Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity.

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

Multicolor-Assay-on-a-Chip Processed by Robotic Operation (MACpro) with Improved Diagnostic Accuracy for Field-Deployable Detection.

The ability to deploy decentralized laboratories with autonomous and reliable disease diagnosis holds the potential to deliver accessible healthcare services for public safety. While microfluidic technologies provide precise manipulation of small fluid volumes with improved assay performance, their limited automation and versatility confine them to laboratories. Herein, we report the utility of multicolor assay-on-a-chip processed by robotic operation (MACpro), to address this unmet need. The MACpro platform comprises a robot-microfluidic interface and an eye-in-hand module that provides flexible yet stable actions to execute tasks in a programmable manner, such as the precise manipulation of the microfluidic chip along with different paths. Notably, MACpro shows improved detection performance by integrating the microbead-based antibody immobilization with enhanced target recognition and multicolor sensing via Cu2+-catalyzed plasmonic etching of gold nanorods for rapid and sensitive analyte quantification. Using interferon-gamma as an example, we demonstrate that MACpro completes a sample-to-answer immunoassay within 30 min and achieves a 10-fold broader dynamic range and a 10-fold lower detection limit compared to standard enzyme-linked immunosorbent assays (0.66 vs 5.2 pg/mL). MACpro extends the applications beyond traditional laboratories and presents an automated solution to expand diagnostic capacity in diverse settings.

Interferon gamma (IFN-γ)-sensitive TB aptasensor based on novel chitosan-indium nano-kesterite (χtCITS)-labeled DNA aptamer hairpin technology.

There has been increasing interest in the use of biosensors for diagnosis of infectious diseases such as tuberculosis (TB) due to their simplicity, affordability, and potential for point-of-care application. The incorporation of aptamer molecules and nanomaterials in biosensor fabrication explores the advantages of high-binding affinity and low immunogenicity of aptamers as well as the high surface-to-volume ratio of nanomaterials, for increased aptasensor performance. In this work, we employed a novel microwave-synthesized copper indium tin sulfide (CITS) substituted-kesterite nanomaterial, together with a natural biopolymer (chitosan), for signal amplification and increased loading of aptamer molecules. Study of the optical properties of CITS nanomaterials showed strong absorption in the UV region characteristic of kesterite semiconductor nanomaterials. X-ray diffraction analysis confirmed the presence of the kesterite phase with average crystallite size of 6.188 nm. Fabrication of interferon-gamma (IFN-γ) TB aptasensor with a chitosan-CITS nanocomposite (χtCITS) increased the aptasensor's electrochemical properties by 77.5 % and improved aptamer loading by 73.7 %. The aptasensor showed excellent sensitivity to IFN-γ concentrations with limit of detection of 6885 fg/mL (405 fM) and linear range of 850-17000 fg/mL (50 - 1000 fM). The aptasensor also exhibited excellent storage and electrochemical stability, with good selectivity towards IFN-γ and possible real sample application.

Periostin in the relation between periodontal disease and atherosclerotic coronary artery disease: A pilot randomized clinical study.

OBJECTIVE: The aim of this study was to analyze the effects of periodontal treatment on markers of atherosclerotic coronary artery disease and circulating levels of periostin. BACKGROUND: Periostin is necessary for periodontal stability, but it is highly present in atherosclerotic plaques. Treatment of periodontal disease, with low levels of local periostin, is thought to reduce systemic levels of periostin. Thus, this may contribute to cardiovascular health. METHODS: A pilot randomized controlled clinical trial was designed to include patients with severe periodontal disease and history of atherosclerotic coronary artery disease. Samples of gingival crevicular fluid (GCF) and serum were collected before and after periodontal treatment by periodontal surgery or non-surgical therapy. The levels of several markers of inflammation and cardiovascular damage were evaluated including CRP, IFN-γ, IL-1ß, IL-10, MIP-1α, periostin, and TNF-α in GCF and CRP, Fibrinogen, IFN-γ, IL-1ß, IL-6, IL-10, L-Selectin, MIP-1α, Periostin, TNF-α, and vWF in serum. RESULTS: A total of 22 patients with an average of 56 years old were recruited for participating in this study. Twenty of them were male. Most of them (82%) had suffered an acute myocardial event and underwent surgery for placing 1, 2, or 3 stents in the coronary arteries more than 6 months ago but less than 1 year. The treatment of periodontal disease resulted in an overall improvement of all periodontal parameters. Regarding the evaluation of GCF and serum, a significant increase of periostin in the GCF was observed after periodontal surgery. In contrast, although other markers in GCF and serum improved, no significant correlations were found. CONCLUSION: Treatment of periodontal disease through periodontal surgery induces a local and transient increase in the levels of periostin in the gingival crevicular fluid. The effects on systemic markers of inflammation and cardiovascular function have not been confirmed.

[Regulation effect of serum IFN-γ and IL-4 expression on immune response in patients with oral lichen planus].

PURPOSE: To investigate the regulation effect of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in the serum of patients with oral lichen planus (OLP) on immune response. METHODS: A total of 83 OLP patients in Wangfujing Branch of Beijing Stomatological Hospital, Capital Medical University from January 2019 to December 2021 were selected as the experimental group, and another 61 healthy volunteers who came to the hospital during the same period were included as the control group. The expression levels of IFN-γ and IL-4 in the serum and the expression of immune function indexes of the two groups were compared. The relationship between the expression levels of IFN-γ, IL-4 and immune function indicators in the experimental group was analyzed. SPSS 22.0 software package was used to analyze the data. RESULTS: The levels of IFN-γ and IL-4 in the experimental group were significantly higher than those in the control group(P＜0.05). There was no significant difference in the levels of IFN-γ between patients with reticulated OLP and erosive OLP in the experimental group (P＞0.05). The level of IL-4 in patients with erosive OLP in the experimental group was significantly higher than that in patients with reticulated OLP(P＜0.05). The levels of CD3+, CD4, CD16++CD56+ and CD4+/CD8+ in the experimental group were significantly lower than those in the control group(P＜0.05), the levels of CD8+ and CD19+ in the experimental group were significantly higher than those in the control group(P＜0.05). The levels of IgG, IgM and CH50 in the experimental group were significantly higher than those in the control group(P＜0.05), and the levels of IgA, C3 and C4 in the experimental group were significantly lower than those in the control group(P＜0.05). Pearson correlation analysis showed that the level of IFN-γ in the experimental group was negatively correlated with CD16++CD56+ and CD4+/CD8+ (P＜0.05), the level of IL-4 in the experimental group was negatively correlated with CD16++CD56+ and CD4+/CD8+(P＜0.05), and the level of IL-4 in the experimental group was positively correlated with IgG (P＜0.05). CONCLUSIONS: Serum IFN-γ and IL-4 in OLP patients are abnormally high, and IL-4 expression is significantly higher in erosive OLP patients. The cellular and humoral immune functions of OLP patients are disordered and related to the regulation of CD16++CD56+, CD4+/CD8+, and IgG immune responses.

Interferon-γ release assay and mantoux response in infants with tuberculous meningitis in low and intermediate burden countries.

AIM: Until now, the performance of interferon-γ release assay (IGRA) and Mantoux tests remains unclear in infant tuberculous meningitis (TBM). Therefore, a systematic review is performed to evaluate the sensitivity of IGRA and Mantoux tests for the diagnosis of infant TBM in low and intermediate tuberculosis (TB) burden countries, while following PRISMA. METHODS: Several databases, including PubMed, EBSCO, Embase, Scopus, Web of Science, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials, were searched. Articles describing the results of IGRA or Mantoux tests among infant TBM were included for analysis. Data, such as age, sex, Mantoux test or IGRA, and cerebrospinal fluid (CSF) microbiological examinations (such as acid-fast bacilli (AFB) smear, TB PCR, and TB culture), were extracted from each study. RESULTS: A total of 31 articles were enrolled for further analysis, including 48 cases. The mean age was 9.4 ± 5.8 months and boys accounted for 57.1% of infants (24/42). Mantoux test was positive in 57.4% (27/47) of tested infants and IGRA was positive in 77.8% (7/9) of infants. In addition, among the infants with confirmed TB, 18 (52.9%, 18/34) of them have positive Mantoux responses and 7 (20.0%, 7/35) have positive IGRA results. CONCLUSIONS: In low or intermediate TB burden countries, the Mantoux test has a poor performance for diagnosing TBM among infants, and IGRAs appear to have a moderate sensitivity for the diagnosis of infant TBM.

Stability-indicative and conformation-specific enzyme linked immunosorbent assay for analysis of recombinant human gamma interferon.

Recombinant human interferon gamma (rhIFN-γ) is a promising molecule for the treatment of several diseases. A pair of conformation-specific monoclonal antibodies (mAbs) against rhIFN-γ was selected from generated hybridoma cell lines to design a sensitive, stability-indicative, sandwich-type ELISA. The main assay parameters were optimized by the checkerboard method for the highest signal-to-noise ratio: assay buffer composition, coating buffer pH and composition, coating temperature-incubation time parameters, and coating mAb concentration and conjugate dilution. Detection and quantification limits were estimated between 0.019 and 0.078 ng/mL, respectively, and recovery values were from 92.03% to 98.40%. The coefficient of variation of intra-assay precision parameters ranged from 2.32% to 9.21% while the inter-analyst variation was between 4.70% and 10.63%, supporting the method's repeatability. The ELISA was specific for correctly folded and non-aggregated molecular species, as compared to intrinsic Trp fluorescence (chemical denaturation) and optical density at 340 nm (thermal aggregation), respectively. However, the method was not sensitive to the small C-terminal degradation of full-length rhIFN-γ1-144 (losses of 6-12 amino acid residues) as compared to results with mass spectrometry and gel electrophoresis. ELISA showed good correlation with rhIFN-γ antiviral biological activity. This method was applied to the stability evaluation of rhIFN-γ in physiological buffer at low concentrations using polypropylene and glass vials also in the presence of adsorption protectant excipients. Furthermore, ELISA could be adapted to other applications such as quantification of IFN-γ in serum samples, Mycobacterium tuberculosis diagnosis, etc.

Efecto del estrés crónico y de la masticación sobre los niveles séricos de interleuquina-6 e interferón-gama / Effect of chronic stress and chewing on serum interleukin-6 and interferon-gamma levels

Introducción: el estrés crónico afecta el equilibrio inmunológico alterando los niveles séricos de interleuquina-6 (IL-6) e interferón gama (INF-γ), dicha alteración afecta al sistema nervioso y al comportamiento humano. La masticación adecuada disminuiría dichos efectos. El objetivo del estudio fue determinar el efecto del estrés crónico y de la masticación sobre los niveles séricos de IL-6 e INF-γ. Métodos: experimento donde se emplearon 64 ratones Balb/c de 8 semanas de edad. Se dividieron en 4 tratamientos: Grupo NE: Masticación normal + estrés, Grupo N: masticación normal sin estrés, Grupo DE: Masticación deficiente + estrés, Grupo D: masticación deficiente sin estrés. Mediante test de ELISA se midió IL-6 e IFN-γ alfinal de la 4ta y de la 8va semana de tratamiento. Resultados: tanto la IL-6 como el IFN-γ fueron mayores en el grupo DE (p<0,05) al final de la 4ta semana. Al evaluarlos al término de la 8va semana se observó que en el grupo NE se incrementó la IL-6 respecto al resto de grupos (p<0,0001), y en el grupo DE fue donde se encontró mayor cantidad de IFN-γ (p<0,0001). Conclusión: el estrés crónico y la masticación deficiente incrementan los niveles séricos de IL-6 e IFN-γ. En cambio, la adecuada masticación disminuye el nivel de tales citoquinas al final de la cuarta semana de tratamiento.

Introduction: chronic stress affects the immune balance by altering the serum levels of interleukin-6 (IL-6) and gamma interferon (INF-γ), this alteration affects the nervous system and human behavior. Appropriate chewing would lessen these effects. The aim of this study was to determine the effect of chewing and chronic stress over serum levels of IL-6 and INF-γ. Methods: experiment in which 64 Balb/C mice of 8 weeks of age were used, they were divided into 4 treatments: Group NE: Normal chewing + stress, Group N: normal chewing without stress, Group DE: Chewing poor + stress, Group D: poor chewing without stress. IL-6 and IFN-γ were measured by ELISA after 4 and 8 weeks of treatment. Results: both IL-6 and IFN-γ were higher in the DE group (p < 0,05) at the end of fourth week of treatment. When evaluating the animals at the end of the eighth week of treatment, it was observed that in the NE group, the IL-6 was increased with respect to the rest (p < 0,0001) and the DE group showed more IFN-γ (p < 0,0001). Conclusion: stress and poor chewing increase serum IL-6 and IFN-γ. In contrast, appropriate chewing decreases the effects of stress on the increase of such cytokines at the end of the fourth week of treatment in animals.

Increased interferon-γ levels and risk of severe malaria: a meta-analysis.

Interferon (IFN)-γ contributes to the pathogenesis of severe malaria; however, its mechanism remains unclear. Herein, differences in IFN-γ levels between patients with severe and uncomplicated malaria were evaluated using qualitative and quantitative (meta-analysis) approaches. The systematic review protocol was registered at PROSPERO (ID: CRD42022315213). The searches for relevant studies were performed in five databases, including PubMed, Scopus, Embase, MEDLINE and Web of Science, between 1 January and 10 July 2022. A meta-analysis was conducted to pool the mean difference (MD) of IFN-γ levels between patients with severe malaria and those with uncomplicated malaria using a random-effects model (DerSimonian and Laird method). Overall, qualitative synthesis indicated that most studies (14, 58.3%) reported no statistically significant difference in IFN-γ levels between patients with severe malaria and those with uncomplicated malaria. Meanwhile, remaining studies (9, 37.5%) reported that IFN-γ levels were significantly higher in patients with severe malaria than those in patients with uncomplicated malaria. Only one study (4.17%) reported that IFN-γ levels were significantly lower in patients with severe malaria than those in patients with uncomplicated malaria. The meta-analysis results indicated that patients with severe malaria had higher mean IFN-γ levels than those with uncomplicated malaria (p < 0.001, MD: 13.63 pg/mL, 95% confidence interval: 6.98-20.29 pg/mL, I2: 99.02%, 14 studies/15 study sites, 652 severe cases/1096 uncomplicated cases). In summary, patients with severe malaria exhibited higher IFN-γ levels than those with uncomplicated malaria, although the heterogeneity of the outcomes is yet to be elucidated. To confirm whether alteration in IFN-γ levels of patients with malaria may indicate disease severity and/or poor prognosis, further studies are warranted.

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine.

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.

Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis: A systematic review and meta-analysis.

OBJECTIVE: Tuberculous meningitis (TBM) is one of the most devastating TB. Accurate identification of TBM is helpful to eliminate TB. Therefore, we assessed the performance of TBAg stimulated IFN-γ (IGRA) and unstimulated IFN-γ in blood and cerebrospinal fluid (CSF) for diagnosing TBM. METHODS: We searched Web of Science, PubMed, Embase and the Cochrane Library databases until March 2022. Bivariate and hierarchical summary receiver operating characteristic models were employed to compute summary estimates for diagnostic accuracy parameters of IGRA and unstimulated IFN-γ in blood and CSF for diagnosing TBM. RESULTS: 28 studies including 1,978 participants and 2,641 samples met the inclusion criteria. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and area under the curve (AUROC) of blood IGRA were separately as 0.73, 0.83, 4.32, 0.33, 13.22 and 0.86, indicating a good diagnostic accuracy of blood IGRA for detecting TBM. The summary sensitivity, specificity, PLR, NLR, DOR and AUROC of CSF IGRA were separately as 0.77, 0.91, 8.82, 0.25, 34.59 and 0.93, indicating good diagnostic accuracy of CSF IGRA for detecting TBM. The summary sensitivity, specificity, PLR, NLR, DOR and AUROC of CSF IFN-γ were separately as 0.86, 0.92, 10.27, 0.16, 65.26 and 0.95, suggesting CSF IFN-γ provided excellent accuracy for diagnosing TBM. CONCLUSIONS: For differentiating TBM from non-TBM individuals, blood and CSF IGRA are good assays and unstimulated CSF IFN-γ is an auxiliary excellent marker.

Highly accurate multiprotein detection on a digital ELISA platform.

The emerging single-molecule detection platform digital enzyme-linked immunosorbent assay (ELISA) can detect numerous proteins simultaneously at serum concentrations as low as picograms per milliliter. We sought to improve cytokine detection with this platform to aid diagnosis of conditions such as allergy and asthma. We developed a multiple single-molecule detection digital ELISA approach, through the application of encoded magnetic microbeads to simultaneously detect three cytokines in one serum sample. We tested the approach's utility to distinguish asthma-related cytokines in children. Concentrations of interleukin-4 (IL-4) and IL-6 were significantly higher in children with asthma than in healthy controls, while the concentration of interferon-γ (IFN-γ) was significantly lower. Our method has higher accuracy than conventional methods, and our results indicate that the proposed improved high-sensitivity digital ELISA-based diagnosis approach can facilitate early detection and treatment of childhood asthma or related diseases.

Clinical utility of immune function based on IFN-γ monitoring of lymphocyte subsets for parvovirus B19 infection in renal recipients.

BACKGROUND: There should be a heightened index of suspicion for Parvovirus B19 (PVB19)-related anemia in organ transplant recipients. Thus far, there is no consensus or recommendation for clinical routine monitoring methods of PVB19 recipients to allow tailoring of immunosuppression. METHODS: We conducted a retrospective study to evaluate the utility of the function (represented by the abilities to secrete IFN-γ) and numbers of lymphocyte subsets in monitoring PVB19 infections in renal recipients posttransplant. The enrolled 109 patients were split into 2 groups according to whether the recipients had an occurrence of PVB19 infection: 37 (33.94%) recipients developed PVB19 infection and 72 (66.06%) immune-stable recipients. RESULTS: The PVB19 infected group had significantly lower absolute counts and functions of different lymphocyte subsets compared with immune-stable recipients. We showed that the frequencies of IFN-γ + CD4 + T cells, IFN-γ + CD8 + T cells, and IFN-γ + NK cells increased markedly after treatment when compared to the occurrence in patients with timepoint before therapy, especially the percentages of IFN-γ + CD4 + T cells were significantly higher. Receiver operating characteristic (ROC) analysis showed that the optimal infection indicator was IFN-γ + NK cells frequency, with an auROC curve of 0.925. Concomitantly, Cox regression analysis indicated that the post-therapy increasing level of IFN-γ secreting function was significantly predictive of recurrent infections (P < 0.001). CONCLUSIONS: We recommend prospective risk stratification for the high-risk population at risk of early-onset PVB19 infection and its recurrence involves screening strategies of immune-based surveillance with the sensitive IFN-γ + secreting monitoring for antiviral prophylaxis and preemptive therapy goal. Clinical Trial Notation: clinical trial registration number: chiCTR-ROC-17010756.

Interferon-γ detection in point of care diagnostics: Short review.

Interferon (IFN)-γ is a cytokine secreted by immune cells. The elevated levels of IFN-γ are an early indicator of multiple diseases such as tuberculosis and autoimmune diseases. This short review focuses on different sensing methods based on optical, electrochemical, and mechanical principles. We explain how specific biorecognition molecules such as antibodies and aptamers are employed in the sensing methods. We also compare different surface functionalization methods and their details. Although the review gives an overview of only IFN-γ sensing, the same strategies can be applied to sensing other analytes with appropriate modifications.

T Cell Response following Anti-COVID-19 BNT162b2 Vaccination Is Maintained against the SARS-CoV-2 Omicron B.1.1.529 Variant of Concern.

The progression of the COVID-19 pandemic has led to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about reduced protective T cell immunity and, consequently, more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type Wuhan-1 SARS-CoV-2 ancestral spike protein and the Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from eight healthy volunteers 4-5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or the Omicron SARS-CoV-2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring cells secreting interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4). For all the examined individuals, comparable levels of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg-secreting cells and only limited numbers of IL-10- and IL-4-secreting cells. The data demonstrate stable T cell activity in response to the emerging Omicron variant in the tested individuals; therefore, the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.

Impairment of CD4+ T and Memory B Cell Responses but Normal Memory CD8+T-Cell Activation on Crohn's Disease after COVID-19 Vaccination: A Twin Case.

Vaccines to prevent the impact of SARS-CoV-2 are now available, including for patients with autoimmune diseases. However, there is no information about how inflammatory bowel disease (IBD) treatment could impact the cellular and humoral immune responses. This study evaluated SARS-CoV-2-specific humoral and cellular responses after vaccination with a two-dose schedule in a Crohn's disease patient treated with Infliximab (10 mg/kg); we included comparisons with a monozygotic twin. The results showed that the Crohn's disease's twin (twin 2) had no antibody detection and reduced activation of CD4+ T cell responses, unlike the twin without the autoimmune disease (twin 1). Twin 2 developed antigen-specific central memory CD8+ T-cells and IFNγ production after the second dose of COVID-19 vaccination, similar to twin 1. These findings elucidated the role of T-cell immunity after COVID-19 immunization on IBD patients despite the lack of antibody production. Finally, our observation supports the consensus recommendation for IBD patients to receive COVID-19 vaccines.

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

The Spleen as a Target to Characterize Immunomodulatory Effects of Down-Stream Processed Cyberlindnera jadinii Yeasts in Atlantic Salmon Exposed to a Dietary Soybean Meal Challenge.

Aquaculture feeds have changed dramatically from being largely based on fishmeal (FM) towards increased use of plant protein sources, which could impact the fish's immune response. In order to characterize immunomodulatory properties of novel functional ingredients, this study used four diets, one based on FM, a challenging diet with 40% soybean meal (SBM), and two diets containing 40% SBM with 5% of Cyberlindnera jadinii yeast exposed to different down-stream processing conditions: heat-inactivated (ICJ) or autolysation (ACJ). The immunomodulatory effects of the diets were analyzed in the spleen of Atlantic salmon after 37 days of feeding, using a transcriptomic evaluation by RNA sequencing (RNA-seq) and the detection of specific immunological markers at the protein level through indirect Enzyme-linked Immunosorbent Assay (indirect ELISA). The results showed that SBM (compared to FM) induced a down-regulation of pathways related to ion binding and transport, along with an increase at the protein level of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). On the other hand, while ICJ (compared to FM-group) maintain the inflammatory response associated with SBM, with higher levels of TNFα and IFNγ, and with an upregulation of creatine kinase activity and phosphagen metabolic process, the inclusion of ACJ was able to modulate the response of Atlantic salmon compared to fish fed the SBM-diet by the activation of biological pathways related to endocytosis, Pattern recognition receptor (PPRs)-signal transduction and transporter activity. In addition, ACJ was also able to control the pro-inflammatory profile of SBM, increasing Interleukin 10 (IL-10) levels and decreasing TNFα production, triggering an immune response similar to that of fish fed an FM-based diet. Finally, we suggest that the spleen is a good candidate to characterize the immunomodulatory effects of functional ingredients in Atlantic salmon. Moreover, the inclusion of ACJ in fish diets, with the ability to control inflammatory processes, could be considered in the formulation of sustainable salmon feed.

Low-Dose Vitamin D3 Supplementation Does Not Affect Natural Regulatory T Cell Population but Attenuates Seasonal Changes in T Cell-Produced IFN-γ: Results From the D-SIRe2 Randomized Controlled Trial.

Background: Seasonal variations have been reported for immune markers. However, the relative contributions of sunlight and vitamin D variability on such seasonal changes are unknown. Objective: This double-blind, randomized, placebo-controlled trial tested whether daily 400 IU vitamin D3 supplementation affected short-term (12 weeks) and long-term (43 weeks) natural regulatory T cell (nTreg) populations in healthy participants. Design: 62 subjects were randomized equally to vitamin D versus placebo in March and assessed at baseline, April (4w), June (12w), September (25w) and January (43w). Circulating nTregs, ex vivo proliferation, IL-10 and IFN-γ productions were measured. Vitamin D metabolites and sunlight exposure were also assessed. Results: Mean serum 25-hydroxyvitamin D (25(OH)D) increased from 35.8(SD 3.0) to 65.3(2.6) nmol/L in April and remained above 75 nmol/L with vitamin D supplementation, whereas it increased from 36.4(3.2) to 49.8(3.5) nmol/L in June to fall back to 39.6(3.5) nmol/L in January with placebo. Immune markers varied similarly between groups according to the season, but independently of 25(OH)D. For nTregs, the mean (%CD3+CD4+CD127lo cells (SEM)) nadir observed in March (2.9(0.1)%) peaked in September at 4.0(0.2)%. Mean T cell proliferation peaked in June (33156(1813) CPM) returning to the nadir in January (17965(978) CPM), while IL-10 peaked in June and reached its nadir in September (median (IQR) of 262(283) to (121(194) pg/ml, respectively). Vitamin D attenuated the seasonal increase in IFN-γ by ~28% with mean ng/ml (SEM) for placebo vs vitamin D, respectively, for April 12.5(1.4) vs 10.0(1.2) (p=0.02); June 13.9(1.3) vs 10.2(1.7) (p=0.02) and January 7.4(1.1) vs 6.0(1.1) (p=0.04). Conclusions: Daily low dose Vitamin D intake did not affect the nTregs population. There were seasonal variation in nTregs, proliferative response and cytokines, suggesting that environmental changes influence immune response, but the mechanism seems independent of vitamin D status. Vitamin D attenuated the seasonal change in T cell-produced IFN-γ, suggesting a decrease in effector response which could be associated with inflammation. Clinical Trial Registration: https://www.isrctn.com, identifier (ISRCTN 73114576).

Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology.

Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.