INF-γ Release Research Articles

Abstract LB040: MACO-355, a unique pan-LILR monoclonal antibody for cancer therapy, re-programs and stimulates immuno-suppressive macrophages in a novel ligand-binding blocking independent manner

Abstract Macrophages populate most solid tumors in large numbers and limit effective anti-tumoral immune responses. The leukocyte immunoglobulin-like receptors (LILR) such as e.g. LILRB1, LILRB2, and others are expressed on tumor associated macrophages. Despite sharing multiple ligands such as major histocompatibility complex class I G (HLA-G), these receptors regulate different macrophage functions such as phagocytosis or cytokine release. To date, therapeutic approaches to targeting LILRs have concentrated on blocking receptor-ligand interactions to relieve ligand-mediated immune suppression. MACO-355 is a fully human antibody binding multiple LILRA (activating) and LILRB (inhibiting) family members including LILRB1, LILRB2, LILRB3, LILRA4 and LILRA6, that does not neutralize ligand (HLA-G, HLA-A, HLA-E) binding to either LILRB1 or to LILRB2. Nevertheless, MACO-355 mediates macrophage reprogramming even under strong immune suppressive conditions, as measured e.g. by stimulation of the TLR agonist induced pro-inflammatory cytokine release in CSF1, TGFβ, IL-10, and IL-4 treated cells. Concordantly, stimulation of immuno-suppressive macrophages with MACO-355 also resulted in significantly higher levels of T cell expansion, proliferation, and INFγ release in a co-culture suppression assay compared to ligand blocking antibodies. Additionally, MACO-355 enhances macrophage phagocytosis irrespective of tumor cell HLA-G ligand status and NK tumor cell killing. All these in vitro activities translate into in vivo activity in A375 melanoma CDX tumor model comparable to that of anti-PD-1 and anti-PD-L1 Abs in humanized mice. Safety studies showed that despite its potent immune stimulatory activity, MACO-355 does not lead to spurious pro-inflammatory cytokine production in in vitro cytokine storm assays. Molecular studies showed MACO-355 binds a novel epitope distant from a ligand binding pocket on LILRs and requires both bi-valent binding and Fc effector function for full activity. This results in pronounced modulation of intracellular kinase signaling. MACO-355 is a unique receptor-ligand non-blocking pan-LILR antibody highly potent in pro-inflammatory reprogramming of immuno-suppressive macrophages, both in vitro and in vivo. This appears to occur via a novel mode of action in which MACO-355 clusters multiple LILRs with Fc-receptors into unconventional signaling hubs. Altogether the data support the future clinical evaluation of MACO-355 as a cancer therapeutic. Citation Format: Krzysztof B. Wicher, Moritz Haneklaus, Martha Lopez-Yrigoyen, Alicia Poindron, Carmen Rodriguez-Seoane, Maikel Fransen, Chantell Payton, Stephane M. Guillame, Luca Cassetta, Stephen Myatt, Carola Ries. MACO-355, a unique pan-LILR monoclonal antibody for cancer therapy, re-programs and stimulates immuno-suppressive macrophages in a novel ligand-binding blocking independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB040.

Abstract 2526: Exploratory study of predicting systemic therapy response by IGRA in advanced stage NSCLC patients

Abstract Purpose: In this study, we aim to prospectively evaluate how patients’ pretreatment PSIG correlated with chemotherapy, immune checkpoint inhibitors (ICI) and epidermal growth factor receptor (EGFR) Tyrosine Kinasae Inhibior (TKI). Also, to assess the change of PSIG during anti-cancer treatment and the relation with clinical condition. Materials and Methods: Newly diagnosed advanced stage non-small cell lung cancer patients were enrolled. The inclusion criteria included pathology or cytology-proved stage IV and non-operable stage III non-small cell lung cancer, treatment-naïve, and planned to receive chemotherapy, EGFR-TKI therapy. Or, planned to receive any line of singe agent immunotherapy. We planned to enroll 30 patients in each treatment group (chemotherapy, immunotherapy, EGFR TKI therapy) with and additional control group of stage I NSCLC.Blood sample from each enrolled patient was obtained for INF-γ release assay (IGRA) within 3 days before the initiation of systemic therapy and 8-12 weeks after treatment. The IGRA was performed with QuantiFERON-TB In-Tube (QFT-GIT; Qiagen, Germany) according to the manufacture’s instruction. The amount of INF-γ produced in response to PHA is determined by the INF-γ level in the PHA-coated tube minus saline-coated tube, and is termed PHA-stimulated INF-γ (PSIG) level. Patients with PSIG level above 7.06IU were defined had high PSIG level according to our previous study. we also performed our in house INF-γ level by ELISA. Tumor response was assessed mainly by chest CT and brain MRI (if available) according to Response Evaluation Criteria in Solid Tumors (RECST). Results: Total 116 patients were enrolled. Demographic distribution was summarized in Table 1. Thirty-five of 116 (30.2%) patients had latent TB infection, while 12 patients (10.3%) had indeterminate OFT test, all due to low mitogen-stimulated INF-γ level. 84 patients had been obtained post-treatment blood test, and the box-plot of in-house INF-γ level were shown in Figure1. For stage I control group, INF-γ level seemed higher after surgery; for chemotherapy and immunotherapy, the INF-γ level also seemed increase among responders (who achieved disease control after treatment). There were no statistically difference of disease control rate between patients with high PSIG level and low PSIG level. Overall survival also showed no statistically difference between 2 PSIV level patient group among advanced stage NSCLC patients. Conclusions:There were no statistically different of disease control rate in PSIG high and low group. The increased trend of INF-γ level after surgery, immunotherapy and chemotherapy may suggested improving immune function after treatment even in the very early disease stage. Citation Format: Hsu Ching Huang, Chi-Lu Chiang, Yung-Hung Luo, Yuh-Min Chen, Chao-Hua Chiu. Exploratory study of predicting systemic therapy response by IGRA in advanced stage NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2526.

The immune and metabolic treatment approach of using testosterone on mice models of liver injury.

Background: Natural killer (NK) cells showed an anti-fibrotic effect; however, their function is thought to be impaired in advanced liver injury. In the current study, we aimed to assess the immune and metabolic impact of testosterone on mice models of liver injury. Methods: Carbon-tetrachloride induced liver fibrosis male mice models was i.p injected for 2 weeks (acute) and 4 weeks (chronic) (n = 36). Testosterone (4mg/kg mouse body weight) was injected i.p. following the first week of the acute model of CCl4 and following the second week of the chronic model of CCl4. At the end of the experiments, mice were sacrificed, and serum was collected for assessing liver enzymes of ALT and AST, as well as inflammatory markers of IL-6, metabolic makers of C-peptide levels, and lipid and glucose profiles. Livers were harvested and used for histological assessments for inflammation and fibrosis. Fibrosis profiles from liver extracts, αSMA and Collagen III, were assessed by RT-PCR. Moreover, liver tissue-resident NK cells were isolated and evaluated for their activity by assessing INF-γ and IL-6 receptors using ELISA and flow cytometry, respectively. Results: Serum ALT, AST, and IL-6, as well as metabolic assessments of cholesterol, triglyceride, C-peptide, fasting blood sugar, and fibrotic profiles, were linearly correlated with disease progressions. Histological characterization of the liver was worsened in the chronic model of liver injury. Testosterone-treated mice exhibit a significant reduction in collagen depositions with less dense fibrosis tissue associated with reduced liver injury enzymes and metabolic markers in both the acute and chronic CCl4 mice models in favor of the latter one (p < 0.05). Moreover, testosterone treatments displayed a significant decrease in serum IL-6 of 2.4-fold (p = 0.0001) and 2.3-fold (p = 0.0003) in the acute and chronic models, respectively (p = 0.002), and data showed an increase in INF-γ release from NK associated with a reduction in their IL-6 receptor expressions (p < 0.05). Conclusion: Our results indicated effects of testosterone on mediating a decreased expressions of NK IL-6 receptors and consequently inducing their activation; which in part, could explain the amelioration of liver injury. Our data suggest an anti-inflammatory and anti-fibrotic treatment approach of using testosterone for delaying disease progressions.

Design of a novel multi-epitope vaccine candidate against Chlamydia trachomatis using structural and nonstructural proteins: an immunoinformatics study

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium which causes eye and sexually transmitted infections. During pregnancy, the bacterium is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to infertility. The aim of our study was design of a multi-epitope vaccine (MEV) candidate against C. trachomatis. After protein sequence adoption from the NCBI, potential epitopes toxicity, antigenicity, allergenicity, MHC-I and MHC-II binding, cytotoxic T lymphocytes (CTLs), Helper T lymphocytes (HTLs) and interferon-γ (IFN-γ)- induction were predicted. The adopted epitopes were fused together using appropriate linkers. In the next step, the MEV structural mapping and characterization, three-dimensional (3D) structure homology modeling and refinement were also performed. The MEV candidate interaction with the toll-like receptor 4 (TLR4) was also docked. The immune responses simulation was assessed using the C-IMMSIM server. Molecular dynamic (MD) simulation verified the structural stability of the TLR4-MEV complex. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach demonstrated the MEV high affinity of binding to the TLR4, MHC-I and MHC-II. The MEV construct was also stable and water soluble and had enough antigenicity and lacked allergenicity with stimulation of T cells and B cells and INF-γ release. The immune simulation confirmed acceptable responses of both the humoral and cellular arms. It is proposed that in vitro and in vivo studies are needed to evaluate the findings of this study. Communicated by Ramaswamy H. Sarma

Humoral and Cellular Response and Associated Variables Nine Months following BNT162b2 Vaccination in Healthcare Workers.

In this study, we aimed to illustrate the trajectory of humoral and cellular immunity nine months after primary vaccination with the BNT162b2 mRNA vaccine among 189 healthcare workers (HCWs). Additionally, we endeavored to identify correlations between immunity parameters and a number of common variables and comorbidities. A total of 189 healthcare workers (HCWs), vaccinated against COVID-19, were finally included in the study. All of the subjects had received two doses of the BNT162b2 vaccine; had undergone antibody tests one, four and nine months post-vaccination; and had completed a medical questionnaire. Further samples taken at nine months were tested for cellular immunity. No participants had evidence of COVID-19 infection pre- or post-vaccination. An anti-S1 receptor binding domain (RBD) antibody assay was used to assess humoral response, and cellular immunity was estimated with an INF-γ release assay (IGRA). Statistical analysis was performed using STATA. We report a statistically significant antibody drop over time. Being above the age of 40 or a smoker reduces the rise of antibodies by 37% and 28%, respectively. More than half of the participants did not demonstrate T-cell activation at nine months. Female gender and antibody levels at four months predispose detection of cellular immunity at nine months post-immunization. This study furthers the qualitative, quantitative, and temporal understanding of the immune response to the BNT162b2 mRNA vaccine and the effect of correlated factors.

An Observational Study on the Humoral and Cellular Immune Response to SARS-CoV-2 mRNA Vaccination in Multiple Sclerosis and Other Autoimmune Neurological Disorders Treated With Anti-CD20 Therapies

Objective To assess adaptive immunity to SARS-CoV-2 in anti-CD20 treated individuals with mRNA vaccination. Background Anti-CD20 therapies attenuate humoral responses to vaccines. However, their effect on T cell responses is less clear. We examined B and T cell responses following COVID-19 vaccination in patients receiving anti-CD20 therapy for multiple sclerosis (MS) and other autoimmune inflammatory neurologic diseases (AINDs, e.g., autoimmune encephalitis, stiff person syndrome, etc.). Design/Methods MS and AIND patients on anti-CD20 therapies were prospectively enrolled for longitudinal analysis of antibody and T cell responses after a 3rd COVID-19 vaccination. Serum antibodies against the receptor-binding domain of the S1 spike protein (RBD-S1 IgG), neutralizing antibodies, and SARS-CoV-2 CD8 T cell responses, using activation-induced markers (AIM) and INF-γ release assays (EUROIMMUN, Germany), were measured at various time points including pre-vaccination, post initial vaccination series, and 4 and 12 weeks after 3rd dose. Results Thirty-four MS and AIND participants are enrolled. Results for these patients (mean age 52 years-old, 79% female, 21 Pfizer, 13 Moderna) demonstrated attenuated RBD IgG antibody responses. However, a robust CD8 T cell response was observed, following a two-dose series, compared to non-immunosuppressed, age-matched vaccinated controls or unvaccinated with severe SARS-CoV-2 infection (p = 0.01). T cell response was sustained long-term (&gt;12 weeks post 3rd dose) in all 11 anti-CD20 patients analyzed thus far. Collections are completed for all participants at 12 weeks and analysis to be completed by 05/15/22. Further analysis includes correlation of the INF- γ release assay compared to RBD-CD8 T cell response detected by AIM assay. Conclusions Results suggest that patients treated with anti-CD20 therapy generate a robust CD8 T cell response to SARS-CoV-2 mRNA after three doses but remain with attenuated humoral immune responses. Our observational study will provide important data to guide vaccine management in patients on or anticipating anti-CD20 therapy.

Abstract 1499: Targeting Ras mutation with TCR therapy

Abstract Ras oncogenes (K/N/HRAS) are frequently mutated in 20% of all human cancers. Globally, approximately 3.4 million new cancer cases are diagnosed annually carrying Ras mutations. There is a large unmet medical need for treating cancers carrying Ras mutations. KRAS mutations account for around 80% of total Ras mutations. Codon 12 and 13 are most frequently mutated in KRAS oncogene. RasG12V and RasG13D represent approximately 22-58% cases of KRAS mutations. RAS mutations are immunogenic and can be recognized by T cell receptors in HLA restricted manner (Steven A. Rosenberg, 2016). Here we report identification of two human TCRs specifically recognizing RasG12V and RasG13D mutations isolated from tumor infiltrating lymphocytes of colorectal cancer patients. Both TCRs show high avidity to RasG12V and RasG13D mutations presented by MHCII alleles widely distributed in human population. Engineered B13.14.1 TCR-T and B8.2.4 TCR-T recognize RASG12V and RASG13D presented on HLA matched tumor cell line and induce strong INFγ release. Furthermore, potent yet specific tumor cell lysis was observed in multiple RASG12V and RASG13D tumor cell lines. Our RasG12V and RasG13D targeted TCR-T therapy could hold great potential for treatment of oncogenic Ras mutation driven cancers. Citation Format: Nan Mou, Yue Yu, Jijun Yuan. Targeting Ras mutation with TCR therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1499.

Morphine-3-glucuronide upregulates PD-L1 expression via TLR4 and promotes the immune escape of non-small cell lung cancer.

Objective:Patients with cancer pain are highly dependent on morphine analgesia, but studies have shown a negative correlation between morphine demand and patient outcomes. The long-term use of morphine may result in abnormally elevated serum morphine-3-glucuronide (M3G) levels. Hence, the effects of M3G on tumor progression are worth studying.Methods:The effects of M3G on PD-L1 expressions in human non-small cell lung cancer (NSCLC) cell lines were first evaluated. Activation of TLR4 downstream pathways after M3G treatment was then determined by Western blot. The effects of M3G on human cytotoxic T lymphocytes (CTL) cytotoxicity and INF-γ release was also detected. Finally, the LLC murine lung adenocarcinoma cell line were used to establish a murine lung cancer model, and the effects of M3G on tumor growth and metastasis were determined.Results:M3G promoted the expressions of PD-L1 in the A549 and H1299 cell lines in a TLR4-dependent manner (P < 0.05). M3G activated the PI3K and the NFκB signaling pathways, and this effect was antagonized by a TLR4 pathway inhibitor. A PI3K pathway inhibitor reversed the M3G-mediated PD-L1 upregulation. M3G inhibited the cytotoxicity of CTL on A549 cells and decreased the level of INF-γ. Repeated M3G intraperitoneal injections promoted LLC tumor growth and lung metastasis through the upregulation of tumor expressed PD-L1 and the reduction of CTL in the tumor microenvironment.Conclusions:M3G specifically activated TLR4 in NSCLC cells and upregulated PD-L1 expression through the PI3K signaling pathway, thereby inhibiting CTL cytotoxicity and finally promoting tumor immune escape.

Treatment with Bifidobacteria can suppress Aβ accumulation and neuroinflammation in APP/PS1 mice.

BackgroundAlzheimer’s disease (AD), being a complex disorder, is affected either by genetic or environmental factors or both. It is observed that there is an excessive accumulation of amyloid β (Aβ) in the extracellular space of the brain. AD is the first neurodegenerative disease in the elderly, and so far there is no effective treatment. In recent years, many studies have reported that Alzheimer’s disease has a relationship with gut microflora, indicating that regulating gut microbiota could offer therapeutic intervention for AD. This study explored the effect Bifidobacteria has in averting AD.MethodsWT and APP/PS1 mice were used for the experiments. The mice were randomly assigned to four groups: WT group, WT + Bi group, AD group (APP/PS1 mouse) and AD + Bi group (Bifidobacteria-treated APP/PS1 mouse). Treatment with Bifidobacteria lasted for 6 months and mice were prepared for immunohistochemistry, immunofluorescence, Thioflavin S staining, Western blotting, PCR and Elisa quantitative assay.ResultsThe results show that after 6 months of treatment with Bifidobacteria signiis to be lesficantly reduces Aβ deposition in cortex and hippocampus of AD mice. The level of insoluble Aβ in the hippocampus and cortex of AD+Bi mice was decreased compared with AD mice. Meanwhile, a significant decrease in the level of soluble Aβ in the cortex of AD+Bi mice but not in the hippocampus was observed. The activation of microglia and the release of inflammatory factors were also determined in this study. From the results, Bifidobacteria inhibited microglial activation and reduced IL-1β, TNF-α, IL-4, IL-6 and INF-γ release. Altogether, these results implied that Bifidobacteria can alleviate the pathological changes of AD through various effects.

Abstract 3265: Anti-GITR agonist antibody for cancer immunotherapy

Abstract Glucocorticoid-induced TNFR-related (GITR) is a gene coding for a member of the TNF receptor superfamily. GITR is expressed at low levels on resting responder T lymphocytes and is up-regulated in T regulatory cells (Treg cells) and in activated T cells. GITR agonist which upregulated co-stimulatory signal to T cells or inhibition of Treg induced suppression which reverts Treg proliferation, is likely to enhance immunity in response to infection and tumor immunity. Therefore, GITR is currently of interest to immunologists as a co-stimulatory immune checkpoint molecule. In this study, various types of GITR immunogens, including gene, protein and cell line, are used for H2L2 mice immunization. Then we generate and characterize panels of anti-GITR monoclonal antibodies which not only could specifically bind to human GITR, but also show cross-reactivity to cyno GITR. Such anti-GITR antibody can increase the expression of NF-κB gene by cross-linking by anti-Fab, and induce the release of INF-γ from T cell. In harsh pH or temperature condition, the anti-GITR antibody can keep the stability. And at high concentration, the viscosity of the anti-GITR antibody is good. Moreover, in vivo study showed that the anti-GITR antibody can effectively inhibit tumor growth. Above results demonstrated that the anti-GITR antibody is expected to develop further for clinical study single or combine with other immune checkpoint antibody. Citation Format: Teddy Yang, Ken Dai, Dongxu Wang, Qianqian Qi, Hu Liu. Anti-GITR agonist antibody for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3265.

Tuberculose pulmonaire sous traitement par immunothérapie type anti-PD1

Immunotherapy is now a standard of care in oncology. There is a need to improve our knowledge about immune-related adverse events, especially infectious diseases. We describe the case of a 49-year old male who received anti-PD1 therapy, to treat metastatic melanoma with pulmonary metastasis. After 3 cycles of nivolumab, computed tomography scanning showed a decrease of the pulmonary metastasis in the upper left lobe, but revealed new pulmonary lesions such as tree-in-bud and a lung cavity in the same lobe. This was diagnosed as pulmonary tuberculosis with no antibiotic resistance identified. The patient continued the immunotherapy and was initiated onto a standard anti-tuberculosis therapy. In the absence of an initial positive IFN-γ release assay (Quantiferon) test, but as there might have been a history of primary infection during childhood, a reactivation of tuberculosis was considered to be likely. This is the ninth case of tuberculosis infection under immunotherapy and it underlines the need to consider infection risks in patients undergoing immunotherapy. An INF-γ release assay screening test should be considered an essential part of the pre-treatment work-up.

Evaluation of comparative intradermal tuberculin and interferon gamma release tests for prediction of tuberculous lesion development in slaughtered cattle

Bovine tuberculosis (BTB) is a chronic contagious disease of cattle which has economical, public health and veterinary importance worldwide. So far, test and slaughter is the most feasible method to control the disease but identification of lesions from slaughtered animals does not always coincide with ante-mortem test results. Fifty four animals which were found positive by comparative intradermal tuberculin test (CIDT) or INF-γ release assay or both were slaughtered and post mortem examination (PME) was conducted to identify tuberculous (TB) lesions. The animals were composed of four age groups (≤ 3; 4-6; 7-9 and ≥ 10 years) the two sexes and two breeds (Boran, Boran-Friesian cross). Out of all the slaughtered animals, lesions were detected from 70.3% (38/54) of them. No significant difference was observed among the four age groups and two sexes of both breeds of animals on the level of TB lesion detection. Higher proportion of the lesion was observed by mediastinal (40.4%) followed by bronchial (34.8%), retropharyngeal (17.9%) and lastly miliary form and mesenteric lymph nodes in equal proportion (3.3%). Among the CIDT positive animals, 59.2% of them had lesions. Similarly, 46.2% of INF-γ release assay positive animals had lesions during postmortem examination. Of the total CIDT (+) and INF-γ (-) animals, postmortem lesions were detected from 22.2% of them, and in the vice-versa, of the CIDT (-) and INF-γ (+) animals, 9.2% of them contain TB lesions. Using the PME as reference test, sensitivity of CIDT was 84.2% and the specificity was 6.2%. Similarly, the sensitivity and specificity of the INF-γ release assay was 65.7 and 37.5% respectively. It is concluded that, both tests predict the development of TB lesions. However, in our condition, CIDT was found more predictable for detection of TB lesions as compared to INF-γ release assay. Key words: Bovine tuberculosis (BTB), comparative intradermal tuberculin test (CIDT), interferon gamma (INF-γ) release, post mortem examination (PME), tuberculous (TB) lesion

Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes.

BackgroundGlioblastoma is one of the most immunosuppressive human tumors. Emerging data suggest that glioblastoma-derived exosomes (GBex) reprogram the tumor microenvironment into a tumor-promoting milieu by mechanisms that not yet understood.MethodsExosomes were isolated from supernatants of glioblastoma cell lines by size exclusion chromatography. The GBex endosomal origin, size, protein cargos, and ex vivo effects on immune cell functions were determined. GBex were injected intravenously into mice to evaluate their ability to in vivo modulate normal immune cell subsets.ResultsGBex carried immunosuppressive proteins, including FasL, TRAIL, CTLA-4, CD39, and CD73, but contained few immunostimulatory proteins. GBex co-incubated with primary human immune cells induced simultaneous activation of multiple molecular pathways. In CD8+ T cells, GBex suppressed TNF-α and INF-γ release and mediated apoptosis. GBex suppressed natural killer (NK) and CD4+ T-cell activation. GBex activated the NF-κB pathway in macrophages and promoted their differentiation into M2 cells. Inhibition of the NF-κB pathway in macrophages reversed the GBex-mediated effects. GBex-driven reprogramming of macrophages involved the release of soluble factors that promoted tumor proliferation in vitro. In mice injected with GBex, the frequency of splenic CD8+ T cells, NK cells, and M1-like macrophages was reduced, while that of naïve and M2-like macrophages increased (P < .05).ConclusionsGBex reprogrammed functions of all types of immune cells in vitro and altered their frequency in vivo. By creating and sustaining a highly immunosuppressive environment, GBex play a key role in promoting tumor progression.

Mitochondrial DNA Mismatches in Thoracic Transplantation: Potential Triggers of Allograft Immunogenicity

Purpose Organ transplant (Tx) recipients of non-European ancestry experience higher rejection rates. Stem cell research shows that single nucleotide variant mismatches (SNVMs) in mitochondria DNA (mtDNA) of the donor and host cells trigger alloimmune responses. However, no prior studies have addressed mtDNA SNVMs in organ transplantation. We hypothesized that SNVMs between donor-recipient (D-R) mtDNA sequences contribute to allograft immunogenicity. Our purpose was to conduct a proof-of-concept study examining the relationship between mtDNA SNVMs and immunogenicity in human thoracic Tx recipients. Methods We assayed 364 mtDNAs in pre-Tx peripheral blood mononuclear cells (PBMCs) samples from 182 Tx recipients (19 heart and 163 lung) and their respective donors, using customized standard techniques for mtDNA next-generation sequencing to a mean depth of 9750x. MtDNA sequence reads for D-R pairs were compared to a reference: rCRS database. SNVMs were identified and analyzed for relationships to donor and recipient self-reported sex and race. To determine if nonsynonymous SNVMs trigger allo-specific immunity, we constructed peptides with and without SNVMs and ran Elispot assays to quantify Interferon gamma (INFγ) release from recipients’ re-activated PBMCs collected 1 year post-transplant. Results We identified on average 46 (SD=20) mtDNA SNVMs per D-R pair, 13 (SD=7) of which were nonsynonymous and could form allo-specific immunogenic proteins. Nonsynonymous SNVMs did not differ significantly in D-R sex-matched (N=133, M=13, SD=6) and sex-mismatched pairs (N=46, M=14, SD=7), t(72)=0.87, p = 0.386. In contrast, nonsynonymous SNVMs were significantly higher in D-R race-discordant transplant pairs (N=50, M=16, SD=9) compared to race-concordant pairs (N=100, M=12, SD=5); t(64)=3.07, p=0.003. This difference persisted even after excluding 69 mtDNA haplogroups hot spots: race-discordant pairs (N=50, M=11, SD=7) and race-concordant pairs (N=100, M=8, SD=4); t(68)=2.14, p=0.029. Elispot data showed 30-200 fold increase in INFγ release from peptides with SNVM compared to control, p Conclusion The higher frequency of D-R mtDNA SNVMs in race-discordant transplant pairs, and the immunogenicity of peptides generated from patients with mtDNA SNVMs supports the hypothesis that SNVMs may contribute to allograft immunogenicity.

Modulation of TNF-α, IL-1Ra and IFN-γ in equine whole blood culture by glucocorticoids

Glucocorticoids are important drugs in the treatment of many inflammatory, autoimmune and allergic diseases in humans and animals. We investigated the effects of hydrocortisone and dexamethasone on TNF-α, IL-1Ra and INF-γ release in stimulated whole blood cell culture from healthy horses. Whole blood cell cultures proved to be useful for the characterization of the anti-inflammatory properties of new drugs.Diluted equine whole blood was exposed to lipopolysaccharide (LPS) and PCPwL (a cocktail consisting of phythemagglutinin E, concanavalin A, pokeweed mitogen and lipopolysaccharide) in the presence or absence of hydrocortisone and dexamethasone (10−12 – 10-5 M). TNF-α and IL-1Ra (LPS) as well as IFN-γ (PCPwL) levels were measured in the supernatants using specific enzyme-linked immunosorbent assay (ELISA).The LPS-induced TNF-α and IL-1Ra as well as the PCPwL-induced IFN-γ levels were more potently suppressed by dexamethasone than by hydrocortisone in a concentration-dependent manner. Dexamethasone inhibited TNF-α, IL-1Ra and IFN-γ with the half maximal inhibition concentration (IC50) values of 0.09 μM, 0.453 μM and 0.001 μM, respectively, whereas hydrocortisone inhibited these cytokines with lower IC50 values of 1.45 μM, 2.96 μM and 0.09 μM, respectively. Our results suggest that the equine whole blood test system is useful and reliable to evaluate drug effects and immunological alterations and offers several advantages including simple and cheap performance in physiological and pathological conditions.

Reacciones adversas cutáneas severas a medicamentos: estado del conocimiento

Las reacciones adversas a medicamentos afectan de manera global entre el 10% y el 20% de los pacientes hospitalizados y el 7% de los pacientes ambulatorios. Aproximadamente, uno de cada mil pacientes hospitalizados sufren reacciones adversas que amenazan la vida. Las reacciones adversas a medicamentos, denominadas como reacciones alérgicas (inmunológicas), se pueden agrupar según la clasificación de Gell y Coombs en: tipo I (mediada por IgE), tipo II (citotóxica), tipo III (por inmunocomplejos) y tipo IV (hipersensibilidad retardada o celular). Actualmente, las reacciones tipo IV se subclasifican según la célula efectora involucrada y sus correspondientes citoquinas. Estos subgrupos se definen como: tipo IVa (con activación del perfil Th1, los macrófagos como células efectoras y liberación de INF-γ y TNF-α), tipo IVb (con activación del perfil Th2, los eosinófilos como células efectoras y liberación de citoquinas como la IL-5, la IL-4 y la IL-13), tipo IVc (con los linfocitos T citotóxicos como células efectoras y liberación de granzimas B y perforinas) y tipo IVd (con los neutrófilos como células efectoras y liberación de CXCL8 y factor estimulante de colonias de granulocitos y macrófagos). La presente revisión se centra en la epidemiología, etiología, fisiopatología y manifestaciones clínicas de las reacciones adversas cutáneas severas a medicamentos como el síndrome de Stevens Johnson, la necrólisis epidérmica tóxica, el síndrome DRESS y la pustulosis exantemática generalizada aguda (PEGA); además de un abordaje terapéutico en cada una de estas enfermedades.

The use of an interferon-gamma release assay as a biomarker of response to anti-TNF-alpha treatment.

Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that plays a central role in the immune system functioning and in the pathogenesis of rheumatoid arthritis (RA). TNF-α inhibition has been demonstrated effective to treat RA; however, response to anti-TNF-α therapies is heterogeneous, with roughly one-third of patients not achieving disease control. Identification of a biological marker to assess the effectiveness of TNF-α inhibition may help to discriminate patients with a reduced response to anti-TNF-α agents. The aim of this study was to assess whether anti-TNF-α treatment was able to modify the cytokine network interfering with interferon gamma (INFγ) release after phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMCs) from RA patients, according to disease activity. We found that RA patients with active disease had low release of INFγ after PHA stimulation, but anti-TNF-α agents were able to modify INFγ production. In anti-TNF-α responders, we observed a higher release of INFγ, achieving levels comparable with those seen in healthy subjects. The ability of PBMCs from RA patients to release INFγ may serve as a biomarker of disease activity and response to anti-TNF-α. Larger studies are needed to validate these data.

Corrigendum

British Journal of HaematologyVolume 162, Issue 3 p. 428-428 CorrigendumFree Access Corrigendum This article corrects the following: Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor Sarah Tettamanti, Virna Marin, Irene Pizzitola, Chiara F. Magnani, Greta M. P. Giordano Attianese, Elisabetta Cribioli, Francesca Maltese, Stefania Galimberti, Angel F. Lopez, Andrea Biondi, Dominique Bonnet, Ettore Biagi, Volume 161Issue 3British Journal of Haematology pages: 389-401 First Published online: February 25, 2013 First published: 11 July 2013 https://doi.org/10.1111/bjh.12430AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat ‘Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor’ by Sarah Tettamanti et al, British Journal of Haematology, 161:3, May 2013, 389-401, the y-axis label ‘pg/ml’ should have been added to Fig 6 and its accompanying legend. The corrected legend should read as below: Fig 6. Cytokines release by anti-CD123. CAR + CIK cells after stimulation with leukaemic targets, HUVEC and monocytes. CIK cells were stimulated with γ-irradiated CEM cells, THP-1 cells, primary AML cells, HUVEC cells and monocytes at 1:5 E:T ratio for 24 h. Release of INF-γ, TNF-α, TNF-β and IL-10 was detected in the culture supernatants by flow cytometry (measured as pg/ml). Data shown are mean ± SE of independent experiments with THP-1 (n = 6), CEM, AML, HUVEC and monocytes (n = 3), and for selected comparisons, P-values of the Mann–Whitney test are reported. Reference Tettamanti, S., Marin, V., Pizzitola, I., Magnani, C.F., Giordano Attianese, G.M.P., Cribioli, E., Maltese, F., Galimberti, S., Lopez, A.F., Biondi, A., Bonnet, D. & Biagi, E. (2013) Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor. British Journal of Haematology, 161, 389– 401. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume162, Issue3August 2013Pages 428-428 ReferencesRelatedInformation

298 Regulation and Function of BMP-4 Expression in the Inflamed Gastric Mucosa

BACKGROUND/AIMS: We previously reported that inhibition of Bone Morphogenetic Protein (BMP) signaling In Vivo, leads to enhancement of Helicobacter pylori (HP)-induced gastric inflammation suggesting that the BMPs can exert novel anti-inflammatory actions in the stomach. The aim of this study was to examine the function and the regulation of BMP-4 expression in gastric inflammation. METHODS: Inflammation was induced by infection of 3 month-old C57BL/6-BMP-4-β-gal/+ mice with HP. Animals were analyzed 2 months after inoculation. Expression of BMP-4 was assessed by X-Gal staining and QRT-PCR. Morphology of the gastric mucosa was examined in sections stained with H&E. Phosphorylation of Smad1-5-8 was determined by immunohistochemistry with anti-phospho-Smad1-5-8 antibodies (abs). Expression of INF-γ was measured by QRT-PCR. Gastric mesenchymal and mucus cells were isolated from the canine gastric mucosa and characterized by staining with anti-vimentin abs and the lectin GSII, respectively. Phosphorylation of STAT1 and ERK2 and expression of BMP-4 in the mesenchymal cells were assessed by western blots. IL-8 expression in the mucus cells was measured by QRT-PCR. INF-γ, IL-17-α, IL-12 and IL23 content in supernatants of cultures of mouse splenocytes and dendritic cells was measured by ELISA. RESULTS: Analysis of mucosal sections of C57BL/6-BMP-4-β-gal/+ mice stained with X-gal, demonstrated that BMP-4 is expressed in the mesenchymal layers of the gastric mucosa. HP induced inflammatory changes in the gastric mucosa and it stimulated the expression of both INF-γ and BMP-4. Phospho-Smad1-5-8 staining of the inflamed gastric mucosa indicated the presence of positively stained nuclei in gastric epithelial cells, but not in mesenchymal cells, or in cells of the inflammatory infiltrate. INF-γ induced the phosphorylation of both STAT-1 and ERK2 and it induced BMP-4 protein expression in the mesenchymal cells. Incubation of the cells with inhibitors of both STAT-1 and ERK2 phosphorylation abrogated the stimulatory effect of INF-γ. BMP-4 failed to affect LPS-induced release of INF-γ, IL-17-α, IL-12 and IL-23 in cultures of mouse splenocytes and dendritic cells. In contrast, BMP-4 potently inhibited both basal and TNF-α stimulated IL-8 gene expression in cultures of isolated mucus cells. CONCLUSIONS: Inflammatory stimuli induce the expression of BMP-4 both In Vivo and In Vitro, through mechanisms that appear to involve the activation of both ERK2 and STAT-1. BMP-4, in turn, inhibits the expression of inflammatory molecules in epithelial, but not in immune cells, suggesting that BMP-4 is a novel anti-inflammatoy peptide released by the mesenchyme to modulate the inflammatory response of the gastric epithelium.

Abstract LB-132: The potential role of the multienzyme aminoacyl-tRNA synthetase complex in Th-1 immunity: implications for cancer immunotherapy

Abstract Recent advances in dendritic cell (DC)-based cancer vaccines have highlighted the potential utility of arming the immune system to target neoplastic self. A strong, yet controlled, TH1 response has typically been the goal of DC-based protocols, as it is cell-based adaptive immunity that possesses the ability to most-efficiently destroy the nascent tumor or occult micrometastases. Nonetheless, many DC-intrinsic factors have impeded the development of a vaccine strong enough to impart meaningful clinical results, and there remains a need for further study of the parameters required for maximal, targeted DC activity. Previously, we have shown that contemporaneous loading of DC with overlapping MHC class I and MHC class II peptide antigens leads to an enhanced TH1 response when compared to canonical DC stimulation as assayed by release of IL-12, sCTLA-4, proliferation of CD8+ CD25+, INF-γ release, and specific killing of target both in vitro and in vivo. These and other data have lead to the hypothesis that DC possess a sensor mechanism capable of discerning amino acid sequence similarities between peptides bound by MHC class I and class II and that tRNA synthetases may serve as critical components of the putative recognition machinery due to their inherent ability to recognize and distinguish between individual amino acid side chains. Here we report that the aminoacyl-tRNA synthetase complex (MACS), a large molecular complex comprised of at least nine aminoacyl-tRNA synthetases, possess interesting characteristics that might link it to this phenomenon. Components of the MACS complex, including the p43 structural subunit, were found in DC exosomes, the contents of which mimic those of the late endosome, a known cross-presentation compartment. MARS (methionyl-tRNA synthetase), a component of the MACS, as well as non-MACS components VARS (valyl-tRNA synthetase) and GARS (glycyl-tRNA synthetase) were found within exosomes as well. UVC cross-linking of exosomal extracts resulted in the incorporation of individual tRNA-synthetase isoforms into a very large complex of indeterminate size. p43 was differentially released from the MACS in response to the loading of DC only with matched class I (GFP mRNA) and class II (recombinant GFP protein) determinants. siRNA knockdown of MACS structural component p38 was able to ablate the ability of doubly-loaded DC to enhance the generation of CD8+CD25+ cells as well as the ability of these cells to secrete IFN-γ. Further, siRNA knockdown of p38 abrogated the release of p43 from DC loaded with matched class I and II determinants. With an enhanced understanding of these regulatory phenomena, the development of small molecule TH1 agonists and/or immunotherapeutic protocols with enhanced clinical efficacy become realistic and achievable goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-132. doi:1538-7445.AM2012-LB-132