Recombinant IFN-γ Research Articles

Platelet Ido1 expression is induced during Plasmodium yoelii infection, altering plasma tryptophan metabolites

AbstractPlatelets are immune responsive in many diseases as noted by changes in platelet messenger RNA in conditions such as sepsis, atherosclerosis, COVID-19, and many other inflammatory and infectious etiologies. The malaria causing Plasmodium parasite is a persistent public health threat and significant evidence shows that platelets participate in host responses to infection. Using a mouse model of nonlethal/uncomplicated malaria, Plasmodium yoelii XNL (PyNL)-infected but not control mouse platelets expressed Ido1, a rate limiting enzyme in tryptophan metabolism that increases kynurenine at the expense of serotonin. Interferon-γ (IFN-γ) is a potent inducer of Ido1 and mice treated with recombinant IFN-γ had increased platelet Ido1 and IDO1 activity. PyNL-infected mice treated with anti-IFN-γ antibody had similar platelet Ido1 and metabolic profiles to that of uninfected controls. PyNL-infected mice become thrombocytopenic by day 7 after infection and transfusion of platelets from IFN-γ–treated wild-type mice but not Ido1−/− mice increased the plasma kynurenine-to-tryptophan ratio, indicating that platelets are a source of postinfection IDO1 activity. We generated platelet-specific Ido1 knockout mice to assess the contribution of platelet Ido1 during PyNL infection. Platelet-specific Ido1−/− mice had increased death and evidence of lung thrombi, which were not present in infected wild-type mice. Platelet Ido1 may be a significant contributor to plasma kynurenine in IFN-γ-driven immune processes and the loss of platelets may limit total Ido1, leading to immune and vascular dysfunction.

HLA class-I antigen presentation machinery (APM) alterations mediate immune evasion in lung cancer brain metastases.

e14014 Background: Although ~30% of patients with lung cancer brain metastasis (LBM) achieve intracranial response with immune checkpoint inhibitors (ICI), most have primary resistance, and those who initially respond develop rapid disease progression. The biological factors contributing to this unfavorable clinical scenario are poorly understood and could include distinct immunologic properties of tumors colonizing the brain. Using annotated tumor/patient cohorts, spatially resolved protein analysis, and in vitro models, we investigated the role of HLA class-I APM defects and interferon (IFN)-γ pathway in LBM. Methods: Using multiplex quantitative immunofluorescence, we measured the levels and spatial distribution of HLA class-I APM markers (β2M, tapasin, calreticulin, ERp57, TAP1, and TAP2), IFN-γ signaling (pSTAT1 and IRF-1), and tumor-infiltrating lymphocytes (TILs) (CD8, CD4, and FOXP3) in 56 LBM, 41 primary lung tumors (PLT), and 10 lung-extracranial metastases (ECM) represented in tissue microarrays. A subset of paired LBM/PLT samples from the same patients were also included (n=8). The spatial analysis of the markers was conducted using fluorescence co-localization and single-cell segmentation strategies. Associations between the markers, clinicopathological characteristics, and survival were studied. Protein expression of β2M and IFN-γ-sensitive markers (total STAT1, pSTAT1, IRF1, and PD-L1) before and after treatment with human recombinant IFN-γ was assessed using Western blot in cultured primary lung H2030 adenocarcinoma cells and their in vivo selected brain metastatic counterpart H2030-BrM3. Results: LBM showed significantly lower β2M, TAP2, and tapasin protein levels than PLT. However, LBM also showed higher expression of pSTAT1 and comparable levels of IRF-1 relative to PLT. Similar results were seen in paired LBM/PLT samples, but not in ECM samples. Tumors with low β2M showed reduced CD8+, CD4+, and FOXP3+ TILs. In LBM no significant association between β2M and IFN-γ pathway markers or major clinicopathologic and molecular patient/tumor features were found. LBM samples from patients progressing after radiotherapy and/or chemotherapy showed significantly lower β2M levels. Reduced β2M expression was associated with shorter overall survival in PLT but not in LBM. The brain metastatic H2030-BrM3 cell line showed lower β2M expression both at baseline and after IFN-γ treatment compared with their parental H2030 counterpart. Conclusions: HLA class-I APM downregulation is a prominent feature of LBM, is independent of local adaptive/inducible IFN-γ stimulation, and is associated with unfavorable clinical features. These findings support that HLA class-I APM deficiency plays a major role in immune evasion and immunotherapy resistance in patients with LBM.

Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

PIM kinase inhibitor AZD1208 in conjunction with Th1 cytokines potentiate death of breast cancer cells in vitro while also maximizing suppression of tumor growth in vivo when combined with immunotherapy

PIM kinases are over-expressed by a number of solid malignancies including breast cancer, and are thought to regulate proliferation, survival, and resistance to treatment, making them attractive therapeutic targets. Because PIM kinases sit at the nexus of multiple oncodriver pathways, PIM antagonist drugs are being tested alone and in conjunction with other therapies to optimize outcomes. We therefore sought to test the combination of pharmacological PIM antagonism and Th1-associated immunotherapy. We show that the pan PIM antagonist, AZD1208, when combined in vitro with Th1 cytokines IFN-γ and TNF-α, potentiates metabolic suppression, overall cell death, and expression of apoptotic markers in human breast cancer cell lines of diverse phenotypes (HER-2pos/ERneg, HER-2pos/ERpos and triple-negative). Interestingly, AZD1208 was shown to moderately inhibit IFN-γ secretion by stimulated T lymphocytes of both human and murine origin, suggesting some inherent immunosuppressive activity of the drug. Nonetheless, when multiplexed therapies were tested in a murine model of HER-2pos breast cancer, combinations of HER-2 peptide-pulsed DCs and AZD1208, as well as recombinant IFN-γ plus AZD1208 significantly suppressed tumor outgrowth compared with single-treatment and control groups. These studies suggest that PIM antagonism may combine productively with certain immunotherapies to improve responsiveness.

Elevated NS1 serum levels reduce CD119 expression and CXCL-10 synthesis in patients with dengue hemorrhagic fever.

The intensity of dengue virus (DV) replication and circulating non-structural protein 1 (NS1) levels may promote changes in the human immune response and favor severe forms of infection. We investigated the correlations between NS1 with CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels, and IFN-γ receptor α chain (CD119) expression, and CXCL10 production by peripheral blood mononuclear cells (PBMCs) stimulated with recombinant IFN-γ in DV-infected patients with different clinical forms. Dengue virus NS1, CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels were measured in 152 DV-infected patients with different clinical forms and 20 non-infected individuals (NI) using enzyme-linked immunosorbent assay (ELISA). In addition, we investigated the CXCL-10 production after in vitro IFN-γ stimulation of PBMCs from 48 DV-infected individuals (with different clinical forms of dengue fever) and 20 NI individuals using ELISA, and CD119 expression on CD14+ cells with flow cytometry. Patients with dengue hemorrhagic fever (DHF) had significantly higher NS1, CXCL-8, and CXCL-10 serum levels than those with classic dengue fever (DF). The response of PBMCs to IFN-γ stimulation was lower in patients with DHF than in those with DF or dengue with complications (DWC), with lower CD119 expression and reduced CXCL-10 synthesis. In addition, these alterations are associated with high NS1 serum levels. Patients with DHF reported high NS1 levels, low CD119 expression, and low CXCL-10 synthesis in PBMCs, which may be associated with infection progression and severity.

Specific Features of Pulmonary Tuberculosis Patients Treatment with Interferon-Gamma Depending on the Nature of the Tissue Inflammatory Reaction

The objective: to evaluate effectiveness of treatment of infiltrative pulmonary tuberculosis patients with exudative or productive types of the tissue inflammatory reaction when additional immunotherapy with recombinant IFN-γ is used.Subjects and Methods. 127 infiltrative tuberculosis patients were enrolled in the study, of them 66 had an exudative type of the inflammatory reaction (2 Groups OG-1 and GS-1 were formed), 61 had a proliferative type (2 Groups OG-2 and GS-2 were formed). Patients from OG-1 and OG-2 Groups additionally received different regimens with recombinant interferon-γ.Result: Comprehensive treatment of infiltrative tuberculosis including intramuscular administration (according to a certain regimen) of recombinant IFN-γ contributes to achievement of pronounced positive changes of clinical and radiological symptoms in patients with both exudative and productive types of the tissue inflammatory reaction. In the groups treated with recombinant IFN-γ, restoration of the cellular immune response, cellular cooperation, normalization of the cytokine profile and increased phagocytosis activity were confirmed by the results of laboratory tests.

Human Synovial Mesenchymal Stem Cells Expressed Immunoregulatory Factors IDO and TSG6 in a Context of Arthritis Mediated by Alphaviruses.

Infection by arthritogenic alphaviruses (aavs) can lead to reactive arthritis, which is characterized by inflammation and persistence of the virus; however, its mechanisms remain ill-characterized. Intriguingly, it has been shown that viral persistence still takes place in spite of robust innate and adaptive immune responses, characterized notably by the infiltration of macrophages (sources of TNF-alpha) as well as T/NK cells (sources of IFN-gamma) in the infected joint. Aavs are known to target mesenchymal stem cells (MSCs) in the synovium, and we herein tested the hypothesis that the infection of MSCs may promote the expression of immunoregulators to skew the anti-viral cellular immune responses. We compared the regulated expression via human synovial MSCs of pro-inflammatory mediators (e.g., IL-1β, IL6, CCL2, miR-221-3p) to that of immunoregulators (e.g., IDO, TSG6, GAS6, miR146a-5p). We used human synovial tissue-derived MSCs which were infected with O'Nyong-Nyong alphavirus (ONNV, class II aav) alone, or combined with recombinant human TNF-α or IFN-γ, to mimic the clinical settings. We confirmed via qPCR and immunofluorescence that ONNV infected human synovial tissue-derived MSCs. Interestingly, ONNV alone did not regulate the expression of pro-inflammatory mediators. In contrast, IDO, TSG6, and GAS6 mRNA expression were increased in response to ONNV infection alone, but particularly when combined with both recombinant cytokines. ONNV infection equally decreased miR-146a-5p and miR-221-3p in the untreated cells and abrogated the stimulatory activity of the recombinant TNF-α but not the IFN-gamma. Our study argues for a major immunoregulatory phenotype of MSCs infected with ONNV which may favor virus persistence in the inflamed joint.

KERS-Inspired Nanostructured Mineral Coatings Boost IFN-γ mRNA Therapeutic Index for Antitumor Immunotherapy.

Tumor-associated macrophage (TAM) reprogramming is a promising therapeutic approach for cancer immunotherapy; however, its efficacy remains modest due to the low bioactivity of the recombinant cytokines used for TAM reprogramming. mRNA therapeutics are capable of generating fully functional proteins for various therapeutic purposes but accused for its poor sustainability. Inspired by kinetic energy recovery systems (KERS) in hybrid vehicles, a cytokine efficacy recovery system (CERS) is designed to substantially augment the therapeutic index of mRNA-based tumor immunotherapy via a "capture and stabilize" mechanism exerted by a nanostructured mineral coating carrying therapeutic cytokine mRNA. CERS remarkably recycles nearly 40% expressed cytokines by capturing them onto the mineral coating to extend its therapeutic timeframe, further polarizing the macrophages to strengthen their tumoricidal activity and activate adaptive immunity against tumors. Notably, interferon-γ (IFN-γ) produced by CERS exhibits ≈42-fold higher biological activity than recombinant IFN-γ, remarkably decreasing the required IFN-γ dosage for TAM reprogramming. In tumor-bearing mice, IFN-γ cmRNA@CERS effectively polarizes TAMs to inhibit osteosarcoma progression. When combined with the PD-L1 monoclonal antibody, IFN-γ cmRNA@CERS significantly boosts antitumor immune responses, and substantially prevents malignant lung metastases. Thus, CERS-mediated mRNA delivery represents a promising strategy to boost antitumor immunity for tumor treatment.

Interferon-γ mediating overexpression of polymeric immunoglobulin receptor in grass carp (Ctenopharyngodon idellus) liver cells

The polymeric immunoglobulin receptor (pIgR) have a vital function in transcytosis of polymeric immunoglobulins in order to defense against invading microorganisms, however, the regulation pathway of pIgR expression in teleosts remains unclear. In this investigation, to examine if the cytokine IFN-γ affected the expression of pIgR, the recombinant proteins of IFN-γ of grass carp was first prepared, after validating that natural pIgR expressed on grass carp (Ctenopharyngodon idellus) hepatocytes (L8824), the L8824 cells were supplemented by different recombinant IFN-γ concentrations at various times, the outcomes revealed a significant dose- and time-dependent increase in pIgR expressions at the gene and secretion component (SC) proteins levels. The levels of pIgR mRNA was measured increasing at 9 h, and increasing most significant during the 9–12 h period, the growth of SC was delayed until 24 h after IFN-γ stimulation. Moreover, protein synthesis inhibitors cycloheximide (CHX) was used to study on whether IFN-γ regulated pIgR expressions through a protein synthesis dependent pathway. Upon inhibitors CHX treatment, the expression of pIgR mRNA were inhibited significantly, and CHX treatment at any time during the first 9 h period demolished the growth in pIgR mRNA that was promoted by IFN-γ, suggesting that IFN-γ is required for the stimulation of pIgR mRNA, which needs de novo protein synthesis. All these outcomes revealed that IFN-γ could upregulate pIgR gene expression, and production of SC, and this IFN-γ stimulated pIgR expression through a protein synthesis dependent pathway, which provided evidences for IFN-γ serves as a regulator for the expression of pIgR, as well as our current knowledge of the expression of pIgR in teleost fish has been improved as a result.

Mendelian susceptibility to mycobacterial diseases: State of the puzzle.

The constant progress of genomics and the establishment of new functional tests have paved the way for identifying monogenic defects conferring a selective predisposition to infections by certain microbes as a new type of inborn errors of immunity (IEIs). Mendelian susceptibility to mycobacterial diseases (MSMD) is the most characterized of these IEIs, with 36 different disorders found in 20 distinct genes (IFNGR1, IFNGR2, IFNG, IL12RB1, IL12RB2, IL23R, IL12B, ISG15, USP18, ZNFX1, TBX21, STAT1, TYK2, IRF8, IRF1, CYBB, JAK1, RORC, NEMO, and SPPL2A) over the last 20 years. MSMD confers a selective susceptibility to infections with weakly virulent mycobacteria, including the M. bovis Bacille Calmette-Guerin (BCG) vaccines and various environmental mycobacteria in patients, primarily children, without classical immune defects. These patients may also present severe forms of tuberculosis, and about half of them might develop non-typhoidal salmonellosis. In some cases, patients also suffer from chronic mucocutaneous candidiasis (CMC), while in others, patients also present severe viral, parasitic, fungal, and/or bacterial diseases. Despite this clinical and genetic heterogeneity, almost all genetic etiologies of MSMD alter the interferon-gamma (IFN-γ)- mediated immunity by impairing or abolishing IFN-γ production or the response to this cytokine. It was proven that the human IFN-γ level is a quantitative trait that defines the outcome of mycobacterial infection. The study of these monogenic defects contributes to understanding the molecular mechanism of mycobacterial diseases in humans and to the development of new diagnostic and therapeutic approaches to improve care and prognosis. For example, MSMD patients with impaired production of IFN-γ may benefit from injections of human recombinant IFN-γ, while for patients with abolished response to this cytokine, hematopoietic stem cell transplantation (HSCT) and promising gene therapy are the only current therapeutic options. These discoveries also bridge the gap between simple Mendelian inheritance and complex human genetics.

T cell-Mediated Development of Stromal Fibroblasts with an Immune-Enhancing Chemokine Profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAF) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA sequencing of CAFs from mouse pancreatic adenocarcinomas identified a subpopulation of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune-suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

Analysis of intestinal epithelial cell responses to IFN-γ during Cryptosporidiuminfection

Abstract Cryptosporidium species are intracellular parasites that infect intestinal epithelial cells (IEC) and the rapid cycle of growth, cell lysis and reinfection every 12 hours can cause severe enteric disease in young or immunocompromised patients. Interferon-γ (IFN-γ) has a crucial role in protective immunity and mice with an IEC-specific deletion of the IFN-γ receptor (IFN-γR ΔIEC) are highly susceptible to infection. However, the dynamics of IFN-γ signalling to IEC and how this leads to parasite control remain poorly understood. Based on the use of a reporter mouse for IFN signalling in vivo, while macrophages showed a transient response to treatment with IFN-γ, IEC exhibited a sustained response to IFN-γ that peaks at 24h. Indeed, the treatment of mice with recombinant IFN-γ transiently limits parasite shedding but takes 24h to show protective activity. Single-cell RNA sequencing of enterocytes from naïve and infected mice identified upregulation of several IFN-γ-inducible effectors but highlighted that induction of these genes was comparable in infected and bystander IEC. Together, these results suggest that while Cryptosporidium-infected cells are responsive to IFN-γ signalling, the protective effects of IFN-γ may be mediated through activation of uninfected bystander enterocytes. These studies provide new insights into the dynamics and function of IFN-γ signalling on IEC that are broadly relevant to other IEC-restricted pathogens. Supported by grants from NIH (R01 AI148249) and post-doctoral fellowships from the Canadian Institutes for Health Research and Fonds de Recherche du Québec – Santé.

A Mouse Monoclonal Antibody Against Human IFN-γ and its Characters

Background and Aims: A monoclonal antibody (mAb) can unambiguously identify, quantify, and purify an antigen or particular epitope at a large scale. The superiority of these antibodies lies in their specificity for the antigenic determinant. So, this study aims to prepare mouse mAb-secreting hybridoma against human gamma interferon (IFN-γ) and determine the produced antibody's characters.
 Materials and Methods: Mouse splenic B lymphocytes immunized with recombinant human IFN-γ were fused with mouse SP2/0 cells. The hybridized cells were selected by hypoxanthine-aminopterin-thymidine and hypoxanthine-thymidine media to obtain monoclonal antibody-producing hybridoma cells. Finally, indirect enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot were used to confirm the creation of antibody-secreting hybridoma cells.
 Results: mAb against IFN-γ were produced by fusing SP2/0 mouse non-secretory myeloma cell line with the spleen cells of immunized mice. This antibody's indirect ELISA optical density was 2.055 on average, and the desired antibody bands were confirmed in SDS-PAGE compared to Septicol® (commercial antibody). Also, in the western blot, the desired antibody could bind to the antigen. IFN-γ transferred on nitrocellulose membrane. In ELISA and western blot tests, anti-mouse IgG conjugated antibodies were used; therefore, the mAb IgG isotype was taken into consideration.
 Conclusion: In this study, a mouse mAb was obtained by immunization of Balb/C mice and fusion of spleen cells of these mice with the SP2/0 cells, which can specifically bind to recombinant human IFN-γ and can be used to detect IFN-γ secretion in all types of intracellular infections, including latent tuberculosis.

Clinical efficacy of recombinant canine interferon-gamma therapy in dogs with cutaneous epitheliotropic T-cell lymphoma.

The antitumour effects of interferon-gamma (IFN-γ) in humans with cutaneous epitheliotropic T-cell lymphoma (CETCL) have been described; however, the efficacy of IFN-γ in dogs has not been investigated. The aim of this study was to evaluate the efficacy of recombinant canine IFN-γ (rCaIFN-γ) therapy in dogs with CETCL. Twenty dogs with CETCL recruited from seven veterinary clinics were enrolled in the study. Fifteen dogs were treated with rCaIFN-γ, and five control dogs were treated with prednisolone. We evaluated survival time, skin lesions (erythema, nodules, ulcers and bleeding), pruritus and general condition (sleep, appetite and body weight). In the rCaIFN-γ group, a questionnaire regarding the therapy was administered to owners after the dogs died. No significant differences existed in the median survival time between the rCaIFN-γ and control groups (log-rank test: p=0.2761, Wilcoxon's rank sum test: p=0.4444). However, there were significant differences in ulcer, bleeding, pruritus, sleep, appetite and body weight between the groups (Wilcoxon-Mann-Whitney U-test: p=0.0023, p=0.0058, p=0.0005, p=0.0191, p=0.0306 and p=0.0306, respectively). Two (40%) of five dogs were euthanised in the control group, compared with none in the rCaIFN-γ group. Fourteen questionnaires were collected, and owners reported that they were satisfied with the rCaIFN-γ treatment. Although the median survival time was not prolonged, rCaIFN-γ could be helpful in maintaining good quality of life for dogs with CETCL.

Production of Soluble Human Gamma Interferon in the <i>Escherichia coli</i> Expression System with a Decrease in Cultivation Temperature

A recombinant strain producing human gamma interferon (IFN-γ) E. coli BL 21/pET-IFN-γ was constructed, providing a high level of its expression. A method has been developed for obtaining a soluble form of recombinant IFN-γ, consisting of the processes of producing a biomass of a producer strain containing a target protein in an amount of 32–37% of the total content of cellular proteins, protein isolation and purification. The purification process included the stages of disintegration, clarification of the cell lysate, chromatographic purification and dialysis. The developed method makes it possible to obtain from 1 g of wet biomass up to 5 mg of the drug with a purity of at least 95% and high specific (antiviral) activity.

Mendelian Susceptibility to Mycobacterial Disease (MSMD): Clinical, Immunological, and Genetic Features of 22 Patients from 15 Moroccan Kindreds.

The first molecular evidence of a monogenic predisposition to mycobacteria came from the study of Mendelian susceptibility to mycobacterial disease (MSMD). We aimed to study this Mendelian susceptibility to mycobacterial diseases in Moroccan kindreds through clinical, immunological, and genetic analysis. Patients presented with clinical features of MSMD were recruited into this study. We used whole blood samples from patients and age-matched healthy controls. To measure IL-12 and IFN-γ production, samples were activated by BCG plus recombinant human IFN-γ or recombinant human IL-12. Immunological assessments and genetic analysis were also done for patients and their relatives. Our study involved 22 cases from 15 unrelated Moroccan kindreds. The average age at diagnosis is 4years. Fourteen patients (64%) were born to consanguineous parents. All patients were vaccinated with the BCG vaccine, and twelve of them (55%) developed locoregional or disseminated BCG infections. The other symptomatic patients had severe tuberculosis and/or recurrent salmonellosis. Genetic mutations were identified on the following genes: IL12RB1 in 8 patients, STAT1 in 7 patients; SPPL2A, IFNGR1, and TYK2 in two patients each; and TBX21 in one patient, with different modes of inheritance. All identified mutations/variants altered production or response to IFN-γ or both. Severe forms of tuberculosis and complications of BCG vaccination may imply a genetic predisposition present in the Moroccan population. In the presence of these infections, systematic genetic studies became necessary. BCG vaccination is contraindicated in MSMD patients and should be delayed in newborn siblings until the exclusion of a genetic predisposition to mycobacteria.

Individual immune cell and cytokine profiles determine platelet-rich plasma composition

ObjectivePlatelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes.DesignFor each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed.ResultsAll three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content.ConclusionsIndividuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual’s immune profile and the concentration method appear to impact the final PRP product.Trial registrationThis study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules

BackgroundTripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PurposeTo identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. Study DesignIn vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. MethodsFlow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. ResultsInterferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 μg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. ConclusionThe TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects

CD4+ T cells drive an inflammatory, TNF-α/IFN-rich tumor microenvironment responsive to chemotherapy

While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4+ Tcells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4+ Tcell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.

IFNγ directly counteracts imatinib-induced apoptosis of primary human CD34+ CML stem/progenitor cells potentially through the upregulation of multiple key survival factors

ABSTRACT Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.