Page 125 - 2021_03-Haematologica-web

HLA-G corrects dysfunction of immune cells in ITP
and only about one-third of ITP patients can achieve long- term remission.5-7 Autoantibodies targeting platelets enhanced T-helper cell activity, initiated abnormal activa- tion of cytotoxic T lymphocytes and disturbed cytokine profiles to accelerate platelet clearance and suppress platelet production in ITP patients.1-4,8 Moreover, the defi- ciency and dysfunction of immunoregulatory cells (i.e., regulatory T cells [Tregs]),9-11 tolerogenic dendritic cells12 and myeloid-derived suppressor cells13 also contribute to the loss of immune tolerance in ITP.
HLA-G is a non-classical major histocompatibility com- plex class I antigen found to be expressed in placental tro- phoblast cells in 1990s.14 There are four membrane-bound (mbHLA-G1 to -G4) and three soluble (sHLA-G5 to -G7) isoforms generated by alternative splicing of the primary transcript.15 It was initially believed that HLA-G in placen- tal cells plays an important role in maternal-fetal immuno- tolerance by interacting with inhibitory receptors (immunoglobulin-like transcript 2 [ILT-2/LILRB1/CD85j], immunoglobulin-like transcript 4 [ILT-4/LILRB2/CD85d] and KIR2DL4 receptor) on leukocytes, which leads to CTL apoptosis, natural killer (NK) cells immobilization, mononuclear cells inhibition and suppressive cytokine environment.16,17 Subsequently HLA-G was found not be restricted to placenta cells, but it is also expressed in human peripheral blood mononuclear cell (PBMC) and plasma.18,19 Additionally to the immune inhibitory capabi- lity mentioned above, HLA-G-ILT2/ILT4 interaction sup- pressed lymphocyte proliferation20,21 and inhibited anti- body secretion of activated B cells.21 Ideas that HLA-G protein facilitates allotransplantation by preventing immune rejection via these actions on immune cells was established. After liver and kidney transplantation, an increased HLA-G level was detected in patients with a reduced incidence of rejection.22-24
Recently, HLA-G-induced immune tolerance has been intensively investigated. The expression of HLA-G (and its cognate receptors, ILT) was correlated with immune escape, which suggests unfavorable outcomes in cancer patients.25,26 On the other hand, HLA-G-induced tolerance can benefit patients with autoimmune diseases. Low so- luble HLA-G (sHLA-G) level was found in patients with rheumatoid arthritis (RA) and multiple sclerosis, and was associated with disease progression in both cases.27,28 Moreover, the decrease of the number and/or function of ILT2 and ILT4 was involved in the pathogenesis of sys- temic lupus erythematosus (SLE).29,30 Another study by Kim et al.31 showed that functional HLA-G 14-bp polymor- phism was associated with susceptibility to RA and SLE. However, the role of HLA-G in ITP remains obscure.
LeMaoult et al.32 reported that both membrane-bound HLA-G (mHLA-G) and secreted HLA-G5 (sHLA-G5) upregulated ILT2/ILT4 expression on APC and CD4+ T cells without the stimulation from exogenous antigens.32 This encouraged the development of recombinant human HLA-G protein (rhHLA-G). Further investigation demon- strated that in vitro synthetic HLA-G protein induced the tolerance of skin grafts in vivo successfully.33 Nevertheless, little has been defined about the effect of rhHLA-G in autoimmune disease.
In this study, we investigated whether there is aberrant HLA/ILT expression and their contribution to the loss of immune tolerance in ITP. Our data showed that the expression of HLA-G and ILT were both decreased in ITP patients, which may result in impaired function of PBMC
and dendritic cells (DC). rhHLA-G upregulated ILT on CD4+ T cells and monocytes, and corrected dysfunction of immune cells. These findings indicate that HLA-G has the potential as a diagnostic marker and therapeutic option for ITP.
Methods
Patients and controls
Plasma samples from 50 newly diagnosed ITP patients were col- lected for the detection of sHLA-G with ELISA. Among them, PBMC from 17 ITP patients were isolated for the in vitro investiga- tion of mHLA-G and ILT (Table 1-2). Samples from 15 healthy vol- unteers were used as controls. The diagnosis of ITP was based on established practice guidelines.1 Fourteen patients with autoanti- body received standard ITP treatment high-dose dexamethasone (HD-DXM; 40 mg/day for 4 consecutive days), as described in the Online Supplementary Appendix and shown in Table 2. Enrollment took place between March 2013 and December 2018. The study was approved by the Medical Ethical Committee of Qilu Hospital, Shandong University. Informed consent was obtained from all patients before collecting samples in accordance with the Declaration of Helsinki.
Reagents
RhHLA-G (Abcam, Cambridge, UK) was dissolved in PBS to prepare a stock solution of 60 g/mL, and stored at -80 °C, accord- ing to the manufacturer’s instructions.
In vitro platelet apoptosis assay
PBMC were cultured with 600 ng/mL rhHLA-G for 3 days.
Freshly isolated human platelets were adjusted to 1x108/mL with 5 M prostaglandin E1. rhHLA-G-modulated PBMC (1×106) were then co-cultured with autologous or allogeneic platelets (1×107) as effector cells and target cells, respectively, and incubated at 37°C for 4 hours (h). Platelet apoptosis was detected using a mitochon- drial membrane potential assay kit (Beyotime Biotechnology, Shanghai, China).
DC suppression assays
Freshly isolated CD4+ T cells were labeled with carboxyfluores- cein diacetate succinimidyl ester (5 mM; Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes (min) at 37°C and quenched by adding 3 mL of cold fetal calf serum (FCS) for 5 min at 4°C. CD4+ cells were washed with RPMI 1640 culture medium three times and seeded at 105 cells/well in U-bottom 96-well plates containing 1 mg/mL anti-human CD3 (Biolegend) and 50 U/mL recombinant human IL-2 (rhIL-2; R&D Systems) in triplicates. DC with or with- out rhHLA-G pre-modulation were added at a 5:1 ratio (T cells : DC). All cells were cultured for 5 days in humidified air with 5% CO2 at 37°C, then collected for flow cytometry analysis.
Animal model and the treatment of HLA-G
Blood was drawn by retro-orbital bleeding. Platelets from C57BL/6 wild-type mice were prepared to immunize C57BL/6 CD61 knockout (KO) mice by transfusion 10 mL of 108 platelets weekly for 4 consecutive weeks.34,35 The immunized CD61 KO mice were sacrificed, and their spleens were homogenized to pre- pare splenocyte suspension. On the day of transplantation, the C57BL/6 SCID mice were subjected to 200 cGy total body irradi- ation, and then injected with the indicated splenocytes (5×104) with or without HLA-G protein administration. Blood from saphenous veins was collected and platelet counts were detected weekly, as described in the Online Supplementary Appendix.34,35
haematologica | 2021; 106(3)
771