Bioengineered extracellular vesicles presenting PD-L1 and Gal-9 to ameliorate new-onset primary ovarian insufficiency (POI)

doi:10.21203/rs.3.rs-4853642/v1

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Bioengineered extracellular vesicles presenting PD-L1 and Gal-9 to ameliorate new-onset primary ovarian insufficiency (POI)

https://doi.org/10.21203/rs.3.rs-4853642/v1

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Primary ovarian insufficiency (POI) is a clinical syndrome of ovarian dysfunction characterized by premature exhaustion of primordial follicles, which causes infertility and dysfunctions of many other organ systems including sexuality, bones, the cardiovascular system, and neurocognition. An important factor in the development of POI is immune dysregulation, which results in augmented T cell response in ovary. Thus, modulation of the ovarian infiltrated T lymphocytes by exogenous PD-L1 or Gal-9 is an ideal approach for treating POI. Herein, we genetic engineered the human umbilical cord mesenchymal stem cells (hucMSCs) derived extracellular vesicles (EVs) overexpressing PD-L1 and Gal-9, which could restrict the activity of ovarian autoreactive T lymphocytes and protect ovarian cells from destruction. Moreover, PD-L1-Gal-9 EVs prominently promoted effector T cell apoptosis in vitro, and reversed the decreased AMH in the POI mice as well as declined proportion of CD8+ T cells infiltrating the ovaries, which contributed to preventing POI progression and preserving the residual ovarian fertility.

Primary ovarian insufficiency (POI) is a clinical syndrome defined as premature exhaustion of the resting pool of primordial follicles before the age of 40 years and characterized by oligo-amenorrhea or amenorrhea for more than 4 months with elevated gonadotrophins (FSH level > 25 IU/l on two occasions over 4 weeks apart)[1, 2]. Approximately 1% of women among women of reproductive age suffer the disturbance of POI, including reproductive health impairment (even infertility), hot flashes, sweating, sleep disorders, vaginal dryness, depression[3], and compromised mental health[4]. Although most cases of POI resulted from genetic[5], autoimmune[6], or iatrogenic factors[7], it is generally acknowledged that the augmented T cell-mediated autoimmunity in the periphery and ovarian microenvironment is the one of the main factors responded for the autoimmune disturbances as well as the follicular maturation dysfunction in POI patients.

Immunotherapy has currently become one of the emerging approaches in the treatment of malignant tumors, with proven efficacy on a wide range of tumors. Immune checkpoint inhibitors (ICIs)[8] including target T-cell receptors like programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)[9, 10] and T cell immunoglobulin mucin 3 (TIM-3)[11, 12] could revitalize exhausted T cells to combat cancers, have becoming a new immunotherapy. However, immune checkpoints blockade can also cause pathological T cells to react with self-antigens and resulting in autoimmune damage to organs. The immune system is critical for optimal ovarian homeostasis and reproductive function. Studies had found that patients with POI had augmented T helper 1 (TH1) responses and regulatory T (Treg) cell deficiency in both the periphery and the ovary compared to the control women[6]. Furthermore, the increased infiltration of TH1 cells in the ovary resulted in follicle atresia and ovarian insufficiency in mice model. The autoimmune response will increase in TH1 inflammation and impair steroidogenesis in GCs (granule cells), which might account for the low estrogen in patients with POI[6, 13, 14]. Thus, exogenous PD-L1 is a promising protein therapeutics for treatment of autoimmune damage. What’s more, blocking the Tim-3/Galectin-9 (Gal-9) signaling pathway significantly enhance the functional activity of T cells. These findings indicate that PD-L1 and Gal-9 might be the beacon of hope for the treatment of POI.

Engineered cell therapies (including MSC based therapies[15–18]) and biomaterials (including hydrogel[19] and ovarian regenerative patch[20]) have been employed in the management of POI. Recently, genetic engineered extracellular vesicles (EVs) have acknowledged as a novel drug delivery approach for some innate advantages including excellent biocompatibility and high stability and possess the unique ability to deliver therapeutic agents to specific locations within the body without triggering immune reactions or unexpected toxicity[21, 22]. Increasing studies has demonstrated that the mesenchymal stem cell therapy could ameliorate the progression POI through diverse mechanisms[23–25], including controlling differentiation, promoting angiogenesis, regulating ovarian fibrosis, inhibiting apoptosis, enhancing autocrine and paracrine effects, suppressing inflammation, modulating the immune system, and genetic regulation. Thus, in this study, we genetically engineered hucMSCs by plasmids transfection to produce and isolate engineered EVs, which presenting two ligands (PD-L1 and Gal-9) as immune inhibitory molecules. And we explored the therapeutic effects of PD-L1-Gal-9 EVs in the immune micro-environment as well as the function of alleviating the progression of POI.

Cell culture

The National Collection of Authenticated Cell Cultures provided human umbilical cord mesenchymal stem cells (hucMSC) were maintained in DMEM (Gibco) with 1% penicillin & streptomycin and 10% fetal bovine serum (FBS). Cl.LY1 + 2−/9 were also obtained from the same source and cultured in RPMI 1640 medium with 1mM sodium pyruvate, 100 IU/mL interleukin-2 (IL-2), 1% penicillin & streptomycin as well as 10% FBS.

Clinical samples

Fresh peripheral blood was collected from patients diagnosed with POI (n = 10) and non-POI donors at Obstetrics and Gynecology Hospital of Fudan University from 2018 to 2023. Separation of peripheral blood mononuclear cells (PBMCs) was performed by EasySep kit (STEMCELL Technologies, Canada), and analyzed by the flow cytometry.

Plasmid design

PD-L1, Gal-9 and Lamp2b transmembrane sequences were synthesized by Maipu Biotechnology (Nanjing, China) and subcloned into a PLV vector. The GFP sequences, mCherry sequences, HiBiT sequences were synthesized by Maipu Biotechnology (Nanjing, China).

Mice

Female C57BL/6 and B6AF1 (6-week-old) were obtained from The Jackson Laboratory. These mice were housed in animal facility of Experimental Animal Center of Fudan University (Shanghai, China), and used for some replication experiments. The immature female C57BL/6 mice (3-week-old) were purchased for GCs isolation. All mice were housed in specific pathogen-free conditions. All animal studies were performed according to the guidelines for the use and care of live animals and were approved by the Animal Care and Use Committees of Fudan University.

Female B6AF1 mice were immunized s.c. with 100 IU ZP3 peptide (amino acids 330–342, NSSSSQFQIHGPR, Invitrogen, Thermo Fisher Scientific) emulsified in complete Freund’s Adjuvant (CFA, Sigma, Louis, MO, USA) on the first day and for the following 14 consecutive days to induce autoimmune POI model.

Isolation of primary mouse T cells

In vivo treatment of PD-L1-Gal-9 EVs in POI mice

The mouse experiments were carried out in compliance with an animal protocol that received approval from the Institutional Animal Care and Use Committee of Fudan University.

The POI mice were left untreated (control group) or injected with PD-L1-Gal-9 EVs (30 mg/kg), PDL1 EVs (30 mg/kg) or Gal-9 EVs (30 mg/kg) every two days through the tail vein.

The AMH levels of POI mice were monitored every two days until the full 60 days, and then the mice were sacrificed for further analysis.

Immunofluorescent assay

To examine the cellular expression and distribution of EGFP-PD-L1 and mCherry-Gal-9, cells (1×10⁵) were plated into confocal well for 24 h. The collected cells had their culture media removed and underwent once PBS wash. The cells were fixed for 20 minutes with 4% paraformaldehyde and then stained for an extra 20 minutes with DAPI. After three PBS washes, the cells were inspected under a Zeiss confocal microscope. For tissues immunofluorescence assay, the tissues were embedded in paraffin and were cut into 5µm sections. The slides were deparaffinized and rehydrated by xylene and different concentration of ethanol (100%, 95%, 80%, 70% and 50%). After the antigen repairment and blocking. Primary antibody and secondary antibody were added at room temperature. Slides were examined under a microscope (Zeiss).

Preparation of PD-L1-Gal-9 EVs

The hucMSCs were seeded into T225 flask and were transfected with “Plex-Lamp2b-PD-L1” and “Plex-Lamp2b-Gal-9” by Polyethylenimine (PEI, Polysciences) according to the protocol provided by the manufacturer (The ratio of PEI to plasmid was 2:1). Six hours after the transfection, the medium was changed as Opti-MEM (Gibco, USA) with 1% penicillin & streptomycin. After 48 h, the conditioned medium (CM) was collected and centrifuged (300 g for 5 min followed by 2,000 g for 10 min). The supernatant was then filtered through a 0.22 µm filter system. Following this, the supernatant was subjected to additional centrifugation at 100,000 g for 60 minutes, which resulted in the isolation of the engineered EVs overexpressing PD-L1 and Gal-9.

Characterization of engineered PD-L1-Gal-9 EVs

The concentrated EVs were collected in maxirecovery 1.5 ml Eppendorf tubes (Axygene, USA) and quantified using transmission electron microscope (TEM) (FEI Tecnai G2 Spirit Twin, Philips, NL), nanoparticle tracking analysis, using NanoSight NS300 (Malvern, Amesbury, GB). And the markers of EVs including CD63, Alix and Tsg101 were detected by Western blot.

Western blot

EVs and cell lysates were prepared to extract proteins by RIPA buffer. Proteins were separated by SDS–polyacylamide gel electrophoresis gel according to the standard procedure. The polyvinylidene difluoride membrane was incubated with 5% BSA for 1.5 hours and then incubated with the primary antibody at 4°C overnight. After washing with 1 × TBST for three times, the membrane was incubated with HRP conjugated secondary antibodies. The membranes were visualized with enhanced chemiluminescence. EVs were detected by CD63 (ab134045), Alix (ab275377) and Tsg101 (ab133586).

In vitro inhibition of T cells

Cl.LY 1 + 2-/9 cells were seeded into the 12-well plates. At the confluent of 60%, cells were treated with PBS, free EVs, PD-L1 EVs, Gal-9 EVs and PD-L1-Gal-9 EVs for 24 h, separately. FITC-Annexin V apoptosis detection kit was used to test the apoptosis of Cl.LY 1 + 2-/9 T cells via flow cytometry. In addition, primary T cells isolated from the spleen were incubated with engineered EVs for a duration of 48 hours and the percentages of FOXP3 + Treg cells in T cell population was examined through flow cytometry.

Flow cytometry

For cell apoptosis assay, 5 × 10⁵ Cl.LY 1 + 2-/9 cells were collected after EVs treatment. Cell apoptosis was analyzed by flow cytometry using FITC-Annexin V and DAPI as recommended by the manufacturer. The cell apoptosis assay was also conducted in the primary mouse T cells. Data from at least 10⁴ cells were acquired using CytoFLEX and the FlowJo software.

Immune cells from ovaries were digested by by collagenase type IV (Solarbio, China) and purified by filtration and centrifugation, followed by staining for flowcytometric analysis with the following antibodies: anti-CD3 (BD Pharmingen), anti-CD4 (BD Pharmingen), anti-CD8 (BD Pharmingen), anti-IFN-γ (BD Pharmingen), anti-GranB (BD Pharmingen), anti-PD-1 (BD Pharmingen) and anti-PD-1 (BD Pharmingen). Cells were also stained for viability using Fixation/Permeabilization Solution KiT (554714, BD). Acquisition and compensation were conducted on a MACS Quant flow cytometry (Miltenyi biotec, Germany). FlowJo (10.8.1) software was used for data analysis.

ELISA assay

Mouse serum was obtained, and the concentration of AMH, FSH and E2 in serum was detected by

ELISA Kit (ALPCO). Blood samples were collected, allowed to clot for 2 hours at room temperature, and then centrifuged at 2000g for 20 min at 4°C.

Immunoreactivity score (MVIS)

The sections were conducted with immunohistochemical staining to detect the expression of Bax and Bcl-2. The sections were evaluated using a double-blind method. The sections were analyzed by two pathologists using a semiquantitative scoring system, according to the staining intensity graded as 3 (brown), 2 (light brown), 1 (light yellow), or 0 (no color), and the number of positive cells graded as 4 (> 75%), 3 (51 ~ 75%), 2 (25 ~ 50%), 1 (5 ~ 25%), or 0 (< 5%). The total score was the sum of the two grades, which was named the immunoreactivity score (MVIS). The median value of the MVIS for each group was calculated.

Statistical Analysis

The mean values of the data were displayed with the appropriate standard deviations. The sample size (n) for each statistical analysis was provided in the figure legends, where required. The statistical analyses were carried out using Prism 10.2.0. Biological replicates were used in all studies, unless otherwise noted. One-way analysis of variance (ANOVA) was conducted in multiple groups comparison, P-values less than 0.05 were regarded as statistically significant: P < 0.05 was denoted by *, P < 0.01 by **, P < 0.001 by ***, and P < 0.0001 by ****.

The dysregulated T cells subpopulation in periphery in patients with POI and in ovarian microenvironment in mice with POI.

To investigate whether dysregulated immunity occurs in POI, we first determined the periphery T cells subpopulation in patients with POI (N = 10) and control women (N = 10) with the flow cytometry assays. Interestingly, POI patients showed significantly increased percentage of CD8 + T cells (Fig. 1B) as well as the increased percentage of PD-1 positive CD8 + T cells (Fig. 1A) in periphery. Then we determined the ovarian T cells subpopulation in mice with POI (N = 5) and control mice (N = 5). POI mice showed significantly increased percentage of CD8 + T cells (Fig. 1D) as well as the increased percentage of PD-1 positive CD8 + T cells (Fig. 1E) in ovaries. Interestingly, there POI mice showed decreased Foxp3 + Treg cells percentage in ovaries (Fig. 1F), while POI patients showed no significantly difference in Foxp3 + Treg cells percentage with normal women (Fig. 1C).

Preparation and identification of PD-L1-Gal-9 EVs.

Following the preparation of PD-L1-Gal-9 EVs from hucMSCs, we examined the collected EVs using confocal microscopy and confirmed the consistent expression of mCherry and GFP fluorescence, as depicted in Fig. 2F. To further exam the characteristics of PD-L1-Gal-9 EVs, we negatively stained EVs and observed through transmission electron microscopy (TEM) and NTA. The results showed that EVs have an obviously cell membrane vesicle-like structure (Fig. 2C), the size distribution of EVs was roughly ranged from 30 to 300 nm (Fig. 2D). According to the results from Western blot, PD-L1-Gal-9 EVs had stable expression levels of classic markers including CD63, Aix and Tsg101 (Fig. 2E, Supplementary S1). Take together, these data demonstrated that we successfully produced and isolated PD-L1-Gal-9-overexpressing EVs.

The effective binding ability of PD-L1-Gal-9 EVs with T cells in vitro.

To utilize the binding effect of PD-L1-Gal-9 EVs with T cells, Cl.LY1 + 2-/9 cell line was used as a mouse CD4 + T cell line, characterized by high surface expression of Tim-3 and PD-1 proteins (Supplementary Figure S6). Then, we produced EVs expressed fluorescent proteins (PD-L1-mNG EVs, Gal-9-mCherry EVs, PD-L1-mNG-Gal-9-mCherry EVs) (Fig. 3A) and EVs expressed HiBiT (PD-L1-HiBiT EVs, Gal-9-HiBiT EVs, PD-L1-Gal-9-HiBiT EVs) (Fig. 3B), and conducted cell binding assays to examine the interaction between engineered EVs and T cells in vitro. As shown in Supplementary Figure S2, under laser scanning confocal microscopy, PKH-26 labeled PD-L1-Gal-9 EVs favorably bound to the primary ovarian cells. Furthermore, the flow cytometry results showed that Cl.LY1 + 2-/9 cells treated with PD-L1-mNG-Gal-9-mCherry EVs could express both GFP and mCherry (Fig. 3E), while cells treated with PD-L1-mNG EVs expressed GFP (Fig. 3F), cells treated with Gal-9-mCherry EVs expressed mCherry (Fig. 3G). In addition, from the HiBiT assay, cells treated with PD-L1-Gal-9-HiBiT EVs expressed higher HiBiT activity than cells treated with PD-L1-HiBiT EVs or Gal-9-HiBiT EVs (Fig. 3D), while there is no significant difference of HiBiT activity between PD-L1-HiBiT EVs, Gal-9-HiBiT EVs, PD-L1-Gal-9-HiBiT EVs (Fig. 3C). Also, the Western blot results showed that PD-L1 and Gal-9 had high expression levels on engineered EVs (Fig. 3H, Supplementary Figure S3). These findings suggested that PD-L1-Gal-9 EVs exhibited the ability to bind to the T cells.

Biological behavior of PD-L1-Gal-9 EVs

To utilize PD-L1-Gal-9 EVs in modulating T cell biological activity, we detected the effect of PD-L1-Gal-9 EVs on Cl.LY1 + 2-/9 cells (Fig. 4A) and primary mouse T cells (Fig. 4B), separately. As depicted in Fig. 4C, after co-incubation with PD-L1-Gal-9 EVs for 24 hours, the proportion of apoptotic Cl.LY 1 + 2−/9 cells significantly increased (Fig. 4C). Meanwhile, the apoptotic proportion is higher than the apoptotic proportion of cells treated with PD-L1 EVs or Gal-9 EVs (Fig. 4F). Similarly, we found the similar results from the engineered EVs treated primary mouse T cells from the flow cytometry detection (Fig. 4D and Fig. 4G). We speculated that the negative immune checkpoints on T cells were recognized and activated by the PD-L1 and Gal-9 ligand on the EVs. Western blot results showed that EVs significantly inhibited the AKT signaling pathway of T cells and induced apoptosis after incubated with EVs for 24 hours (Fig. 4I, Supplementary Figure S4). Furthermore, the Foxp3 expression in primary mouse T cells decreased after the co-incubation with PD-L1-Gal-9 EVs for 48 hours (Fig. 4E and Fig. 4H).

Therapeutic efficacy of PD-L1-Gal-9 EVs in POI mice

To explore the therapeutic ability of the PD-L1-Gal-9 EVs in the POI process in POI mice in vivo, we used pZP3 to establish POI model mice. Then we injected PD-L1-Gal-9 EVs, PD-L1 EVs, Gal-9 EVs and PBS into POI mice via tail-vein injection (Fig. 5A). As shown in the Fig. 5B and Fig. 5B, PD-L1-Gal-9 EVs treatment significantly restored the ovary weight of POI mice to normal levels. Moreover, compared to the untreated control group, the number of preovulatory, secondary, primary and primordial follicles was significantly lower in the POI group (the numbers of atretic follicles in POI mice after PD-L1-Gal-9 EVs treatment were comparable to untreated control mice (Fig. 5F and Fig. 5G). The engineered PD-L1-Gal-9 EVs could increase the the number of preovulatory, secondary, primary and primordial follicles in the POI mice. In addition, when compared to the control group, the AMH levels (Fig. 5H) and E2 level (Fig. 5J) were significantly lower in the POI group, while the FSH levels were significantly higher (Fig. 5I). The PD-L1-Gal-9 EVs treatment could increase the AMH levels and E2 level, while decrease the FSH level (Fig. 5H, Fig. 5I and Fig. 5J).

Subpopulation and activity of ovaries-infiltrating T cells in the POI mice with engineered EVs treatment

We further investigated whether EVs could protect the residual ovarian cells.

We collected ovaries from the POI mice subjected to various treatments as specified, and then we conducted immunofluorescence analysis to detect the PD-1 expression and TIM-3 expression. In the untreated mice with normal AMH levels, there was a notable absence of PD-1 + cells or TIM-3 + cells within the ovaries, while here was a notable infiltration of PD-1 + cells or TIM-3 + cells within the ovaries in the POI mice (Fig. 6A). These results indicate that abundant infiltrating of immune toxicity T cells to destroy the ovarian cells within the POI mice ovaries. From the flow cytometry analysis, the PD-L1 EVs and Gal-9 EVs treatment could decrease the expression of PD-1 + cells (Fig. 6B and Fig. 6D) or TIM-3 + cells (Fig. 6C and Fig. 6E) to a certain extent compared with the PBS group in POI mice. Encouragingly, the PD-1 + cells or TIM-3 + cells infiltrating in ovaries remarkably decline in PD-L1-Gal-9 EVs treated mice (Fig. 6B and Fig. 6C).

Ovarian cells are acknowledged to be eliminated by activated CD8 + cytotoxic T lymphocytes by the secretion of cytokines such as granzyme B and IFN-γ. Therefore, we assessed the proportion of activated CD8 + T cells that secreting these cytokines within the ovaries. Virtually, flow cytometry results revealed an enhancement in the proportion of IFN-γ + CD8 + T cells (Fig. 6G and Fig. 6H) and GanB + CD8 + T cells (Fig. 6F and Fig. 6I) within the ovaries in POI mice. Notably, the reduction was significantly greater in the PD-L1-Gal-9 EVs treatment group compared to PD-L1 EVs treatment or Gal-9 EVs treatment. This effect results in the low apoptosis effect of GCs in the ovaries (Supplementary Figure S5). These data indicated that PD-L1-Gal-9 EVs had the promising potential to effectively suppress the activity of infiltrating CD8 + T cells within the ovaries, which was correlated with the restoration of ovarian function in POI mice.

In the present study, we designed specific plasmids to transfect hucMSCs, and subsequently produced and isolated endogenous engineered EVs, overexpressing immunosuppressive ligands PD-L1 and Gal-9 on the membrane based on the EVs biogenesis mechanism. In vitro, engineered PD-L1-Gal-9 EVs exhibited the ability to induce inhibitory and apoptotic responses in T cells in vitro and ex vivo. Our in vitro studies showed that PD-L1-Gal-9 EVs were capable of triggering apoptosis in T cells, which contributed to the regulation of immune dysfunction. Furthermore, engineered EVs were able to decrease the proportion of the ovarian infiltrating T cells, as well as to preserve the ovarian cells and to increase the AMH levels. These lead to the postponement of the POI progression. In the presented delicate algorithm, the engineered EVs were produced to bind “auto-reactive T cells” and induce them apoptosis. Consequently, the engineered EVs could protect ovarian cells from destroying and could postpone the progression of POI.

In this study, we select the hucMSCs to produce the EVs. It is reported that transplanting MSCs into POI mice could delay the progression of POI, suggesting the critical role of MSCs in the POI 's treatment[17, 26]. MSCs could play a role in suppressing inflammation, promoting tissue remodeling, and regulating the immune system[27]. Increasing studies reveals that MSCs could promote ovarian function preservation, thereby suppressing the development of POI[17, 28]. Since the critical roles of MSCs in POI therapy field, we leveraged them to produce engineered extracellular vesicles that exhibit overexpression of Gal-9 and PD-L1 through genetic endogenous engineering.

Extracellular vesicles serving as therapeutic agents and drug carriers, present numerous advantages compared to conventional medications[29]. The hucMSCs derived engineered EVs could also avoid the potential immunogenicity and security issues, which might be the best origins of EVs for the ovarian disorders. What’s more, we produced the EVs through the plasmids transfection method and collected the endogenous EVs from the supernatant. Different from the extrusion methods used by some previous studies[30–32], the endogenous method could isolate pure EVs without cells component of cytoplasm (organelles, nucleus and cytoplasmic components). According to the EVs endogenous process[33, 34], after the transfection of the plasmids containing fusing proteins, the EVs sorting domain[21, 35] (transmembrane domain including CD63[36, 37], CD9[38], CD81 and Lamp2b[39, 40], etc.) could link to the proteins of interests and express outside the EVs membrane. This principle could help researchers produce the endogenous ligands on the surface of the EVs and apply this therapeutic approach to diverse diseases. However, in our research findings, the PD-L1-Gal-9 EVs outperformed the PD-L1 EVs or Gal-9 EVs for therapeutic efficacy. This may be because, as depicted in Fig. 3E and 3H, PD-L1-Gal-9 EVs could express both PD-1L and Gal-9, while PD-L1 EVs and Gal-9 EVs could only express a certain endogenous PD-L1 or Gal-9. Furthermore, the effect of PD-L1-Gal-9 EVs are relatively better than the single expression group. Considering all the ligands are linked to the transmembrane domain “Lamp2b” in the outside of EVs membrane, the limited capacity of cellular membrane biology could only support a certain number of Lamp2b and Lamp2b linked ligands. The EVs expressing both PD-L1 and Gal-9 could demonstrate superior effectiveness to inhibit the toxic T cells.

Immune checkpoint blockade therapy has got intensive attention and interest in cancer treatment[41, 42]. Nevertheless, these negative regulatory immune checkpoints also serve a vital function in preserving immune tolerance within healthy tissues and organs. The PD-1/PD-L1 signaling axis also exerts a significant influence on peripheral tissue organ tolerance, and disrupting this immune inhibitory mechanism can trigger the occurrence and progression of various autoimmune diseases[43, 44]. Therefore, harness the inhibitory ligands of immune checkpoints to suppress lymphocyte activity developing a new therapeutic approach for autoimmune diseases[45]. It was reported that the inhibitory immune checkpoint ligands such as PD-L1 and Gal-9 could act as immune suppressive molecules to suppress autoreactive immune cells and subsequently could alleviate the type 1 diabetes[30, 46]. Previous study also showed that the activation of the Gal-9/Tim-3 pathway inhibits Th1 effector function and encourages the participation of Treg cells in the immune evasion process observed in chronic lymphocytic leukemia (CLL)[47].

There are also several limitations of this study. Firstly, engineered EVs specifically express PD-L1 and Gal-9 could not only display with ovarian infiltrating TIM-3 + PD-1 + T cells, but also might inhibit the normal PD-1 + TIM-3 + immune cells around the whole body. Secondly, whether the engineered EVs could influence the function of the bone marrow derived cells in the long run, need to be evaluated. Nonetheless, considering the various advantages of EVs, the engineered EVs are considered potential candidates for disease therapies.

Totally, this study would expand the application of genetic endogenous engineered EVs inevitably. The engineered EVs not only could deliver the proteins of interest (including RNA and proteins), but also could express the ligands of interest on the surface to display with cells in immune system. Furthermore, the delicate design could suppress the POI progression in mice model by promoting the apoptosis of autoreactive T cells and subsequently restore ovarian cells from autoimmune attack. This effective approach obtains promising potential in alleviating and suppressing of the progression of POI.

Ethics approval and consent to participate

All animal procedures were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee at the Fudan University (SYXK2020-0032).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This study was supported by the Project of National Natural Science Foundation of China (No.81771524, No.81471416, No.32060208, No.82260532), General program of Guangxi Natural Science Foundation (No.2019JJA140071).

Authors’ Contributions

G.N.Z. and Y.Y.G. conceived and performed experiments, wrote the manuscript. J.X.D. analyzed the data. G.M.L. provided reagents. K.Q.H. and G.N.Z. provided supervision.

Acknowledgements

We gratefully thank all colleagues in the Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases. This study was supported by the Project of National Natural Science Foundation of China (No.32060208, No.82260532), General program of Guangxi Natural Science Foundation (No.2019JJA140071).

Declaration of interests

The authors declare no competing interests.

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No competing interests reported.

SupplementaryFigureS1.tif
Supplementary Figure S1. The expression of markers in PD-L1-Gal-9 EVs. The original membrane of CD63, Tsg101 and Alix expression in PD-L1-Gal-9 EVs through Western blot.
SupplementaryFigureS2.tif
Supplementary Figure S2. The images of PKH-26 (red) labeled EVs entered FITC (green) labeled actin in primary ovarian cells. The nucleus of cells was stained with DAPI, the cells were stained with FITC-Actin Tracker, the EVs were labeled with PKH-26.
SupplementaryFigureS3.tif
Supplementary Figure S3. The expression of engineered molecules in diverse EVs. The original membrane of PD-L1 and Gal-9 expression in PD-L1-Gal-9 EVs, PD-L1 EVs, Gal-9 EVs and free EVs through Western blot.
SupplementaryFigureS4.tif
Supplementary Figure S4. The expression of apoptotic pathway molecules in diverse EVs treated cells. The original membrane of Akt, Bcl-2 and GAPDH expression in PD-L1-Gal-9 EVs, PD-L1 EVs, Gal-9 EVs and free EVs treated Cl.LY1+2−/9 cells through Western blot.
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Supplementary Figure S6. Flow cytometry evaluation to test the expression of PD-1, and Tim-3 proteins on Cl.LY1+2-/9 cells. Flow cytometry evaluation to test the expression of PD-1, and Tim-3 proteins on Cl.LY1+2-/9 cells.
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Reviews received at journal
26 Aug, 2024
Reviewers agreed at journal
13 Aug, 2024
Reviewers invited by journal
10 Aug, 2024
Editor assigned by journal
06 Aug, 2024
Submission checks completed at journal
06 Aug, 2024
First submitted to journal
03 Aug, 2024

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Bioengineered extracellular vesicles presenting PD-L1 and Gal-9 to ameliorate new-onset primary ovarian insufficiency (POI)

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Abstract

Figures

Introduction

Methods and materials

Cell culture

Clinical samples

Plasmid design

Mice

Isolation of primary mouse T cells

In vivo treatment of PD-L1-Gal-9 EVs in POI mice

Immunofluorescent assay

Preparation of PD-L1-Gal-9 EVs

Characterization of engineered PD-L1-Gal-9 EVs

Western blot

In vitro inhibition of T cells

Flow cytometry

ELISA assay

Immunoreactivity score (MVIS)

Statistical Analysis

Results

Biological behavior of PD-L1-Gal-9 EVs

Therapeutic efficacy of PD-L1-Gal-9 EVs in POI mice

Subpopulation and activity of ovaries-infiltrating T cells in the POI mice with engineered EVs treatment

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1