CHBP Induces Stronger Immunosuppressive CD127+ M

Li et al. Cell Death and Disease (2021) 12:177 https://doi.org/10.1038/s41419-021-03448-7 Cell Death & Disease

ARTICLE Open Access CHBP induces stronger immunosuppressive CD127+ M-MDSC via erythropoietin receptor Jiawei Li1,2,GuoweiTu3, Weitao Zhang1,2,YiZhang2,4,XuepengZhang2,3,YueQiu2,3,JiyanWang2,5,TianleSun6, Tongyu Zhu1,2,7, Cheng Yang 1,2,8 and Ruiming Rong1,2,9

Abstract Erythropoietin (EPO) is not only an erythropoiesis hormone but also an immune-regulatory cytokine. The receptors of EPO (EPOR)2 and tissue-protective receptor (TPR), mediate EPO’s immune regulation. Our group firstly reported a nonerythropoietic peptide derivant of EPO, cyclic helix B peptide (CHBP), which could inhibit macrophages inflammation and dendritic cells (DCs) maturation. As a kind of innate immune regulatory cell, myeloid-derived suppressor cells (MDSCs) share a common myeloid progenitor with macrophages and DCs. In this study, we investigated the effects on MDSCs differentiation and immunosuppressive function via CHBP induction. CHBP promoted MDSCs differentiate toward M-MDSCs with enhanced immunosuppressive capability. Infusion of CHBP-induced M-MDSCs significantly prolonged murine skin allograft survival compared to its counterpart without CHBP stimulation. In addition, we found CHBP increased the proportion of CD11b+Ly6G−Ly6Chigh CD127+ M-MDSCs, which exerted a stronger immunosuppressive function compared to CD11b+Ly6G−Ly6Chigh CD127− M-MDSCs. In CHBP induced M-MDSCs, we found that EPOR downstream signal proteins Jak2 and STAT3 were upregulated, which had a strong relationship with MDSC function. In addition, CHBP upregulated GATA-binding protein 3 (GATA-3) protein translation level, which was an upstream signal of CD127 and regulator of STAT3. These effects of CHBP could be reversed if Epor was deficient. Our novel findings identified a new subset of M-MDSCs with better immunosuppressive capability, which was induced 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; by the EPOR-mediated Jak2/GATA3/STAT3 pathway. These results are beneficial for CHBP clinical translation and MDSC cell therapy in the future.

Introduction mainly consisted of EPO receptor (EPOR) and β common Erythropoietin (EPO), as an endogenous protein for red receptor, mediates EPO-involved anti-inﬂammation, anti- blood cell production, has been discovered for over a apoptosis, and immune regulation functions1. Thus, EPO century. Although the name of EPO refers mainly to the is a multifaceted protein due to its nonerythropoietic function of erythropoietic hormone, this molecule also effects2. However, the very high dosage required to exerts important effects as a cytokine and growth factor achieve that goal would cause unfavorable side effects, that affect multiple organs, including the immune system. including hypertension and thrombosis. A series of EPO The unique tissue-protective receptor (TPR), which is derivant have been developed including carbamoylated EPO and peptides3. Our group ﬁrstly synthesized and reported a novel proteolysis-resistant cyclic helix B pep- Correspondence: Cheng Yang ([email protected]) or Ruiming Rong tide (CHBP) with improved metabolic stability and tissue- ([email protected]) protective potency4. 1Department of Urology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Theoretically, CHBP inherits EPO-mediated immune 2Shanghai Key Laboratory of Organ Transplantation, Shanghai 200032, regulation effects because of they share TPR. In fact, China CHBP reveals excellent cell, tissue, and organ protective Full list of author information is available at the end of the article These authors contributed equally: Jiawei Li, Guowei Tu, Weitao Zhang property such as mesenchymal stem cell, kidney, and Edited by Y. Wang

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– + heart4 6. Cravedi et al. found that EPO/EPOR signal CHBP significantly increased this CD127 M-MDSCs inhibited conventional T-cell proliferation in vitro via subset proportion via EPOR. tyrosine phosphatase SHP-1–dependent uncoupling of IL-2Rβ signaling. Conversely, EPO-initiated signals Results facilitated Treg proliferation by augmenting IL-2Rγ sig- CHBP induced M-MDSCs differentiation and enhanced M- naling and maintaining constitutively quenched IL-2Rβ MDSCs immunosuppressive function signaling. Furthermore, EPO prolonged heart allograft To understand whether the presence of CHBP directly survival, whereas pharmacologic downregulation of alters the development of MDSCs, we detected the + kidney-derived EPO abrogated kidney allograft accep- CD11b cells differentiation, numbers, phenotypes, and tance7. These results suggest manipulating the EPO/ immunosuppressive function of bone marrow cells which EPOR signaling axis could be exploited to prevent were cultured with granulocyte-macrophage colony-sti- transplant rejection. In our previous study, we found mulating factor (GM-CSF) or GM-CSF + CHBP for CHBP administration significantly reduced kidney allo- 7 days. We added CHBP with different dosages (0.2, 20, graft acute rejection in a rat model. CHBP inhibited and 2000 nM) during MDSCs induction in vitro. Firstly, + dendritic cell (DC) maturation via Jak2/STAT3 pathway, we found that despite CHBP decreased CD11b cells which was a classical downstream signal upon EPOR8. proportion and number, the proportion of M-MDSCs was Taken into these two types of research together, EPOR significantly increased with a reduction of G-MDSCs ratio signal, either stimulated by EPO or CHBP, can modulate (Figs. 1A, B and S1), suggesting that CHBP regulates both innate and adaptive immunity2. MDSCs differentiation toward M-MDSCs. Targeting adaptive immune response is not the only focus To determine the immunosuppressive effect of GM- when prevention and treatment of allograft rejection. Innate CSF + CHBP induced G-MDSC and M-MDSC on T-cell immune response provocation and regulation are to be of activation, we detected cell proliferation productions of importance in the pathological process9,10.Asakindof T cells stimulated by ConA in the presence of the induced innate immune cell, myeloid-derived suppressor cells MDSCs at different CHBP doses. The addition of GM- (MDSCs) share a common myeloid progenitor with DC. CSF induced M-MDSCs markedly inhibited the pro- + MDSCs are not a terminally differentiated population of cells. liferative response of CD4 T cells comparing to G- + + In mice, MDSCs are defined as CD11b Gr1 cells. Usually, MDSCs (Fig. 1C). However, GM-CSF + CHBP-induced MDSCs can be divided into two major groups, which can be G-MDSC showed lessened suppressive function, but GM- identified by a combination of specificmarkers.Granulocytic CSF + CHBP induced M-MDSC possessed stronger + + MDSCs (G-MDSCs) are defined as CD11b Ly6G Ly6Clow immunosuppressive ability on T-cell proliferation than cell and monocytic MDSCs (M-MDSCs) are defined as GM-CSF-induced M-MDSCs, especially in the 20 nM + − CD11b Ly6G Ly6Chigh cells in mice. The main character- CHBP group (Fig. 1C). Thus, CHBP could increase M- istic of MDSCs is immunosuppression in terms of suppres- MDSCs proportion and enhance the immunosuppressive sion of T- and B-cell proliferation, induction of T-cell function of GM-CSF-induced M-MDSCs. apoptosis, inhibition of DC development and macrophages cytokine production, impairment of the effect of natural killer Infusion of CHBP-induced M-MDSCs prolonged skin cells on alloantigens and promotion of regulatory T cells allograft survival (Tregs)11. It is reported that MDSCs reduced allograft Since CHBP-induced M-MDSCs exerted enhanced – rejection as well as induced tolerance in transplantation12 14, immunosuppressive function, we employed the BALB/c and M-MDSCs are more suppressive than G-MDSCs on a (H2-D) alloskin-grafted B6 (H2-B) mouse model and per-cell basis15. Therefore, it might be more effective to infused these cells into the recipient mice. We took pic- induce more M-MDSCs in vivo during tolerance induction tures at different times to evaluate the rejection degree or prevention of allograft rejection. In addition, whether the (Fig. 2A). M-MDSCs with CHBP treatment significantly EPOR signal regulates MDSCs differentiation and function is extended the skin allograft survival compared to the M- unknown. MDSCs without CHBP induction (Fig. 2B). The rejection In the current study, we found CHBP induced more M- and tissue scores were also reduced in the CHBP-induced MDSCs rather than G-MDSCs. The M-MDSCs induced M-MDSC group (Fig. 2C). Thus, GM-CSF + CHBP- by CHBP showed a more potent immunosuppressive induced M-MDSC could increase the allograft survival capability. Infusion of CHBP-induced M-MDSCs ame- and promote tissue healing. liorated skin allograft rejection and prolonged allograft survival. Furthermore, we identified a new M-MDSCs Infusion of CHBP-induced M-MDSCs decreased CD4+, subset with CD127 positive, which revealed a strong CD8+ T cells and increased Tregs in peripheral and skin − immunosuppressive function than CD127 M-MDSCs. To investigate the influence on T cell number in the peripheral and infiltration in the skin, we examined the

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A B Control CHBP (nM) CHBP 0.2 nM CHBP 20 nM Control 0.2 20 2000 CHBP 2000 nM

70.3 60.8 56.9 58.0 p = 0.002 p = 0.001 p = 0.004 p = 0.002 p < 0.001 50 60 p = 0.004 100 p = 0.003 p < 0.001

CD11b 80 40 40 cells + 60 30 32.1 26.8 28.1 30.3 40 20 20 41.3 45.6 44.2 43.2 20 10 % of CD11b % of G-MDSC cells% of G-MDSC % of M-MDSC cells

Ly6G 0 0 0 Ly6C

Control C CHBP 0.2 nM p = 0.001 CHBP 20 nM Gate on CD4 p < 0.001 CHBP 2000 nM p = 0.001 p < 0.001 p < 0.001 18,000 80,000 G-MDSC 15,000 60,000 12,000

9,000 40,000 p = 0.047 6,000 20,000 M-MDSC 3,000 Median Fluorescence Intensity Median Fluorescence Intensity 0 0 G-MDSC:CD4+ T=1:1 M-MDSC:CD4+ T=1:1 CFSE-FITC

Fig. 1 CHBP induced M-MDSCs differentiation and enhanced M-MDSCs immunosuppressive function. A Detection of CD11b+ myeloid cells, CD11b+Ly6G−Ly6Chigh M-MDSCs, and CD11b+Ly6G+Ly6Clow G-MDSCs by ﬂow cytometry. B Quantiﬁcation analysis of CD11b+ myeloid cells, M-MDSCs and G-MDSCs. C T-cell proliferation detected by CFSE. The ratio of G-MDSCs, M-MDSCs, and T cells was 1:1.

+ + CD4 ,CD8 T cells, and Tregs in the blood, spleen, and The effects of CHBP on MDSCs were dependent on EPOR skin. Flow cytometry analysis demonstrated the infusion of As we mentioned in the introduction, CHBP shares CHBP-induced M-MDSCs decreased the proportion of EPOR with EPO, on which their immune regulation + + CD4 ,CD8 T cells and increased Tregs in the blood (Fig. functions are dependent. In this study, we generated △lyz 3A), as well as in the spleen (Fig. 3B). Similar results were Epor mice, whose myeloid-derived cells lack EPOR. In observed in the skin (Fig. S2b). These results suggested that in vitro experiments, the MDSCs deficient of EPOR had a infusion of CHBP-induced M-MDSCs ameliorated effector slight influence on differentiation in terms of more G- T cells response and favorited Tregs. However, the per- MDSCs and fewer M-MDSCs. In CHBP treatment + centage of CD3 cells in blood showed no significant dif- groups, deficiency of EPOR abolished CHBP effects, ferences between groups (Fig. S2a), which suggested that especially on M-MDSCs induction (Fig. 4A, B). Next, we neither M-MDSC nor CHBP-induced M-MDSC had an performed a T-cell proliferation assay to examine the influence on circulating T cells. To determine whether immunosuppressive function of G-MDSCs and M- CHBP could induce activated T cells into Tregs in vitro, we MDSCs. We use different ratios between MDSCs and performed the Transwell test and found that M-MDSC T cells. Although T cell proliferation was not inhibited by induced by CHBP induced more Tregs than their coun- G-MDSCs at a 1:1 ratio in the above result (Fig. 1D), terparts without CHBP stimulation (Fig. S2c). CHBP-induced G-MDSCs still acquired enhanced immunosuppressive function. Knockout of Epor in G- MDSCs reduced CHBP effect (Fig. 4C). The results of M-

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Fig. 2 Infusion of CHBP-induced M-MDSCs prolonged skin allograft survival. A Skin pictures at different days post transplantation. B The survival rates. C H&E staining of the skin (POD 14). Scale bars: 1st row 1 mm, 2nd row 250 μm, and 3rd row 100 μm.

MDSCs were amazing. CHBP increased 1.1–4.5 folds M-MDSCs with or without CHBP treatment using mRNA immunosuppressive function of M-MDSCs at 1:16, 1:8, sequencing (Fig. 5A). We found that the mRNA expression fl/fl △lyz 1:4, and 1:2, respectively (Epor + CHBP vs. Epor + of GATA-binding protein 3 (GATA3) was slightly CHBP, Fig. 4D). increased with an IL-7R increasing by CHBP. String ana- To confirm whether the inhibitory function of CHBP lysis showed the protein–protein interaction and we found induced M-MDSCs, we detected the mRNA and protein that there was a strong connection between GATA3, expressions of inducible NO synthase (iNOS) and STAT3, and IL-7R (Figs. S3a and 5B). Hoyler et al. reported + − arginase-1 (Arg-1) in sorted CD11b Ly6G Ly6Chigh that GATA3 controlled IL-7R expression16, we speculate cells. Both of iNOS and Arg-1 were significantly CHBP regulates GATA3 expression and then induces IL- fl/fl increased by CHBP in Epor M-MDSCs, but reversed in 7R expression. Therefore, we further examined the Jak2/ △lyz Epor M-MDSCs (Fig. 4E, G). A similar result was GATA3/STAT3 protein level in M-MDSCs. We found that observed in Arg-1 activity (Fig. 4F). These results indicate Jak2, GATA3, p-STAT3, and STAT3 protein level was fl/fl that CHBP enhances M-MDSCs immunosuppressive significantly increased by CHBP in Epor M-MDSCs, and function through EPOR. the effect was abolished when M-MDSCs deficient of Epor (Fig. 5C, D). Next, we extracted membrane proteins of M- CD127+ M-MDSCs induced by CHBP exerted stronger MDSCs and performed a protein array to further check IL- immunosuppressive function 7R protein level (Figs. S2b and 5E). Molecular function To further investigate the mechanism that CHBP enrichment showed that CHBP-induced MDSCs had enhanced M-MDSCs immunosuppressive function, we intense function with receptor-ligand and cytokine recep- analyzed the mRNA differences between the two groups of tor activity (Fig. 5F). In addition, biological process and

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p < 0.001 CHBP-induced A Blood Control M-MDSC 30 p = 0.001 p < 0.001 M-MDSC Control Gated on M-MDSC 17.8 CD45+ 23.6 14.4 20 CHBP-induced M-MDSC T cells +

10 % of CD4

0 p < 0.001 CD4 80 p < 0.001 p < 0.001 Gated on 68.2 24.0 11.1 CD45+ 60 T cells + 40

20 % of CD8

0 CD8 p < 0.001 Gated on p < 0.001 p < 0.001 CD4+CD25+ 0.72 3.33 9.43 10 8 Treg +

6 Foxp3 + 4

2 % of CD4 Foxp3 0

p < 0.001 CHBP-induced 40 B Spleen Control M-MDSC M-MDSC p < 0.001 p < 0.001 Control Gated on 30 28.2 25.5 14.0 M-MDSC CD45+ T cells + 20 CHBP-induced M-MDSC

10 % of CD4

0 p < 0.001

CD4 p = 0.002 p = 0.001 40 Gated on 27.2 20.3 17.7 CD45+ 30 T cells + 20

10 % of CD8

0 CD8 p < 0.001 Gated on p < 0.001 p < 0.001 9.29 13.5 21.6 30 CD4+CD25+ Treg + 20 Foxp3 + 10

Foxp3 % of CD4 0

C CD4CD8 Foxp3

Control

M-MDSC

CHBP-induced M-MDSC

100 μm

p < 0.001 p < 0.001 p < 0.001 Control p = 0.006 p = 0.001 25 40 30 p < 0.001 p < 0.001 p < 0.006 p < 0.001 M-MDSC 20 30 Treg CHBP-induced M-MDSC + T cells T cells + + 20 15 20 10 10 10 5 Number of CD4 of Number Number of CD8 Number of Foxp3 0 0 0

Fig. 3 Infusion of CHBP-induced M-MDSCs decreased CD4+, CD8+ T cells, and increased Tregs in peripheral and skin. A Detection of the proportion of CD4+, CD8+ T cells, and Foxp3+ Tregs in the blood. B Detection the proportion of CD4+, CD8+ T cells, and Foxp3+ Tregs in the spleen. C CD4, CD8, and Foxp3 immunohistochemistry staining in the alloskin with semi-quantiﬁcation analysis (POD 14). Scale bar: 100 μm.

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Eporfl/fl fl/fl fl/fl fl/fl A Epor Epor +CHBP B Epor +CHBP Gated on Epor䕧lyz 32.1 30.8 CD11b+ p = 0.007 lyz Epor䕧 +CHBP p = 0.002 p = 0.006

55.9 62.6 p p p = 0.001 p < 0.001 40 < 0.001 = 0.010 80

lyz lyz Eporȏ Eporȏ +CHBP 30 60

31.9 34.5 20 40

51.0 54.2 10 20 % of G-MDSC cells% of G-MDSC % of M-MDSC cells

Ly6G Ly6C 0 0 C G-MDSC:CD4+ T 1:2 1:4 1:8 1:16 1:32 Eporfl/fl fl/fl Epor +CHBP fl/fl Epor Epor䕧lyz lyz Epor䕧 +CHBP 150,000

*** fl/fl *** Epor +CHBP 100,000

50,000

Eporȏlyz 0 1:2 1:4 1:8 1:16 1:32

Median fluorescence intensity (MFI) fluorescence intensity Median G-MDSC:T cells * Eporfl/fl+CHBP vs. Eporȏlyz+CHBP Eporȏlyz+CHBP

D M-MDSC:CD4+ T Eporfl/fl fl/fl Eporfl/fl Epor +CHBP Epor䕧lyz lyz Epor䕧 +CHBP

150,000 Eporfl/fl+CHBP *** *** 100,000 ***

Eporȏlyz 50,000 ***

0 1:2 1:4 1:8 1:16 1:32

Median fluorescence intensity (MFI) fluorescence intensity Median M-MDSC:T cells Eporȏlyz+CHBP * Eporfl/fl+CHBP vs. Eporȏlyz+CHBP

Fig. 4 (continued)

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E Eporfl/fl F Eporfl/fl p < 0.001 +CHBP p < 0.001 p < 0.001 Epor䕧lyz lyz p < 0.001 p < 0.001 Epor䕧 +CHBP p < 0.001 p < 0.001 p < 0.001 p < 0.001 300 800 1500

Eporfl/fl fl/fl 600 Epor +CHBP 200 1000 Epor䕧lyz Epor䕧lyz 400 +CHBP

100 500 iNOS mRNA Arg-1 mRNA 200 Arginase activity (mU) 0 0 0

G Eporfl/fl fl/fl Epor +CHBP iNOS 130 kD Epor䕧lyz lyz Arg-1 40 kD Epor䕧 +CHBP p = 0.018 β-actin 42 kD p = 0.046 p = 0.023 p = 0.036 p = 0.043 1.5 1.0 -actin) -actin) 0.8 1.0 0.6

0.4 0.5 0.2

0.0 0.0 iNOS protein (against iNOS protein Arg-1 protein (against Arg-1 protein

Fig. 4 The effects of CHBP on MDSCs were dependent on EPOR. A Detection of CD11b+Ly6G−Ly6Chigh M-MDSCs and CD11b+Ly6G+Ly6Clow G-MDSCs by ﬂow cytometry. B Quantiﬁcation analysis of the G-MDSCs and M-MDSCs proportions. C T-cell proliferation detected by CFSE. The ratios of G-MDSCs and T cells were 1:2, 1:4, 1:8, 1:16, and 1:32. D T-cell proliferation detected by CFSE. The ratios of M-MDSCs and T cells were 1:2, 1:4, 1:8, 1:16, and 1:32. E The expressions of iNOS and Arg-1 by qPCR. F The arginase-1 activity. G The protein expressions of iNOS and Arg-1 by western blot.

KEGG pathway also demonstrated CHBP induced MDSCs Discussion have a close relationship with cell proliferation and In recent years, numerous studies have shown that EPO cytokine–cytokine receptor interaction (Fig. S3c, d). GO acts far beyond erythropoiesis. The immune regulatory and KEGG pathway analyses revealed that CHBP increased function of EPO endows EPO and its derivant-based drug M-MDSCs cell proliferation and receptor activity. IL-7R, development more application possibilities17.Therearetwo also named as CD127, is a membrane receptor protein. We kinds of key receptors combined with EPO (EPOR)2 and + examined the proportion of CD127 M-MDSCs using flow TPR. (EPOR)2 and TPR are expressed on a variety of cytometry (Fig. 5G). These results demonstrated that immune cells, such as macrophages, DCs, mast cells, and + CHBP significantly upregulated CD127 M-MDSCs in lymphocytes18. An increasing body of evidence demonstrates total M-MDSCs, whose effect was dependent on EPOR. At that EPO and its derivatives can directly affect the manner by + last, we sorted these CD127 M-MDSCs from CHBP- which immune cells exert their immunoregulatory effects. induced M-MDSCs and compared their immunosuppres- For instance, our group found that EPO ameliorated acute − sive function to CD127 M-MDSCs using CFSE-labeled kidney injury by reducing macrophage infiltration and pro- + T cells proliferation assay. The results showed CD127 M- moting M2 phenotype polarization in vivo19. EPOR is also MDSCs had prominent stronger immunosuppressive expressed on DC20.WepreviouslydemonstratedthatCHBP − capacity than CD127 M-MDSCs (Fig. 5H). These results could ameliorate acute rejection in a rat kidney transplan- suggest that CHBP enhances M-MDSCs immunosup- tation model via inhibition of DC maturation8.Thisstudy pressive function might be attributed to the induction of confirmed that CHBP-regulates MDSCs differentiation and + CD127 M-MDSCs.

Ofﬁcial journal of the Cell Death Differentiation Association Page 8 of 12 11 10.5 10 9.5 9 8.5 8 Osteoactvin Testican3 LOX−1 Endocan FetuinA Marapsin IL−7R TIM−1 CCL6 Chemerin CD80 EDAR CD48 BAFFR Betacellulin MBL−2 PLGF−2 Limitin Kremen−1 CD6 OX40L H60 Periostin IL−33 C5a DLL4 Meteorin TRAIL DAN P−Cadherin Nope +CHBP +CHBP lyz lyz CHBP fl/fl fl/fl 䕧䕧 = 0.001 p

Epor Epor Epor Epor Control +CHBP = 0.007 cation of the CD127-positive cells in +CHBP p lyz lyz ﬁ fl/fl fl/fl 䕧䕧 = 0.003 p The Jak2 and GATA3 protein expression by Epor Epor Epor Epor M-MDSC C M-MDSC RNA-seq detected the different RNA expressions - +

40 30 20 10

cells in M-MDSC in cells

= 0.020 CD127 of % p + CD127 CD127 = 0.001 p = 0.038 +CHBP p +CHBP lyz lyz fl/fl fl/fl 䕧䕧 32.0 B +CHBP

1.2 0.8 0.4 0.0

+CHBP

Epor Epor Epor Epor

Detection and quanti -actin) (against protein GATA-3 β lyz 2.5 2 1.5 1 0.5 0 −0.5 fl/fl Ƹ G < 0.001 p Protein array compared the difference in M-MDSCs with or without E Epor Epor lyz 27.9 23.4 25.9 0 fl/fl Il7r Gatad2a Gata1 Gata3 Gata4 Il7 Gata6 Gatad2b Gatad1 Gata2 Ƹ

80 60 40 20 = 0.006 < 0.001

140 120 100 p p Median fluorescence intensity (MFI) intensity fluorescence Median < 0.001 p Epor Epor < 0.001 CHBP p

Gated on CD11b+Ly6G-Ly6C+ on CD11b+Ly6G-Ly6C+ Gated = 0.033 CD127

< 0.001

p SSC- H

The String analysis of mRNA sequencing. 0.4 0.3 0.2 0.1 0.0 1.0 0.8 0.6 0.4 0.2 0.0

-actin) (against protein Jak2 p-STAT3 protein (against STAT3) (against protein p-STAT3 β B G

Control M-MDSC + 86 kD 42 kD 48 kD 42 kD 125 kD 86 kD 10 8 6 4 2 0 (2021) 12:177 (2021) 12:177 ra Arf1 Il7r Vps26a Fkbp1a Uqcr11 Tmem164 Tmem9b Schip1 Gatad2b Golph3l Kif13b Slc8a1 Ms4a2 Mdfic Tlr8 Atp7a Lmbrd2 Nr2c2 Kmt2a Slc37a2 Birc6 Utrn Herc2 Vps13a Senp5 Ulbp1 Nfya Uprt Gse1 Hmgb1−ps8 Prrc2c Tr pm 7 Sertad2 Slc36a1 Btbd7 Ipmk Plagl2 St6galnac6 Mapkapk5 Sigmar1 Gpatch4 Pja1 Tr i m 26 Bcl2l1 Pex11b Chpf2 Ak4 Pdf Ap1ar Creb5 Gata1 Slc36a3os Sh2d5 Ugt1a6b Ggnbp1 Tspan2 Ccl5 Ccdc137 Akt1s1 Nudt1 Gvin1 Tnfsf15 Csnk1g1 Slc9a7 Jmjd4 Micu3 Ro Ppp1r12b Ttc21a Rab2b Ttll4 Bhlhb9 Tnfsf10 Cit Amn1 Kantr Pou2f1 Nfix Cdc14b Tpk1 Dgkq Foxn3 Nav1 Dst Prrg4 Homez Adamts6 Clnk Cav1 Trp53i11 Apitd1 Ppp1cc Nr4a3 Umad1 Afmid Sgsm3 Arfgef3 Arhgap28 Enpp1 Rfx3 B3gnt5 Plag1 Nos1ap Shroom4 Etv1 Abi2 Prokr1 Cdkl5 Pgr Cfh Erc1 Aak1 Rasal2 Tgif2 Gzmk Emid1 Kcnab3 Il7 Gata4 Gata6 Gata3 Bach2 Eml6 Atm Dip2a Dpm1 Mga Hipk2 Plekhg1 Bola1 Prrc2b Gga3 Rapgef6 Peak1 Kmt2c Suv39h1 Kcna3 Cep85l Abl2 Mfsd7b Soga1 Prdm10 Clock Fbxo48 Cep170 Rreb1 Flrt2 Lcor Ttpal Ptar1 Foxp1 Gdap10 Nfat5 Arl5b Ahcyl2 Cbfa2t2 Mdn1 Dmxl1 Klhl11 Met Lpin2 Rere Traf3 Phc3 Arid4b Asxl2 Xpo4 Abca13 Tet2 Ndufa12 Fen1 Orai1 Xiap Gatad1 Mpc1 Limd2 Gata2 Idi1 Ss18 Stra13 Gatad2a Gxylt1 Kat6a Vps13c Tiam1 Tnks Rnf213 Atad2b Macf1 Smg1 Brox Mycbp2 Tmem131 Usp9x Epb41 Mef2a Itgb3 Nfe2l1 Xist Myadm Tlr4 Bcap29 Ugt1a1 Egr2 Smek2 Tnpo1 Etohi1 Inhba Lnpep Lyst Atf5 Rassf5 Wdfy2 T-cell proliferation detected by CFSE. The ratio of G-MDSCs and T cells was 1:1. M-MDSC CD127 CHBP H - The molecular function enrichment of protein array. F Control CD127 Jak2 STAT3 β-actin β-actin GATA3 The p-STAT3 and STAT3 protein expression by western blot. A C p-STAT3 F H DE M-MDSCs induced by CHBP exerted stronger immunosuppressive function. A ow cytometry. D ﬂ + Cell Death and Disease cial journal of the Cell Death Differentiation Association ﬁ Fig. 5 CD127 western blot. in M-MDSCs with or without CHBP treatment in vitro. M-MDSCs by CHBP treatment in vitro. Of Li et al. Li et al. Cell Death and Disease (2021) 12:177 Page 9 of 12

function via EPOR signal. This is the firstreportabout responding to IL-7-promoted T-cell survival in vitro. As a EPOR-mediated immune regulation on MDSCs. matter of fact, GATA3 could bind to the Il7r locus27. It is encouraging that CHBP promotes MDSCs to dif- Here, we found that GATA3 protein translation could be ferentiate toward M-MDSCs and empowers M-MDSCs up-regulated upon EPOR signal stimulation rather than with stronger immunosuppressive function than G- its mRNA transcription. Then the increased GATA3 MDSCs. Although both G-MDSCs and M-MDSCs have might promote more CD127 expression in M-MDSCs. the immunosuppressive ability, the strength and STAT3 is probably one of the main transcription factors mechanisms are different. It is now accepted that M- that regulate MDSC function. Recent works have high- MDSCs are more suppressive than G-MDSCs on a per- lighted the importance of signaling pathways downstream cell basis15. Therefore, CHBP demonstrates a bright of STAT3 that are responsible for MDSC differentiation prospect clinical translation in M-MDSCs-based cell in tumor models28. Besides Il7r, GATA3 also bounds to therapy. To be in fact, our data showed CHBP also the −1710 to −1530 region of STAT3 promoter and enhanced G-MDSCs immunosuppressive function repressed its transcription29. Therefore, GATA3 might + according to Fig. 4C. It seems that there were opposite play a central role in the new CD127 M-MDSCs subset results in Figs. 1D and 4C, our explanation is the differ- differentiation and function. ence ratio between G-MDSCs and T cells. In conclusion, the EPO derivant peptide CHBP induces The most important finding in our research is that CHBP M-MDSCs differentiation and enhanced M-MDSCs + induced CD127 M-MDSCs. CD127 is the α chain of IL-7R. immunosuppressive function via EPOR-mediated Jak2/ It is well-known that low expression of CD127 is a cell GATA3/STAT3 pathway. Infusion of these induced M- surface marker of human Tregs which could be instead of MDSCs prolongs skin allograft survival. CHBP induces + + + − + intracellular Foxp3. So, the CD4 CD25 CD127low T cells more CD11b Ly6G Ly6ChighCD127 M-MDSCs, which represent human Tregs. However, some recent studies have more potent immunosuppressive function compared − demonstrated an opposite expression pattern of CD127 in to CD127 counterparts. Our novel findings identified a innate immune regulatory cells. In 2016, Björklund et al.21 new subset of M-MDSCs with the better immunosup- found a heterogeneity of CD127 in innate lymphoid cells pressive capability and demonstrated EPOR-mediated (ILCs) using single-cell RNA sequencing. Next, Fan’sgroup Jak2/GATA3/STAT3 pathway. These results are benefit − + + identified a novel subset of ILCs, Lin CD45 CD127 IL- for CHBP clinical translation in the future. + 10 ILCreg. The ILCreg secrets IL-10 and TGF-β1, with immunosuppressive function22. Autoimmune regulator Materials and methods (AIRE) plays a key role in central and periphery immune Animal strains tolerance. A recent study confirmed these AIRE expression C57BL/6 (B6) and BALB/c mice were purchased from + + + cells outside the thymus are CD127 CCR7 PD-L1 DCs23. the Slac. Company (Shanghai, China). The myeloid- Taken these studies together, CD127 might be an important specific EPOR conditional knockout mice (Lyz-Cre/ cell surface marker of some innate immune regulatory cells. Eporloxp/loxp) under C57BL/6 genetic background were Our results for the first time identified a new subset of obtained by crossing Eporloxp/loxp mice with mice + − + MDSCs, CD11b Ly6G Ly6ChighCD127 M-MDSCs. The expressing Cre recombinase under the control of the M-MDSCs with CD127 expression reveal more immuno- Lysozyme promoter (Lyz-Cre). Eporloxp/loxp littermates suppressive ability than those without CD127 expression. served as the WT control. Lyz-Cre mice were kindly However, the mechanism is still unknown. Does CD127 provided by Professor Yong Zhao from the Institute of directly combine with target immune cells or indirectly Zoology, Chinese Academy of Sciences (Beijing, China). initiate other signal transductions? This question might be Eporloxp/loxp mice were generated with Cyagen Bios- answered in a future experiments. ciences Inc. (Shanghai, China). Mice were used between 6 We elucidated that GATA3 is the key transcription and 8 weeks of age. Experimental protocols were factor of which CHBP induced CD127 expression in M- approved by the Animal Ethics Committee of Zhongshan MDSCs. GATA3 is a double zinc-finger transcription Hospital Fudan University (Shanghai, China). factor that is required for the effector fate decision of Th2 cells24,25. GATA3 integrates diverse upstream signals to Generation of Eporloxp/loxp mice control target gene expression and cellular functions26. Epor floxed mice were generated by Cyagen Biosciences. Hoyler et al. demonstrated that GATA3 is essential for Briefly, the Epor gene was located on chromosome 9 in ILC2 fate decisions, whose cell surface marker contains mice. The linearized vector was subsequently delivered to CD12716. CD127 expression was significantly lower in ES cells (C57BL/6) via electroporation, followed by drug + GATA3-deficient CD8 T cells than in wild-type (WT) selection, PCR screening, and Southern Blot confirmation. + counterparts. In addition, compared to WT CD8 T cells, After gaining 94 drug-resistant clones, we have confirmed + GATA3-deficient CD8 T cells were defective in 12 potentially targeted clones, 6 of which were expanded

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for Southern Blotting. After confirming correctly targeted (clone: FJK-165) (all antibodies were purchased from BD ES clones via Southern Blotting, we selected some clones Biosciences, eBioscience or Biolegend company) on BD for blastocyst microinjection, followed by chimera pro- FACSCallibur (BD Biosciences, San Diego, CA, USA) and duction. Founders were confirmed as germline-transmitted analyzed by Flow JoX software. via crossbreeding with WT. In the end, four male and four female F1 heterozygous mutant mice confirmed as the final T-cell proliferation assay deliverables for this project. A single-cell suspension was prepared from spleens of C57BL/6 (B6). Naïve T cells were isolated from mono- Mice skin transplantation model nuclear cells by magnetic-activated cell sorting according Recipient B6 mice were injected with i.v. 2 × 106 MDSC to the manufacturer’s instructions (Miltenyi Biotec, or CHBP induced MDSC and the matched controls were Auburn, CA, USA). Cells were labeled with 2 μM CFSE given equal volumes of 0.9% saline solution. Full- (Invitrogen) for 5 min in PBS at 37 °C and washed twice thickness tail skin tissue of BALB/c mice was grafted on with PBS. The labeled cells were then stimulated with the dorsal part of the C57BL/6 (B6) recipients on day 0, ConA (eBioscience) (2 μg/ml) in the presence of different according to the procedure as described previously30. doses of MDSCs as indicated for 72 h. The cell pro- Photographs were taken daily with a digital camera until liferation was then determined by flow cytometry after the graft was rejected completely. Fourteen days later, the staining with anti-CD4 antibody. + + + + + percentages of CD4 , CD8 , and CD4 CD25 Foxp3 cells in the peripheral blood and spleens were detected by Real-time quantitative PCR (qPCR) flow cytometry. Total RNA isolation was performed using TRIzol reagent according to the manufacturer’s instructions Induction of MDSCs from bone marrow cells (Invitrogen). The quality and integrity of RNA were Bone marrow cells from tibias and femurs of C57BL/6 evaluated via A260/A280 ratio and 18s/28s band by △lyz and Epor mice (6–8 weeks) were flushed with PBS and agarose electrophoresis. Then total RNA was reversed to the red blood cells were lysed by RBC lysis buffer (BD the first-strand cDNA using RevertAid First Strand cDNA Biosciences, CA, USA). Totally, 2 × 106 non-adherent Synthesis Kit (Thermo Fisher Scientific, Inc.). RT-qPCR ™ bone marrow cells were cultured with 50 ng/ml GM-CSF was performed in duplicate using All-in-One qPCR Mix (PeproTech, RH, USA) or 50 ng/ml GM-CSF + 20 μM (GeneCopoeia, Inc., Maryland, USA). An Eppendorf CHBP, respectively, in 6 cm dishes (Corning, USA) in 3 ml Mastercycler Realplex PCR system was used for quanti- of complete RPMI 1640 medium (containing 10% FBS, 1% tative PCR under the following conditions: initial dena- MEM nonessential amino acids (NEAA) solution, 1% turation was performed at 95 °C for 10 min, followed by sodium pyruvate, 1% streptomycin and penicillin, and 2 μl 40 cycles of denaturation at 95 °C for 10 s, annealing at 2-Mercaptoethanol) (RPMI 1640 medium, FBS, MEM 60 °C for 20 s and extension at 72 °C for 15 s. GAPDH was NEAA, sodium pyruvate, and streptomycin and penicillin used as an internal control to normalize differences in the were purchased from Gibco, NY, USA; 2- amount of total RNA in each sample. The threshold cycle Mercaptoethanol was purchased from Sigma-Aldrich, St. (Ct) values were analyzed using the comparative Ct Louis, USA) at 37 °C, 5% CO2 for 7 days. (−ΔCt) method. The expression level of target genes was obtained by normalizing to the endogenous reference and Cell staining and flow cytometry relative to control. Primer sequences are listed in Table After 7-day induction, induced MDSCs were tested or S1. sorted on Beckman Coulter MoFlo AstriosEQ (Beckman Coulter, CA, USA) or FACS AriaIII (BD Biosciences, San Arginase-1 activity Diego, CA, USA) by using mAb CD11b (clone: M1/70), A total of 1 × 106 cells were mixed with 100 µl of 0.1% Ly-6G (clone: 1A8), Ly-6C (clone: AL-21), and CD127 Triton X-100. After 30 min of incubation on a shaker, (clone: A7R34) (all antibodies were purchased from BD 100 μl of 25 mM Tris-HCL and 20 ul of 10 mM MnCl2 Biosciences or eBioscience company). Flow cytometry were added to the sample. The arginase was then acti- verified that all the isolated MDSCs yielded a pure vated by heating the sample for 10 min at 56 °C, and population of more than 90%. The sorted MDSCs were arginine hydrolysis was conducted by incubating the stored in PBS + 10% FBS for further experiments. sample with 100 μl of 0.5 M L-arginine (pH 9.7) at 37 °C Splenocytes and peripheral blood mononuclear cells for 60–120 min. The reaction was stopped with 900 ul of were obtained from spleens and peripheral blood of mice H2SO4 (96%)/H3PO4 (85%)/H2O, and the sample was by Ficoll density gradient centrifugation. Cells were tested mixed with 40 μl of 9% isonitrosopropiophenone. Absor- by using mAb CD45 (clone: 30-F11), CD3 (clone: 17A2), bance was read at 540 nm in the microplate reader. All CD4 (clone: GK1.5), CD8 (clone: 53-6.7), and Foxp3 samples were read in triplicate.

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Western blot Protein array Twenty micrograms of proteins from the MDSCs Membrane proteins of GM-CSF and GM-CSF + CHBP homogenates were separated on 15% or 10% (wt/vol) induced MDSC were measured with an AAM-CYT-7 polyacrylamide denaturing gels and electro-blotted onto RayBio Mouse Cytokine Antibody Array. Differential polyvinylidene fluoride membranes. The primary anti- expression proteins with a P value ≤ 0.05 and fold bodies used were anti-iNOS (1:1000, abcam, USA), Arg-1 change ≥ 1.2 or ≤0.83 were considered to be upregulated (1:1000, Cell Signaling Technology, Danvers, MA, USA), and downregulated, respectively. Gene ontology analysis, Jak2 (1:1000, Cell Signaling Technology), p-STAT3 pathway annotation, transcription factor enrichment, and (1:1000, Cell Signaling Technology), STAT3 (1:1000, Cell comparison with various immunological and oncogenic Signaling Technology) and GATA3 (1:1000, Cell Signaling gene signatures were performed with the use of the Technology). The semiquantitative analysis (AlphaView DAVID knowledge base. Software 3.3, Cell Biosciences, Inc.) results were expressed as the optical volume densities (OD × mm2) normalized to Statistics GAPDH (1:1000 dilution, Cell Signaling Technology) or Data were analyzed using GraphPad Prism 8 software or R β-actin (1:10,000 dilution, Abcam, Cambridge, UK). language. Packages such as Limma, Annotation, ggplot2, enrichGo were used to analyze the RNA-sequencing and HE staining and pathological assessment array results. Quantitative variables were analyzed by one- Mice skin tissues were fixed with 10% formalin and way ANOVA (among three or more groups), two-tailed bedded with paraffin and were cut into 4 μm thick slices. independent t test (between two groups) and expressed as After dewaxing, HE staining was performed. HE staining means ± standard deviation (S.D.). P < 0.05 was considered was observed under microscopy at 200× to evaluate tissue statistically significant. damage. Harvested mice skin tissues were fixed overnight in 10% Acknowledgements fi μ Funding This study was supported by the National Key R&D Program of China formalin, embedded in paraf n, and cut into 4 m sec- (2018YFA0107501 to Ruiming Rong, 2018YFA0107502 to Cheng Yang), tions. Sections for CD4, CD8, and Foxp3 staining were National Natural Science Foundation of China (81770746 to Cheng Yang, incubated with 3% hydrogen peroxide to block endogen- 81770747 and 81970646 to Ruiming Rong), Shanghai Rising‐Star Program (19QA1406300 to Cheng Yang), Medical and Health Talents Training Plan for ous peroxidase for 20 min at 37 °C, and Ags were retrieved the Excellent Youth of Shanghai Municipal (2018YQ50 to Cheng Yang), 2019 using a high-pressure method in citrate buffer. The sec- Shanghai Youth Talent Development Program (to Cheng Yang), Project ELITE: tions were incubated with an anti-CD4 mAb (1:5000; A Special Supportive Program for Organ Transplantation by COTDF (2019JYJH05 to Cheng Yang) and the Science and Technology Commission of ab183685, Abcam), an anti-CD8 mAb (1:2000; ab217344, Shanghai Municipality (16431902300 to Tongyu Zhu). Abcam) or anti-Foxp3 mAb (1:100; 12653, Cell Signaling Technology) at 4 °C overnight. The sections with mice Author details CD4, CD8, or Foxp3 Abs were rinsed three times in TBST 1Department of Urology, Zhongshan Hospital, Fudan University, Shanghai 2 and incubated with an HRP-conjugated rabbit anti-goat 200032, China. Shanghai Key Laboratory of Organ Transplantation, Shanghai 200032, China. 3Department of Critical Care Medicine, Zhongshan Hospital, IgG H&L (1:2000; ab205718, Abcam) at room tempera- Fudan University, Shanghai 200032, China. 4Zhongshan Hospital Institute of ture for 45 min. DAB was added for a coloration for 5 min Clinical Science, Zhongshan Hospital, Fudan University, Shanghai 200032, 5 immunoreactivity was analyzed by image-pro plus. China. Department of Urology, Shanghai Public Health Clinical Center, Shanghai 201508, China. 6Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou RNA sequencing 215006, China. 7Shanghai Public Health Clinical Center, Shanghai 201508, 8 GM-CSF and GM-CSF + CHBP induced MDSC RNA China. Zhangjiang Institute of Fudan University, Shanghai 201203, China. 9Department of Transfusion, Zhongshan Hospital, Fudan University, Shanghai were extracted with Trizol (15596-026, Invitrogen). RNA 200032, China sequencing was employed and single-end 75-bp-length reads were generated. Data were aligned to the mouse Author contributions genome (mm9 version) with the TopHat2 algorithm. HT- Jiawei Li and Cheng Yang drafted the paper. Jiawei Li, Cheng Yang and Guowei Tu revised the manuscript. Cheng Yang, Tongyu Zhu and Ruiming seq and DESeq algorithms were used in order to measure Rong conceived and designed the study. Jiawei Li, Weitao Zhang and Yi Zhang gene expression and identify differential expression between participated in the protocol design. Jiyan Wang provided the HE and the two groups of cells. Genes with a P value ≤ 0.05 and fold immunochemistry staining. Weitao Zhang, Yue Qiu and Guowei Tu performed ≥ ≤− the animal models. Jiawei Li, Yi Zhang, Xuepeng Zhang and Weitao Zhang change 1.5 or 1.5 were considered to be upregulated participated in the flow cytometry, western blot, PCR, RNA-seq, and other and downregulated, respectively. Gene ontology analysis, molecular function examinations. Tianle Sun provided the mouse feeding and pathway annotation, transcription factor enrichment, and the identification of knockout mice. All authors participated in paper writing fi comparison with various immunological and oncogenic and approved the nal version of the paper. gene signatures were performed with the use of DAVID Data availability knowledge base, Ingenuity Pathway Analysis software, and Raw data files for RNA-seq of MDSCs can be found at NCBI Gene Expression Molecular Signature Database from Broad Institute. Omnibus Accession number: GEO: GSE160822.

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