7.

Wild-type (WT) C57BL/6 mice received skin grafts from BALB/c donor mice. Skin allotransplantation was performed as described in previous studies (20, 21). Briefly, full-thickness trunk skin sized approximately 1 cm² was grafted onto the right dorsal flank of a recipient and covered with a sterile bandage (Johnson & Johnson). The skin graft was monitored daily after removal of the bandage at eight days posttransplantation. Skin allograft rejection was defined as graft necrosis of larger than 90% of the graft area.

Cervical heterotopic heart transplantation in recipient mice was performed. Briefly, the right cervical common carotid artery and the external jugular vein of recipients were incised, with vascular cuffs being made by 24-G and 22-G intravenous catheters (BD Biosciences), respectively, and connected to the ascending aorta and the pulmonary artery of a donor heart (22). Survival of heart allograft was monitored by visual observation and palpation, while allograft rejection was defined as the cessation of cardiac contractions.

Skin allografts were fixed in 4% paraformaldehyde for 18 hours and then embedded in paraffin after dehydration. Paraffin slides with 3-μm thickness were made, deparaffinized and stained with hematoxylin and eosin (H&E staining). For immunohistochemistry, sections were incubated with primary anti-CD3 (clone: E4T1B, 1:1000; CST) or ani-Foxp3 (clone: D608R, 1:1000; CST) Ab at 4˚C overnight, and then with secondary Ab HRP–anti-rabbit IgG (Maxim) at room temperature for 30 min. Finally, the slides were colored with diaminobenzidine (DAB, Maxim). For quantitative analysis, slides for H&E were imaged at a magnification of 100× and those for IHC were 200x. The cellular infiltration and integrated optical density (IOD) of CD3⁺ or Foxp3⁺ staining in the images were measured using software ImageJ.

Draining lymph node and spleen cells were harvested and stained with anti-CD4-FITC or APC (clone: RM4.5), CD8-FITC or PE (clone: 53-6.7), CD44-PerCP-Cyanine5.5 (clone: IM7), CD62L-PE-Cy7 (clone: MEL-14), CD11C-PE (clone: HL3), CD80-FITC (clone: HL3), CD86-APC (clone: GL-1), IFN-γ-APC (clone: XMG1.2), IL-17-PE (clone: TC11-18H10) and FoxP3-PE (clone: FJK-16s) mAbs (eBioscience or BD Biosciences). To analyze intracellular IFN-γ, cells were cultured with PMA and ionomycin in the presence of monensin for six hours before the intracellular staining. To stain for intracellular FoxP3 and IFN-γ, cells were fixed, permeated using the Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kits (eBioscience), stained with anti-FoxP3 or anti-IFN-γ Ab, and analyzed using FACSAria III (BD Biosciences). To purify CD4⁺CD25^- conventional T cells for Treg induction in vitro, splenocytes were stained with anti-CD4 PerCP-Cyanine5.5 (clone: RM4-5, eBioscience, USA) and anti-CD25-PE Abs (clone: 3C7, eBioscience, USA), and then CD4⁺CD25^- T cells were sorted using FACSAria III (BD Biosciences). The purity of the sorted cells was typically >97%.

FACS-sorted CD4⁺CD25^- T cells (2 x 10⁶/ml) were cultured in the complete RIPM 1640 medium (10%FBS, 2mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin) and stimulated with anti-CD3/anti-CD28 Abs (5 μg/ml) in the presence of rIL-2 (2ng/ml, Peprotech) for four days. Cells were also treated with ESAT-6 (0.5, 1 and 2 μg/ml, respectively), CFP-10 (1 μg/ml), rapamycin (0.1μM), or E/Z-IT-603 (an inhibitor of IĸBα/c-Rel signaling, 10 μM) in some groups. Tregs then were quantified using flow cytometry. To measure conventional T cell proliferation in a relatively physiological setting, primarily nylon wood-enriched T cells were stained with CFSE (5 μM) at room temperature for 15 min, washed and cultured for four days, and finally analyzed through a flow cytometer (NovoCyte Quanteo, Agilent, USA).

The proteins of IFN-γ and IL-10 in the supernatant of the culture were measured using ELISA kits according to the manufacturer’s instructions (Boster, China), while the absorbance was read at 450 nm in a microplate spectrophotometer (Thermo Fisher Scientific, USA).

Whole protein was obtained from RIPA buffer after lysing cells, while the nuclear protein of the cells was extracted by the Nuclear and Cytoplasmic Protein Extraction Kits (Beyotime, China). The concentration of protein sample was measured using a BCA protein assay kit (Thermo Fisher Scientific, USA). Proteins were run in 10% SDS-PAGE gels and transferred to a PVDF-membrane. After blocking in Tris-buffered saline with Tween-20 containing 5% (w/v) BSA at room temperature for 1-h, the membrane was incubated with rabbit primary Abs, including anti–p-IKBα (clone: 14D4, 1:1000, CST), anti-IKBα (clone: 44D4, 1:1000, CST), anti-α-tubulin (clone: 11H10, 1:1000; CST), anti–c-Rel (clone: EPR25178-58, 1:1000, Abcam) and anti-Histone H3 (clone: AFB7534, 1:3000, Affinity) Abs, at 4˚C overnight. After incubation, the membrane was washed with TBST and then incubated with a secondary antibody, HRP-conjugated goat anti-rabbit IgG or anti-mouse IgG (1:2000, CST, USA) for one hour. Blot signals were detected by a Bio-Rad Gel imaging system and analyzed with ImageJ software.

Statistical comparisons of the means were performed using one-way ANOVA for multiple groups or Student t-test for two groups only. Data were analyzed using GraphPad Prism 8. The analysis of graft survival was performed using Kaplan–Meier method (log-rank test). A value of P < 0.05 was considered statistically significant.

ESAT-6 has been shown to regulate immune responsiveness. To determine the effects of ESAT-6 protein on allograft survival, C57BL/6 mice were transplanted with skin derived from BALB/c mice and treated with ESAT-6, culture filtrate protein-10 (CFP-10), rapamycin (Rapa) or ESAT-6 plus Rapa. As shown in Figure 1A , ESAT-6 prolonged skin allograft survival in recipient mice compared to the control, with a statistical significance (median survival time: MST = 20 vs. 12 days). As a positive control, Rapa also prolonged the allograft survival (MST = 22 vs. 12). Moreover, the combined treatment with ESAT-6 and Rapa further extended allograft survival compared to the treatment with either ESAT-6 alone (MST = 34 vs. 20) or Rapa alone (MST = 34 vs. 22 days). However, CFP-10 did not significantly alter allograft survival time. Like ESAT-6, CFP-10 is a protein also expressed in RD1 region of Mtb and thus can serve as a control for ESAT-6. Taken together, our findings suggest that ESAT-6 significantly suppresses allograft rejection.

We then asked if ESAT-6 could diminish cellular infiltration in a transplant, as determined by H&E and IHC stainings after allotransplantation. Representatives of rejected or accepted skin transplants are shown in Figure 1B . H&E staining displayed marked cellular infiltration in untreated and transplanted control mice, while much less cellular infiltration was found in recipient mice treated with ESAT-6 or Rapa ( Figures 1C, D ). Treatment with both ESAT6 and Rapa further suppressed cellular infiltration compared to Rapa alone ( Figures 1C, D ). Similarly, ESAT-6 or Rapa also inhibited CD3⁺ T cell infiltration in allografts compared with the control group ( Figure 1E ), while a further decrease in CD3⁺ T cells was observed in the group treated with ESAT6 plus Rapa compared to Rapa alone.

CD4⁺Foxp3⁺ Tregs are essential for allograft survival or immune tolerance (23, 24). Therefore, we further analyzed Foxp3 expression in a skin allograft through IHC staining. We found that either ESAT-6 or Rapa enhanced Foxp3 expression in skin transplants ( Figure 1F ). Importantly, treatment with ESAT-6 plus Rapa induced even more Foxp3 expression in an allograft compared to that with Rapa treatment alone.

C57BL/6 mice were transplanted with a BALB/c heart and treated with Rapa and/or ESAT-6. Spleen and draining cervical LN cells were isolated seven days posttransplantation, while CD4⁺Foxp3⁺ Tregs were quantified by flow cytometric analysis. Treatment with ESAT-6 or Rapa alone increased the percentages of CD4⁺Foxp3⁺ Tregs in the spleen and LNs ( Figures 2A, B ) compared to the control group, whereas CFP-10 failed to do so. Importantly, the treatment with ESAT-6 plus Rapa further increased the frequency of Tregs compared to Rapa alone. On the other hand, cardiac allograft survival was significantly prolonged by ESAT-6 (MST= 21 vs. 6 days) or Rapa alone (MST= 27 vs. 6 days), while combined treatment further extended allograft survival compared to Rapa alone (MST= 41 vs. 27 days) ( Figure 2C ). However, treatment with depleting anti-CD25 Ab reversed the EAST-6-mediated extension of allograft survival ( Figure 2C ). Our findings suggest that ESAT-6 also prolongs cardiac allograft survival by increasing CD4⁺Foxp3⁺ Tregs.

T helper 1 (Th1) cells play a pivotal role in mediating alloimmune responses or allograft rejection. Since we found that ESAT-6 alleviated CD3⁺ T cell infiltration in a skin allograft, we then assessed if it regulated the Th1 response in vivo. Recipient mice receiving a skin allograft were treated with ESAT-6 and/or Rapa, and then draining lymph node (LN) and spleen cells from recipient mice were harvested and analyzed by FACS 10 days after transplantation. As shown in Figure 3 , either ESAT-6 or Rapa reduced the frequency of CD4⁺IFN-γ⁺ Th1 cells in both spleens ( Figures 3A, B ) and LNs ( Figures 3A, C ) of recipient mice. The percentage of Th1 cells in LNs of recipients was further decreased by treatment with both ESAT-6 and Rapa ( Figure 3C ). These results suggest that ESAT-6 indeed inhibits Th1 differentiation after allotransplantation.

Since activation and maturation of DCs are important for T cell differentiation/activation, we then examined whether ESAT-6 would affect the maturation of DCs ten days after skin transplantation. Using flow cytometric analyses, we found that ESAT-6 did not significantly alter the frequency of CD11c⁺CD86⁺ or CD11c⁺CD80⁺ cells in the spleen of recipient mice ( Supplementary Figure S1 ), indicating that the maturation of DCs, unlike Th1 differentiation, is not particularly affected by the treatment with ESAT-6 in the face of vigorous alloimmune responses.

We further asked whether it would reduce effector CD4⁺ and CD8⁺ T cells in vivo. Draining LN and spleen cells from recipient mice receiving skin allografts were isolated 10 days posttransplantation, and the frequency of CD44^highCD62L^low effector CD4⁺ and CD8⁺ T cells were analyzed by FACS. Compared to control group, ESAT-6 or Rapa alone reduced the frequency of CD4⁺CD44^highCD62L^low ( Figures 4A–C ) and CD8⁺D44^highCD62L^low ( Figures 4D–F ) effector T cells (Teff) in both spleen and LNs of the recipient mice. Moreover, compared to the treatment with Rapa alone, combined treatment with both ESAT-6 and Rapa further decreased the percentage of CD4⁺CD44^highCD 62L^low Teff in both spleen and LNs ( Figures 4B, C ) as well as the percentage of CD8⁺CD44^highCD62L^low Teff in LNs only ( Figure 4F ). These results indicate that ESAT-6 inhibits the generation of both CD4⁺ and CD8⁺ effector T cells in the context of allotransplantation.

The c-Rel in NF-ĸB pathway is critical for the transcriptional regulation of expression of forkhead box P3 (FoxP3), which governs the Treg development (25). Since we found that ESAT-6 increased CD4⁺Foxp3⁺ Tregs, we hypothesized ESAT-6 might promote Treg development by acting on IĸBα/c-Rel signaling in CD4⁺ T cells. The proteins of IκBα, phospho-IκBα and c-Rel isolated from conventional CD4⁺CD25^- T cells, which were cultured and stimulated by anti-CD3/CD28 Abs for two days, were measured via Western blotting. As shown in Figure 5A , ESAT-6 augmented phosphorylation of IκBα and promoted nuclear c-Rel accumulation, seemingly in a dose-dependent manner (ES: 0.5, 1, 2 μg/ml), compared to the control group. As controls, neither Rapa nor CFP-10, which is a chaperone protein of ESAT-6 and also important for Mtb virulence (26), enhanced c-Rel expression in the nucleus. These findings indicate that ESAT-6, but not CFP-10, enhances IĸBα/c-Rel signaling in conventional CD4⁺CD25^- T cells, resulting in generation of more Tregs.

To further confirm that ESAT-6 promotes Treg development by acting on c-Rel signaling, FACS-sorted CD4⁺CD25^- conventional T cells were cultured with anti-CD3/anti-CD28 Abs in the absence or presence of ESAT-6 (1 μg/ml) for four days. In some groups, cells were treated with ESAT-6 plus E/Z-IT-603 that blocks c-Rel signaling. What we found was that ESAT-6 induced CD4⁺Foxp3⁺ Tregs in vitro, and so did Rapa ( Figure 5B ) as a positive control, while blocking c-Rel with E/Z-IT-603 reversed the effects of ESAT-6 on the Treg generation.

To determine an effect of ESAT-6 on conventional T cell activation or function, nylon wood-enriched T cells from splenocytes were stained with CFSE and then cultured with anti-CD3/anti-CD28 Abs in the presence of ESAT-6 (0.5, 1, 2 μg/ml, respectively) for four days. As shown in Figures 6A, B , either ESAT-6 or Rapa significantly inhibited both CD4⁺ and CD8⁺ T cell proliferation in vitro. We also found that either ESAT-6 or Rapa inhibited IFN-γ secretion ( Figure 6C ) while increasing IL-10 secretion ( Figure 6D ) in the culture of T cells. Joint treatment with both ESAT-6 and Rapa resulted in a further decrease in IFN-γ and increase in IL-10 production compared to ESAT-6 or Rapa treatment alone. These data suggest that ESAT-6 inhibits conventional T cell proliferation and activation.