CD8+ T cells exhaustion induced by myeloid‐derived suppressor cells in myelodysplastic syndromes patients might be through TIM3/Gal‐9 pathway

Abstract CD8+ T cells play a central role in antitumour immunity, which often exhibit ‘exhaustion’ in the setting of malignancy and chronic viral infection due to T cell immunoglobulin and mucin domain 3 (TIM3) and myeloid‐derived suppressor cells (MDSCs). Our team previously found that overactive MDSCs and exhausted TIM3+CD8+ T cells were observed in myelodysplastic syndromes (MDS) patients. However, it is not obvious whether MDSCs suppress CD8+ T cells through TIM3/Gal‐9 pathway. Here, Gal‐9, as the ligand of TIM3, was overexpressed in MDSCs. The levels of Gal‐9 in bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients were elevated. CD8+ T cells from MDS or normal controls produced less perforin and granzyme B and exhibited increased early apoptosis after co‐culture with MDSCs from MDS. Meanwhile, the cytokines produced by CD8+ T cells could be partially restored by TIM3/Gal‐9 pathway inhibitors. Furthermore, CD8+ T cells produced less perforin and granzyme B after co‐culture with excess exogenous Gal‐9, and the function of CD8+ T cells was similarly restored by TIM3/Gal‐9 pathway inhibitors. Expression of Notch1, EOMES (associated with perforin and granzyme B secretion), p‐mTOR and p‐AKT (associated with cell proliferation) was decreased in CD8+ T cells from MDS after co‐culture with excess exogenous Gal‐9. These suggested that MDSCs might be the donor of Gal‐9, and TIM3/Gal‐9 pathway might be involved in CD8+ T cells exhaustion in MDS, and that TIM3/Gal‐9 pathway inhibitor might be the promising candidate for target therapy of MDS in the future.


| INTRODUC TI ON
Myelodysplastic syndromes (MDS) are malignant bone marrow disorders characterized by ineffective haematopoiesis and high risk to transformation into acute myeloid leukaemia. Increased malignant clones and cellular immunodeficiency might contribute to the pathogenesis of MDS. 1-5 Cytotoxic T lymphocytes (CTLs) play a central role in tumour immunity. 6 Adoptive T cell immunotherapy in neoplastic disorders has been restricted by CD8 + T cells 'exhaustion' due to some immune checkpoint inhibitors, such as T cell immunoglobulin and mucin domain 3 (TIM3), programmed death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphocyte-activation gene 3 (LAG3). 7,8 Among these immune checkpoints, the interaction of TIM3 with its ligand galectin-9 (Gal-9) promotes Tc1 cells 9 apoptosis, CD8 + T cells exhaustion, 10,11 malignant cell proliferation 12 and myeloid-derived suppressor cells (MDSCs) amplification. 13 In humans, MDSCs are usually defined as CD14 − CD11b + cells or the cells which express the myeloid marker (CD33) and are lack of lineage maturation markers and HLA-DR 14 (Lin − HLA-DR − CD33 + cells). In previous research, increased Lin − HLA-DR − CD33 + MDSCs in MDS patients were observed, and this cell population was obviously separated by flow cytometry than other labelled cells. We also found that CD8 + T cells could be inhibited by MDSCs, which were 'crazy' in MDS patients. CD8 + T cells with less INF-γ overexpressed TIM3 in MDS patients. Further, TIM3 + CD8 + T cells produce less perforin and granzyme B. 1, 2 The hypothesis that CD8 + T cells exhaustion might be induced by MDSCs through TIM3/Gal-9 pathway drove us to detect Gal-9 expression in MDSCs, and the level of perforin and granzyme B in CD8 + T cells after co-culture with MDSCs or excess exogenous Gal-9 or TIM3/Gal-9 pathway inhibitors. We also explored Notch1, eomesodermin (EOMES), phospho-mTOR (p-mTOR) and phospho-AKT (p-AKT) proteins, which are related to the function and proliferation of CD8 + T cells, 15,16 to verify that TIM3/Gal-9 pathway might be involved in CD8 + T cells exhaustion induced by MDSCs in MDS patients.

| Patients
Fifty-two treatment-naive Chinese MDS patients (32 males and 20 females, median age 64, Table 1) who were newly diagnosed according to the WHO classification 17  Thirty-one healthy volunteers (mean age 44.48 ± 14.87) were enrolled. There was no difference in the sex ratio or age between MDS patients and normal healthy volunteers (P > .05).
The study was approved by the Ethics Committee of Tianjin Medical University General Hospital. Informed written consents were obtained from all the patients and normal controls following the Declaration of Helsinki.

| Membrane markers and intracellular markers detection
One hundred microlitres of peripheral blood and bone marrow samples from MDS patients and healthy volunteers were placed in heparin-containing anticoagulant tubes. The specimens were incubated with 15 µL of Lin/HLA-DR/CD33 and their isotype control antibodies at 4°C for 30 minutes. After incubation, erythrocytes were lysed with 2 mL of erythrolysin for 10 minutes, centrifuged at 400 g for 5 minutes and washed twice with phosphate buffered saline (PBS). After permeabilizing the cell membrane using an IntraSure Kit (BD Biosciences), 5 μL galectin-9 monoclonal antibody was added to the cells, incubated for 20 minutes at 4°C in the dark and washed twice with PBS. Finally, 5 × 10 5 cells per tube were detected by flow cytometry. After CD8 + T cells and MDSCs were co-cultured, they were co-incubated with CD3/CD8 antibodies in the same way. For intracellular staining, the samples were incubated with perforin and granzyme B antibodies after permeabilizing the cell membrane. The phenotype of MDSCs was analysed for the cell surface markers Lin, HLA-DR and CD33. Intracellular expression of galectin-9 was determined. Perforin or granzyme B expression in co-cultured CD8 + T cells was analysed. All data were collected on a flow cytometer The above antibodies were added as described by the manufacturer.

| Sorting CD8 + T cells and MDSCs
Ten millilitres of fresh peripheral blood or bone marrow was ob-

| Culture MDSCs
MDSCs were sorted by flow cytometry and then cultured with 10% foetal bovine serum (FBS) (containing 60 mg/L penicillin and 100 mg/L streptomycin) (Gibco) in the presence of 50 ng/mL recombinant human (rh) granulocyte-monocyte colony-stimulating factor (GM-CSF) (PeproTech Inc) at 37°C in a 5% CO 2 incubator. The culture supernatants were collected after 48 hours for ELISA.

| Co-culture of CD8 + T cells with exogenous Gal-9
CD8 + T cells selected by magnetic beads were co-cultured with 2 ng/mL exogenous recombinant human Gal-9 (R&D Systems) with 10% FBS (containing 60 mg/L penicillin and 100 mg/L streptomycin) at 37°C in a 5% CO 2 incubator for 24-48 hours. Then, the expression of perforin, granzyme B and pathway-related proteins in CD8 + T cells were detected with or without TIM3/Gal-9 inhibitors.

| Real-time PCR
Expression of the Gal-9 gene in MDSCs was detected by real-

| Enzyme-linked immunosorbent assay (ELISA)
Bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients and normal controls were harvested. The levels of Gal-9 were measured by using a Human Galectin-9 ELISA Kit (KAMIYA Biomedical Company) according to the manufacturer's instructions.

| Western blot analysis
CD8 + T cells cultured alone and co-cultured with exogenous Gal-9 were collected and lysed in RIPA buffer supplemented with complete protease inhibitors and phosphatase inhibitors (Roche). Protein levels were quantified using a BCA Kit. Proteins were separated by electrophoresis on 4%-12% precast gels (Bio-Rad Laboratories) and

| Statistical analysis
Prism statistical software (v7.00; GraphPad Software, Inc, La Jolla, CA, USA) was applied for data analysis. Statistical differences between two groups were analysed using Student's t test. One-way ANOVA was used to compare three groups or more. All data are presented as the mean ± standard error of the mean (normal distribution data) or median (25% percentile-75% percentile) (non-normal distribution data).

| RE SULTS
3.1 | Galectin-9 was highly expressed in MDSCs, and Gal-9 was also elevated in bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients compared to those from normal controls  that MDSCs might release Gal-9 into the bone marrow microenvironment and exert immunosuppressive effects.  (Figure 2A was an upward trend in co-cultured group as seen in the flow cytogram, although there was no significant difference( Figure 2E).

| Decreased perforin and granzyme B in CD8 + T cells after co-cultivation with excess exogenous Gal-9
We speculated that MDSCs might be a source of Gal-9 due to its overexpression; therefore, we took excess exogenous Gal-9 in the place of MDSCs and co-cultured CD8 + T cells with excess exogenous Gal-9 (recombinant human Gal-9 [R&D Systems]) and TIM3/Gal-9 pathway inhibitors.
Increasing malignant clones and cellular immunity defects are involved in the pathogenesis of MDS, 1-5 which is a malignant haematological disease. Daver et al 27 found that the efficacy of hypomethylating agent combination with immune checkpoint inhibitors (PD-1 inhibitor) in MDS was better than hypomethylating agents alone.
TIM3, as one of the important immunity checkpoints, interacts with its ligand Gal-9 24 could promote Tc1 cell 9 apoptosis, CD8 + T cell exhaustion, 10,11 malignant cell proliferation 12 and MDSC proliferation. 13 MDSCs, which are powerful cellular immunity suppressor, could badly inhibit the antitumour immune response mediated by the effector T cells and lead to tumour cells immunological surveillance escape. 28,29 Here, we found that Gal-9 was also highly expressed in MDSCs were much lower than that in real infectious diseases. These suggested that cellular immunity was insufficient for malignant clonal cells due to cellular immunity tolerance, both in low-risk and highrisk MDS groups. We found that there was no difference of Gal-9 in MDSCs of high-risk and low-risk MDS patients, which was consistent with our hypothesis. Therefore, further exploration of the molecular mechanism of Gal-9 up-regulation is needed.
Perforin and granzyme B are the most important cytokines in CD8 + T cells against infectious and malignant cells. MDSCs can inhibit CD8 + T cells in esophageal squamous cell carcinoma. 8 Here, perforin, granzyme B and proliferation decreased and the early apoptosis of CD8 + T cells increased after CD8 + T cells from controls were co-cultured with MDSCs. These data implied that MDSCs with excessive Gal-9 might induce TIM3 overexpression in CD8 + T cells, leading to a downward trend of perforin and granzyme B after CD8 + T cells from MDS were co-cultured with MDSCs. We hypothesized that MDSCs might be a source of Gal-9. Therefore, we replaced MDSCs with excessive exogenous Gal-9 to repeat the above procedure and found that CD8 + T cells indeed produced less perforin and granzyme B after co-cultivation with excessive exogenous Gal-9, similar to that after co-cultivation with MDSCs from MDS patients. The secretion of perforin and granzyme B by CD8 + T cells could be partially restored by TIM3/Gal-9 pathway inhibitors.
Meanwhile, the early apoptosis of CD8 + T cells after co-culture with MDSCs was higher than that of CD8 + T cells alone and could be partially restored by TIM3/Gal-9 pathway inhibitors. It suggested that CD8 + T cells exhaustion might be induced by MDSC through TIM3/Gal-9 pathway.
The T-box transcription factor eomesodermin (EOMES) plays an important role in the development and maturation of CTL. 31 Notch1 could regulate perforin and granzyme B by affecting EOMES. 15,32 Here, we found that Notch1 and EOMES decreased after co-cultivation with excessive exogenous Gal-9. It indicated that the TIM3/ Gal-9 pathway might induce CD8 + T cells 'exhaustion' by down-regulating Notch1 and EOMES.
Mammalian target of rapamycin (mTOR) is a serine/threonine (Thr)-protein kinase that plays a central role in regulating cell growth F I G U R E 4 Early apoptosis of CD8 + T cells from MDS patients after co-culture with MDSCs or TIM3/Gal-9 inhibitors. (A) Scattered dots in the red rectangle represent early apoptosis of CD8 + T cells from MDS patients in the five co-culture systems. The a. b. c. d. and e. experimental groups represent CD8 + T cells from MDS, CD8 + T cells+MDSCs from MDS, CD8 + T cells+MDSCs+F38-2E2, CD8 + T cells+MDSCs+9M1-3 and CD8 + T cells+MDSCs+F38-2E2+9M1-3, respectively. (B) The histogram represents early apoptotic cells. *P < .05, **P < .01, NS = not significant F I G U R E 5 Perforin and granzyme B in CD8 + T cells from MDS patients after co-culture with excess exogenous Gal-9 or TIM3/Gal-9 inhibitors. (A) Flow cytometry scatter diagrams of perforin and granzyme B expression in CD8 + T cells from MDS. a: CD8 + T cells alone; i: CD8 + T cells with excess exogenous Gal-9; j: CD8 + T cells from MDS with excess exogenous Gal-9 and F38-2E2; and k: CD8 + T cells from MDS with excess exogenous Gal-9 and 9M1-3. (B and C) The histogram represents the expression of perforin and granzyme B in the four groups. *P < .05, **P < .01, ***P < .001, NS = not significant and proliferation. 33 Overactivation of AKT/mTOR after phosphorylation is associated with over-proliferation of leukaemia cells, 16,34 as well as tumour cells such as liver cancers and pancreatic neuroendocrine tumours. 35,36 Our study found that p-mTOR and p-AKT decreased in CD8 + T cells after co-culture with exogenous Gal-9, indicating that TIM3/Gal-9 pathway might suppress CD8 + T cells through down-regulating AKT/mTOR ( Figure 6C).
In conclusion, CD8 + T cells 'exhaustion' could be induced by up-regulating Gal-9 in MDSCs and TIM3 in CD8 + T cells of MDS patients, which was partially restored by TIM3/Gal-9 pathway inhibitors. TIM3/Gal-9 pathway may be involved in CD8 + T cells exhaustion induced by MDSCs in MDS, leading to malignant MDS clone immunological surveillance escape and over-proliferation.
Therefore, TIM3/Gal-9 pathway inhibitors might be the promising candidate for target therapy of MDS in the future.