IL-10-Engineered Dendritic Cells Modulate Allogeneic CD8+ T Cell Responses.

Tolerogenic dendritic cells (tolDC) play a central role in regulating immune homeostasis and in promoting peripheral tolerance. These features render tolDC a promising tool for cell-based approaches aimed at inducing tolerance in T-cell mediated diseases and in allogeneic transplantation. We developed a protocol to generate genetically engineered human tolDC overexpressing IL-10 (DCIL-10) by means of a bidirectional lentiviral vector (LV) encoding for IL-10. DCIL-10 promote allo-specific T regulatory type 1 (Tr1) cells, modulate allogeneic CD4+ T cell responses in vitro and in vivo, and are stable in a pro-inflammatory milieu. In the present study, we investigated the ability of DCIL-10 to modulate cytotoxic CD8+ T cell responses. We demonstrate that DCIL-10 reduces allogeneic CD8+ T cell proliferation and activation in primary mixed lymphocyte reactions (MLR). Moreover, long-term stimulation with DCIL-10 induces allo-specific anergic CD8+ T cells without signs of exhaustion. DCIL-10-primed CD8+ T cells display limited cytotoxic activity. These findings indicate that stable over-expression of IL-10 in human DC leads to a population of cells able to modulate cytotoxic allogeneic CD8+ T cell responses, overall indicating that DCIL-10 represent a promising cellular product for clinical applications aimed at inducing tolerance after transplantation.

Combined plasma levels of IL-10 and testosterone, but not soluble HLA-G5, predict the risk of death in COVID-19 patients.

BACKGROUND: The identification of biomarkers correlated with coronavirus disease 2019 (COVID-19) outcomes is a relevant need for clinical management. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by elevated interleukin (IL)-6, IL-10, HLA-G, and impaired testosterone production. OBJECTIVES: We aimed at defining the combined impact of sex hormones, interleukin-10, and HLA-G on COVID-19 pathophysiology and their relationship in male patients. MATERIALS AND METHODS: We measured by chemiluminescence immunoassay, electrochemiluminescent assays, and enzyme-linked immunosorbent assay circulating total testosterone, 17ß-estradiol (E2 ), IL-10, and -HLAG5 as well as SARS-CoV-2 S1/S2 Immunoglobulin G from 292 healthy controls and 111 COVID-19 patients with different disease severity at hospital admission, and in 53 COVID-19 patients at 7-month follow-up. RESULTS AND DISCUSSION: We found significantly higher levels of IL-10, HLA-G, and E2 in COVID-19 patients compared to healthy controls and an inverse correlation between IL-10 and testosterone, with IL-10, progressively increasing and testosterone progressively decreasing with disease severity. This correlation was lost at the 7-month follow-up. The risk of death in COVID-19 patients with low testosterone increased in the presence of high IL-10. A negative correlation between SARS-CoV-2 Immunoglobulin G and HLA-G or IL-10 at hospitalization was observed. At the 7-month follow-up, IL-10 and testosterone normalized, and  HLA-G decreased. CONCLUSION: Our findings indicate that combined evaluation of IL-10 and testosterone predicts the risk of death in men with COVID-19 and support the hypothesis that IL-10 fails to suppress excessive inflammation by promoting viral spreading.

Natural history of type 1 diabetes on an immunodysregulatory background with genetic alteration in B-cell activating factor receptor: A case report.

The immunological events leading to type 1 diabetes (T1D) are complex and heterogeneous, underscoring the necessity to study rare cases to improve our understanding. Here, we report the case of a 16-year-old patient who showed glycosuria during a regular checkup. Upon further evaluation, stage 2 T1D, autoimmune thrombocytopenic purpura (AITP), and common variable immunodeficiency (CVID) were diagnosed. The patient underwent low carb diet, losing > 8 kg, and was placed on Ig replacement therapy. Anti-CD20 monoclonal antibody (Rituximab, RTX) was administered 2 years after diagnosis to treat peripheral polyneuropathy, whereas an atypical mycobacteriosis manifested 4 years after diagnosis and was managed with prolonged antibiotic treatment. In the fifth year of monitoring, the patient progressed to insulin dependency despite ZnT8A autoantibody resolution and IA-2A and GADA autoantibody decline. The patient had low T1D genetic risk score (GRS = 0.22817) and absence of human leukocyte antigen (HLA) DR3/DR4-DQ8. Genetic analysis identified the monoallelic mutation H159Y in TNFRSF13C, a gene encoding B-cell activating factor receptor (BAFFR). Significant reduced blood B-cell numbers and BAFFR levels were observed in line with a dysregulation in BAFF-BAFFR signaling. The elevated frequency of PD-1+ dysfunctional Tfh cells composed predominantly by Th1 phenotype was observed at disease onset and during follow-up. This case report describes a patient progressing to T1D on a BAFFR-mediated immunodysregulatory background, suggesting a role of BAFF-BAFFR signaling in islet-specific tolerance and T1D progression.

HLA-G Genotype/Expression/Disease Association Studies: Success, Hurdles, and Perspectives.

The non-classical HLA-G is a well-known immune-modulatory molecule. In physiological condition, HLA-G surface expression is restricted to the maternal-fetal interface and to immune-privileged adult tissues, whereas soluble forms of HLA-G are detectable in various body fluids. HLA-G can be de novo expressed in pathological conditions including tumors, chronic infections, or after allogeneic transplantation. HLA-G exerts positive effects modulating innate and adaptive immune responses and promoting tolerance, or detrimental effects inducing immune escape mechanisms. HLA-G locus, in contrast to classical HLA class I gene, is highly polymorphic in the non-coding 3' untranslated region (UTR) and in the 5' upstream regulatory region (5' URR). Variability in these regions influences HLA-G expression by modifying mRNA stability or allowing posttranscriptional regulation in the case of 3' UTR or by sensing the microenvironment and responding to specific stimuli in the case of HLA-G promoter regions (5' URR). The influence of genetic variations on the expression of HLA-G makes it an attractive biomarker to monitor disease predisposition and progression, or response to therapy. Here, we summarize the current knowledge, efforts, and obstacles to generate a general consensus on the correlation between HLA-G genetic variability, protein expression, and disease predisposition. Moreover, we discuss perspectives for future investigation on HLA-G genotype/expression in association with disease predisposition and progression.

Protocol to assess the suppression of T-cell proliferation by human MDSC.

Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye.

Inhibition of iNKT Cells by the HLA-G-ILT2 Checkpoint and Poor Stimulation by HLA-G-Expressing Tolerogenic DC.

Invariant Natural Killer T (iNKT) cells are a small and distinct population of T cells crucial in immunomodulation. After activation by alpha-GalactosylCeramide (αGC), an exogenic glycolipid antigen, iNKT cells can rapidly release cytokines to enhance specific anti-tumor activity. Several human clinical trials on iNKT cell-based anti-cancer are ongoing, however results are not as striking as in murine models. Given that iNKT-based immunotherapies are dependent mainly on antigen-presenting cells (APC), a human tolerogenic molecule with no murine homolog, such as Human Leucocyte Antigen G (HLA-G), could contribute to this discrepancy. HLA-G is a well-known immune checkpoint molecule involved in fetal-maternal tolerance and in tumor immune escape. HLA-G exerts its immunomodulatory functions through the interaction with immune inhibitory receptors such as ILT2, differentially expressed on immune cell subsets. We hypothesized that HLA-G might inhibit iNKT function directly or by inducing tolerogenic APC leading to iNKT cell anergy, which could impact the results of current clinical trials. Using an ILT2-transduced murine iNKT cell line and human iNKT cells, we demonstrate that iNKT cells are sensitive to HLA-G, which inhibits their cytokine secretion. Furthermore, human HLA-G+ dendritic cells, called DC-10, failed at inducing iNKT cell activation compared to their autologous HLA-G‒ DCs counterparts. Our data show for the first time that the HLA-G/ILT2 ICP is involved in iNKT cell function modulation.

Role of myeloid regulatory cells (MRCs) in maintaining tissue homeostasis and promoting tolerance in autoimmunity, inflammatory disease and transplantation.

Myeloid cells play a pivotal role in regulating innate and adaptive immune responses. In inflammation, autoimmunity, and after transplantation, myeloid cells have contrasting roles: on the one hand they initiate the immune response, promoting activation and expansion of effector T-cells, and on the other, they counter-regulate inflammation, maintain tissue homeostasis, and promote tolerance. The latter activities are mediated by several myeloid cells including polymorphonuclear neutrophils, macrophages, myeloid-derived suppressor cells, and dendritic cells. Since these cells have been associated with immune suppression and tolerance, they will be further referred to as myeloid regulatory cells (MRCs). In recent years, MRCs have emerged as a therapeutic target or have been regarded as a potential cellular therapeutic product for tolerance induction. However, several open questions must be addressed to enable the therapeutic application of MRCs including: how do they function at the site of inflammation, how to best target these cells to modulate their activities, and how to isolate or to generate pure populations for adoptive cell therapies. In this review, we will give an overview of the current knowledge on MRCs in inflammation, autoimmunity, and transplantation. We will discuss current strategies to target MRCs and to exploit their tolerogenic potential as a cell-based therapy.

DHRS9 Is a Stable Marker of Human Regulatory Macrophages.

BACKGROUND: The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs. METHODS: The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry. RESULTS: DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14 monocytes and was further enhanced by IFN-γ treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs. CONCLUSION: DHRS9 is a specific and stable marker of human Mregs.

HLA-G expression on blasts and tolerogenic cells in patients affected by acute myeloid leukemia.

Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4(+) T cells in the peripheral blood of AML patients at diagnosis. Moreover, we explored the possible influence of the 3' untranslated region (3'UTR) of HLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4(+) T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4(+) T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the host's immune system. Further studies on larger populations are required to verify our findings.

The cellular amount of the common gamma-chain influences spontaneous or induced cell proliferation.

Mutations of the IL2RG encoding the common gamma-chain (gamma(c)) lead to the X-linked SCID disease. Gene correction through ex vivo retroviral transduction restored the immunological impairment in the most of treated patients, although lymphoproliferative events occurred in five of them. Even though in two cases it was clearly documented an insertional mutagenesis in LMO2, it is conceivable that gamma(c) could have a role per se in malignant lymphoproliferation. The gamma(c) is a shared cytokine receptor subunit, involved also in growth hormone (GH) receptor signaling. Through short interfering RNA or using X-linked SCID B lymphoblastoid cell lines lacking gamma(c), we demonstrate that self-sufficient growth was strongly dependent on gamma(c) expression. Furthermore, a correlation between gamma(c) amount and the extent of constitutive activation of JAK3 was found. The reduction of gamma(c) protein expression also reduced GH-induced proliferation and STAT5 nuclear translocation in B lymphoblastoid cell lines. Hence, our data demonstrate that gamma(c) plays a remarkable role in either spontaneous or GH-induced cell cycle progression depending on the amount of protein expression, suggesting a potential role as enhancing cofactor in lymphoproliferation.

Functional interaction of common gamma-chain and growth hormone receptor signaling apparatus.

We previously reported on an X-linked SCID (X-SCID) patient, who also had peripheral growth hormone (GH) hyporesponsiveness and abnormalities of the protein phosphorylation events following GH receptor (GHR) stimulation. In the present study, we examined a potential role of common cytokine receptor gamma-chain (gammac) in GHR signaling using EBV-transformed lymphocytes from healthy subjects and gammac-negative X-SCID patients. We demonstrated that the proliferative response to GH stimulation of the B cell lines of gammac-negative patients was impaired despite a comparable cellular expression of GHR molecules to controls. In patients, after GH stimulation, no phosphorylation of STAT5 was observed. In addition, the molecule localization through confocal microscopy revealed that in B cell lines of patients no nuclear translocation of STAT5b following GH stimulation occurred differently from controls. Biochemical analysis of the nuclear extracts of gammac-negative cell lines provided further evidence that the amount of STAT5b and its phosphorylated form did not increase following GH stimulation. In patients, cells reconstituted with wild-type gammac abnormal biochemical and functional events were restored resulting in nuclear translocation of STAT5. Confocal experiments revealed that GHR and gammac were colocalized on the cell membrane. Our study demonstrates the existence of a previously unappreciated relationship between GHR-signaling pathway and gammac, which is required for the activation of STAT5b in B cell lines. These data also confirm that growth failure in X-SCID is primarily related to the genetic alteration of the IL2RG gene.