The HLA-G Immune Checkpoint Plays a Pivotal Role in the Regulation of Immune Response in Autoimmune Diseases

The human G-leukocyte antigen (HLA-G) molecule is a non-classical major histocompatibility complex (MHC) class I molecule. The pertinence of HLA-G has been investigated in numerous studies which have sought to elucidate the relevance of HLA-G in pathologic conditions, such as autoimmune diseases, cancers, and hematologic malignancies. One of the main goals of the current research on HLA-G is to use this molecule in clinical practice, either in diagnostics or as a therapeutic target. Since HLA-G antigens are currently considered as immunomodulatory molecules that are involved in reducing inflammatory and immune responses, in this review, we decided to focus on this group of antigens as potential determinants of progression in autoimmune diseases. This article highlights what we consider as recent pivotal findings on the immunomodulatory function of HLA-G, not only to establish the role of HLA-G in the human body, but also to explain how these proteins mediate the immune response.

The Immunomodulatory Function of Vitamin D, with Particular Reference to SARS-CoV-2

Vaccines are the only way to reduce the morbidity associated to SARS-CoV-2 infection. The appearance of new mutations urges us to increase the effectiveness of vaccines as a complementary alternative. In this context, the use of adjuvant strategies has improved the effectiveness of different vaccines against virus infections such as dengue, influenza, and common cold. Recent reports on patients infected by COVID-19 reveal that low levels of circulating vitamin D correlate with a severe respiratory insufficiency. The immunomodulatory activity of this micronutrient attenuates the synthesis of pro-inflammatory cytokines and at the same time, increases antibody production. Therefore, the present review proposes the use of vitamin D as adjuvant micronutrient to increase the efficacy of vaccines against SARS-CoV-2 infection.

Macrophage-derived Apoptotic Vesicles Regulate Fate Commitment of Mesenchymal Stem Cells via miR155

Background In tissue engineering, mesenchymal stem cells (MSCs) are common seed cells because of abundant sources, strong proliferation ability and immunomodulatory function. Numerous researches have demonstrated that MSC-macrophage crosstalk played a key role in the tissue engineering. Macrophages could regulate the differentiation of MSCs via different molecular mechanisms, including extracellular vesicles. Apoptotic macrophages could generate large amounts of apoptotic vesicles (apoVs), whereas the functions of macrophage-derived apoVs remain largely unknown. There was no research to clarify the role of macrophage-derived apoVs in MSC fate choices. In this study, we aimed to characterize macrophage-derived apoVs, and investigate the roles of macrophage-derived apoVs in the fate commitment of MSCs. Methods We characterized macrophage-derived apoVs, and investigated their role in MSC osteogenesis and adipogenesis in vitro and in vivo. Furthermore, we performed microRNA loss- and gain- of function experiments and western blot to determine the molecular mechanism. Results We found that macrophage-derived apoVs inhibited osteogenesis and promoted adipogenesis in vitro and in vivo. In mechanism, apoVs regulated osteogenesis and adipogenesis of MSCs by delivering microRNA155 (miR155). Conclusions Macrophage-derived apoVs could regulate the osteogenesis and adipogenesis of MSCs through delivering miR155, which provided novel insights for MSC-mediated tissue engineering.

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells. Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs). Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3. Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. Disclosures Battula: Tolero Pharmaceuticals: Research Funding.

SARS-CoV-2 Exposed Mesenchymal Stromal Cell from Congenital Pulmonary Airway Malformations: Transcriptomic Analysis and the Expression of Immunomodulatory Genes

The inflammatory response plays a central role in the complications of congenital pulmonary airway malformations (CPAM) and severe coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the transcriptional changes induced by SARS-CoV-2 exposure in pediatric MSCs derived from pediatric lung (MSCs-lung) and CPAM tissues (MSCs-CPAM) in order to elucidate potential pathways involved in SARS-CoV-2 infection in a condition of exacerbated inflammatory response. MSCs-lung and MSCs-CPAM do not express angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TRMPSS2). SARS-CoV-2 appears to be unable to replicate in MSCs-CPAM and MSCs-lung. MSCs-lung and MSCs-CPAM maintained the expression of stemness markers MSCs-lung show an inflammatory response (IL6, IL1B, CXCL8, and CXCL10), and the activation of Notch3 non-canonical pathway; this route appears silent in MSCs-CPAM, and cytokine genes expression is reduced. Decreased value of p21 in MSCs-lung suggested no cell cycle block, and cells did not undergo apoptosis. MSCs-lung appears to increase genes associated with immunomodulatory function but could contribute to inflammation, while MSCs-CPAM keeps stable or reduce the immunomodulatory receptors expression, but they also reduce their cytokines expression. These data indicated that, independently from their perilesional or cystic origin, the MSCs populations already present in a patient affected with CPAM are not permissive for SARS-CoV-2 entry, and they will not spread the disease in case of infection. Moreover, these MSCs will not undergo apoptosis when they come in contact with SARS-CoV-2; on the contrary, they maintain their staminality profile.

Calcium silicate enhances immunosuppressive function of MSCs to indirectly modulate the polarization of macrophages

Bioactive silicate ceramics (BSCs) have been widely reported to be able to induce bone tissue regeneration, but the underlying mechanisms have not been fully elucidated. Previous studies have reported that ionic products of BSCs can promote bone regeneration by directly simulating osteogenic differentiation of MSCs and modulating the polarization of macrophages to create a favorable inflammation microenvironment for initiating bone regeneration cascades. However, the immunomodulatory ability of MSCs also plays a critical role in bone regeneration but the effects of BSCs on the immunomodulatory ability of MSCs have been rarely investigated. This study aims to investigate the effects of ionic products of BSCs on the immunoregulatory ability of MSCs to further understand the mechanism of BSCs enhancing bone regeneration. Results showed that ionic products of calcium silicate (CS), one of the representative BSCs, could enhance the immunosuppressive function of human bone marrow mesenchymal stem cells (HBMSCs) by up-regulating the expression of immunosuppressive factors in HBMSCs via NF-κB pathway. In addition, CS-activated HBMSCs showed stronger stimulatory effects on M2 polarization of macrophages than CS ionic products. Furthermore, the macrophages educated by CS-activated HBMSCs showed stronger stimulatory effects on the early osteogenic differentiation of HBMSCs than the ones regulated by CS ionic products. These results not only provide further understanding on the mechanism of BSCs enhancing bone regeneration but also suggest that it is critical to consider the effects of biomaterials on the immunomodulatory function of the tissue forming cells when the immunomodulatory function of biomaterials is investigated.

Immunomodulatory Function of Polyvinylpyrrolidone (PVP)-Functionalized Gold Nanoparticles in Vibrio-Stimulated Sea Urchin Immune Cells

We investigated the role of the gold nanoparticles functionalized with polyvinylpyrrolidone (PVP–AuNPs) on the innate immune response against an acute infection caused by Vibrio anguillarum in an in vitro immunological nonmammalian next-generation model, the sea urchin Paracentrotus lividus. To profile the immunomodulatory function of PVP–AuNPs (0.1 μg mL−1) in sea urchin immune cells stimulated by Vibrio (10 μg mL−1) for 3 h, we focused on the baseline immunological state of the donor, and we analysed the topography, cellular metabolism, and expression of human cell surface antigens of the exposed cells, as well as the signalling leading the interaction between PVP–AuNPs and the Vibrio-stimulated cells. PVP–AuNPs are not able to silence the inflammatory signalling (TLR4/p38MAPK/NF-κB signalling) that involves the whole population of P. lividus immune cells exposed to Vibrio. However, our findings emphasise the ability of PVP–AuNPs to stimulate a subset of rare cells (defined here as Group 3) that express CD45 and CD14 antigens on their surface, which are known to be involved in immune cell maturation and macrophage activation in humans. Our evidence on how PVP–AuNPs may stimulate sea urchin immune cells represents an important starting point for planning new research work on the topic.

The Immunoregulatory Effect of Mechanical Stress on Three-dimensional Cultured Mesenchymal Stem Cells After Extensive Expansion

Background: Mesenchymal stem cells (MSCs) have been used to treat immunopathy, and three-dimensional (3D) cultured MSCs show enhanced immunomodulatory property compared with those in two-dimensional (2D) culture. However, both the regulatory mechanisms remain unclear. The aim of the study was to investigate the role of mechanical stress in maintaining the immunomodulatory function of 2D and 3D cultured MSCs.Methods: Umbilical cord mesenchymal stem cells (UC-MSCs) were plated on tissue culture plastic (TCP) as 2D culture and 3D cultured UC-MSCs were seeded in matrigel. Surface markers, clonogenicity, proliferation and immunoregulatory property of UC-MSCs were evaluated. Meanwhile, we established the mouse models of colitis and type 1 diabetes mellitus (T1DM) to reveal the pharmacotherapeutic effects of 3D cultured MSCs in vivo. The effect of changing mechanical stress by modulating Yes-associated protein (YAP) on immunomodulatory function of 2D and 3D cultured UC-MSCs was evaluated by immunofluorescent analysis, real-time quantitative polymerase chain reaction (qPCR) and western blot.Results: We verified early passage UC-MSCs in 2D and 3D cultures exhibited stemness, immunomodulatory property and therapeutic efficacy against immunopathy. However, these characteristics of 2D cultured UC-MSCs were impaired after extensive expansion, whereas 3D culture extended them for several passages by activating YAP. Moreover, prostaglandin E2 (PGE2) could up-regulate YAP to improve the immunomodulatory ability of 2D cultured UC-MSCs after extensive expansion. Conclusions: This work found for the first time that the significance of mechanical stress in maintaining immunoregulatory function of 2D and 3D cultured UC-MSCs, providing a new idea for improving the efficacy of MSCs-based immunotherapy.