Previous studies have suggested that missense muta- tions are associated with partial, as opposed to com- plete, IFNγR1 deficiency (13). Our study demonstrates that the identification of a recessive missense muta- tion or an in-frame deletion in IFNGR1 is insufficient to exclude complete deficiency. Functional assays should be performed, such as quantification of TNFα production by freshly prepared blood cells stimulated with IFN-γ (5, 12), detection of STAT-1 phosphoryla- tion in stimulated freshly prepared monocytes (43), detection of STAT-1 nuclear translocation in stimu- lated EBV-B cells (13, 17), or detection of HLA-DR induction on the surface of stimulated SV40-trans- formed fibroblasts (11, 13, 17). Cellular responses to low and high concentrations of IFN-γ should be test- ed to differentiate complete and partial IFNγR1 defi- ciency. Accurate molecular diagnosis by biochemical, functional, and genetic studies is of the utmost impor- tance for predicting clinical outcome and guiding the treatment of patients. Partial and complete IFNγR1 deficiency differ markedly in prognosis and treatment. IFN-γ treatment may be offered to patients with par- tial IFNγR1 deficiency, whereas bone marrow trans- plantation is the only curative treatment available for patients with complete IFNγR1 deficiency. This study stresses the importance of accurate molecular diagno- sis of the underlying inherited disorder for the ration- al treatment of patients with mycobacterial disease.
An interesting finding in naive genomic APOL1 G1–transgenic mice was the elevation in ALT in mice from 2 different APOL1 G1 founder lines. While APOL1 risk alleles have not been reported to be associated with liver injury, the elevations observed in these mice are modest and variable, such that they may be overlooked in humans as within normal range. As a result, a close analysis of ALT levels in APOL1 risk allele carriers in com- parison to those that lack these alleles has likely not been performed and may be warranted. Interestingly, hydro- dynamic gene delivery (HGD) of APOL1 G1 to mice resulted in widespread liver necrosis and macrophage infiltration, while delivery of APOL1 G0 did not drastically affect liver pathology (50). How APOL1 risk variants cause toxicity remains unclear; however, several different mechanisms have been proposed, including altered autophagic flux due to effects on endolysosomal trafficking (16, 17, 31, 32, 51). Our data support this hypothesis, as we observed enhanced suppression of autophagy following IFN-γ challenge in APOL1 G1 mice in compar- ison to APOL1 G0 mice. This effect is clearer in the liver than in the kidney, likely due to heterogeneity in the kidney. Future studies are needed to elucidate this further. Importantly, reduction of APOL1 with IONIS-APOL- 1 Rx reduces plasma ALT elevations and suppression of autophagy upon IFN-γ challenge in liver, indicating that ALT elevations and enhanced suppression of autophagy in APOL1 G1 mice is APOL1 dependent. While other mechanisms of APOL1 risk allele–mediated toxicity, including inflammasome activation and altered electrolytic flux, have also been proposed, IONIS-APOL1 Rx , by inhibiting APOL1 itself, will likely be able to suppress all modes of toxicity, including PKR activation by APOL1 G1/G2 RNA stem loops (52).
In addition, NK cells elaborate TNF-α con- tributed to elimination of intracellular patho- gens (11, 22, 23). TNF-α has a potentfunction against mouse model of leishmaniasis (24). Other investigators showed that treatment with TNF-α led to reduction of lesion size and parasitic burden (25, 26). Furthermore, admin- istration of anti TNF-α neutralizing antibody was associated with a transient aggravation of the disease (27, 28). Bogdan et al. (29) showed that TNF-α contributes to protective immuni- ty by synergizing with IFN-γ to activate mac- rophages. IFN-γ and TNF-α stimulate macro- phages to produce NO as an important effec- tor molecule of innate and adaptive responses to L. major (30-32). We found that rLPG3 but not NT and CT fragments can stimulate NK cells to produce TNF-α. TNF-α production by NK cells only achieved at highest concentra- tion of LPG3 (20µgr/ml). On the other hand, LPG3 fragments (NT and CT) were not able to stimulate human NK cells to produce TNF-α indicating the necessity of whole LPG3 for TNF-α production.
We found that IFN-γ with different concentrations can increase the percentage of CD4 + CD25 + Tregs in the whole cell system. Because the purity of CD4 + CD25 − T cells be- fore stimulating is above 96 % and the viability of all the cells both before and after stimulating is above 95 %, we can confirm that the increased percentage of CD4 + CD25 + Tregs is due to the conversion of CD4 + CD25 − T cells by IFN-γ. Especially, in the presence of anti-CD3 and anti- CD28 antibodies only, CD4 + CD25 + T cells can also be in- duced. It indicates anti-CD3 and anti-CD28 antibodies play an important role in helping IFN-γ to induce CD4 + CD25 + T cells. Maybe anti-CD3 antibody provides a first signal through the TCR to activate CD4 + CD25 − T cells and anti- CD28 antibody provides a costimulatory signal in this process. Moreover, induction ration is associated with the concentration of IFN-γ. When the concentration of IFN-γ is 40 ng/ml, the ration reaches the peak. The possible rea- son is that IFN-γ is a “double-edged sword”. It has regula- tory and cytotoxic function. When the concentration of IFN-γ is between 20 and 40 ng/ml, it plays a regulatory role. Once it is above 40 ng/ml, it plays a cytotoxic role. In our study we show that 40 ng/ml IFN-γ has the most powerful ability to induce CD4 + CD25 + Tregs in MG pa- tients while the most suitable concentration of IFN-γ is 20 ng/ml in HCs. With IFN-γ of any concentration, the conversion rate of HCs is higher than that of MG patients. When anti-CD3 and anti-CD28 antibodies are added only, the rate of HCs is still higher than that of MG patients. It reveals that because the micro environments are different in MG patients and HCs, there may be some difference of sensitivity to IFN-γ in CD4 + CD25 − T cells between MG patients and HCs or among the MG patients with different severity. And CD4 + CD25 − T cells from HCs are also more sensitive to anti-CD3 and anti-CD28 antibodies. On the other side, the concentration of IFN-γ in plasma of MG may affect the function of CD4 + CD25 + Tregs. The ques- tions raised above will be studied in our subsequent experi- ments. The results from different age stages are also compared and these results show rarely any statistical dif- ference. But there are other factors such as the severity of MG, sex and the duration of disease involved, so it is desir- able to perform further study in the future to examine the exact effect of age to the induction.
dent responses, including p38 mitogen-activated protein (MAP) kinase, phosphatidylinositol 3 (PI3)-kinase, and protein kinase C isoforms [48-51]. Through alteration of these modulating pathways, CSE could indirectly affect IFN-γ-mediated immunity. Furthermore, there are multi- ple interferon pathways that control antiviral defense that could be affected by CSE. Recent reports indicate that type I interferon production, signal transduction, and antiviral effects are impaired in cells exposed to cigarette smoke condensate or conditioned media [47,52,53]. Altered interferon responses after cigarette smoke expo- sure may not be limited to epithelial cells. For example, macrophage and fibroblast cell lines exposed to cigarette smoke preparations in media and alveolar macrophages isolated from individuals that smoke cigarettes have reduced responsiveness to interferons [47,52,54-56]. Thus, cigarette smoke appears to affect multiple compo- nents of interferon-dependent antiviral defense. It is important to note that IFN-γ has other important func- tions in tissues besides antiviral defense. For example, IFN-γ is important for immune surveillance against malignant cells, and inhibition of interferon effects could
This study highlights the utility of mass cytometry coupled with multiparameter phospho-specific antibodies in analyzing signal differences in small-volume patient samples. Using this approach, dif- ferences in STAT1 and/or STAT3 phosphorylation in subsets of CD4 T cells and classical monocytes following IFN-γ stimulation were identified in treatment-naive polyarticular JIA patient compared with control samples. The enhanced responsiveness of CD4 T cells to IFN-γ stimulation correlated with increased expression of JAK1 and SOCS1 in treatment-naive JIA patients. These results suggest that drugs that attenuate IFN-γ signaling (e.g., JAK inhibitors) may be useful in treating polyarticular JIA patients and provide a potential mechanistic rationale for anecdotal observations regarding the effective- ness of JAK inhibitors in refractory polyarticular JIA patients (including the case series of 3 refractory polyarticular JIA patients treated with tofacitinib, a JAK inhibitor, in the Supplemental Data) and for an ongoing clinical trial (NCT02591434) of tofacitinib in polyarticular JIA patients. Interestingly, one of patients in the case series (case 2) was included in the mass cytometry analysis as a treatment-naive sample (patient 7 in Table 1), and a follow up sample was subsequently obtained while the patient was in clinical remission on tofacitinib (after completion of the initial analysis). Naive CD4 T cell p-STAT1 and p-STAT3 following IFN-γ stimulation were lower in the remission sample while on tofacitinib (as well as leflunomide and hydroxychloroquine) compared with the paired treatment-naive samples (Sup- plemental Figure 13). Future studies will focus on more detailed interrogation of specific CD45RA +
at 1 μg/ml (BD Biosciences). Cultures were incubated for 20 hours at 37°C, in an atmosphere of 5% CO 2 . Brefeldin A at 1 μg/ml (GolgiPlug, BD Biosciences) was then added and culture reincubated for a further 4 hours at 37°C, 5% CO 2 . At the end of stimulation, rbcs were lysed and lymphocytes were fixed simul- taneously with BD Lyse/Fix solution (BD Biosciences), followed by lymphocyte permeabilization using BD Perm 2 solution (BD Biosciences) according to the manufacturer’s instructions. Cells were then stained with 50 μl of an antibody cocktail containing anti–human CD4-APC (BD Biosciences), anti–human CD8- APC-H7 (BD Biosciences), anti–human KLRG1-PE (BioLegend), anti–human IFN-γ-FITC (BD Biosci- ences), anti–human IL-2-AF700 (BioLegend), and anti–human TNF-PE-Cy7 (BD Biosciences) at previ- ously determined optimum dilution, along with 1 μl of human Fc receptor blocking solution (Human TruStain FcX, BioLegend) for 30 minutes at room temperature. It was washed and resuspended in buffer (PBS with 0.5% BSA and 2 mM EDTA) until acquisition. Further details on antibody clones are available in Supplemental Table 2. Acquisition was performed with BD LSR Fortessa 4 (BD Biosciences) and DIVA software. Boolean gates and cytokine positive cell frequencies were determined with FlowJo. Background was determined as the frequency of cytokine positive T cells after uninfected rbc extract stimulation. Poly- functionality index was calculated as previously described (40).
including IFN-γ (56). As JUNB can be phosphorylated and activated by JNK in immune cells (20), our data imply that JUNB, rather than c-JUN, might be the relevant target downstream of JNK in immune cells modulating IFN-γ expression during acute hepatitis. Hepatitis viruses have evolved to suppress the IFN antiviral response (57), and impaired IFN signaling is a common immune defect in human cancer (58). This might explain the rather limited benefit of IFN-based therapies in the clinic (14). However, recent data indicate that NK and NKT cells are part of the immune sup- pressive tumor microenvironment (59, 60) and that T cell–derived IFN-γ and TNF-α efficiently induce tumor arrest and senescence (61), prompting interest in exploring innovative cancer therapies based on the manipulation of T, NK, or NKT cells. Since a better understanding of the functions of NK and NKT cells is crucial for their rational use in tumor immunotherapies, our findings could well be relevant for cancer research.
IFN-γ induces the expression and membrane translocation of hWARS, which subsequently sensitizes cells to infection. hWARS was previously shown to be upregulated by IFN-γ in several epithelial and fibroblast cell lines (21). We hypothesized that IFN-γ produc- tion elicited by innate immunity during primary infection could induce the expression of hWARS in certain cell types. To test this, we selected a human neuron-committed teratocarcinoma cell line (NT2), primary human neuron stem cells (HNSCs), and primary human bronchial epithelial cells (HBEpCs), because neuronal and pulmonary cells represent the most physiologically relevant cell models for studying EV-A71, which is known to be associated with severe neurological diseases and pulmonary edema. NT2 is known to be poorly susceptible to EV-A71 infection under unstim- ulated conditions. We stimulated NT2 cells, as well as RD cells with different doses of IFN-γ. Under low-titer infection conditions (50% tissue culture infectious dose [TCID 50 ] per milliliter = 1), a small percentage of NT2 and RD cells were infected by EV-A71, as shown by the weak detection of viral protein using Western blot analysis (Figure 4A, lanes 1 and 5). When cells were cultured in the presence of IFN-γ, mimicking cytokine production as part of the host response to EV-A71 infection, we detected enhanced protein and mRNA expression of hWARS in an IFN-γ–dependent manner (Figure 4A; see Supplemental Figure 6, A and B for the expression of hWARS in response to treatment of proinflammatory cytokines in HNSCs and HBEpCs, respectively). More interestingly, we also observed plasma membrane translocation of hWARS upon IFN-γ stimulation (Figure 4B). The surface expression of hWARS in mock- and IFN-γ–treated NT2 and RD cells was further examined hWARS was further demonstrated in the orthogonal view of the
chronic OVA-induced model, a role for IFN-γ in airway inflamma- tion was also observed, in our SA model, a role for IFN-γ in airway inflammation was not evident, but instead, IL-17 was involved in promoting airway inflammation. In a study of wound healing in skin, Slpi-deficient mice were found to generate active TGF-β, which played a role in wound healing (43). Since TGF-β is a cen- tral mediator of airway remodeling (44), SLPI deficiency may also contribute to airway remodeling in asthma, another hallmark of SA and chronic asthma, which is believed to be responsible for per- sistent AHR (45). It is interesting to note that multiple allergens have protease activities as do different cell types, such as neutro- phils and mast cells. Thus, it is possible that SLPI may play a fun- damental role in inhibiting both allergen- and cell-associated pro- teases such that its downregulation promotes the SA phenotype via effects on AHR, FeNO levels, and airway remodeling. It will be useful to develop an inducible airway–specific SLPI transgenic mouse system to study effects of SLPI expression on allergic air- way disease — this approach may inhibit AHR to a greater degree due to local overexpression in airway epithelial cells where SLPI is normally expressed.
Asthma is considered a Th2 cell–associated disorder. Despite this, both the Th1 cell–associated cytokine IFN-γ and airway neutrophilia have been implicated in severe asthma. To investigate the relative contributions of different immune system components to the pathogenesis of asthma, we previously developed a model that exhibits several features of severe asthma in humans, including airway neutrophilia and increased lung IFN-γ. In the present studies, we tested the hypothesis that IFN-γ regulates mast cell function in our model of chronic asthma. Engraftment of mast cell–deficient Kit W-sh/W-sh mice, which develop markedly attenuated features of
We found that the immune system functions less efficiently in the elastic media of the great vessels than in small vessels, the adventitia of the great ves- sels, or visceral organs such as spleen, liver, and lung. For this reason the immune system, rather than viral inter- actions with cellular receptors, deter- mines tropism of γHV68 for the great elastic arteries during chronic disease. These findings make the important point that chronic diseases of the great vessels may be due to immunoprivilege of the elastic media, resulting in per- sistence of pathogens or antigens in this site, in turn leading to chronic inflammation. Lack of effective immune clearance was independent of IFN-γ, although IFN-γ prevented the establishment of medial infection and regulated the severity of disease once infection was established. This essential protective role of IFN-γ contrasts with studies emphasizing promotion of vas- cular pathology by IFN-γ (reviewed in ref. 7), underscoring the fact that the nature of the vascular insult determines whether specific immune functions are helpful or harmful.
FIGURE 4 | Anti-proliferative and pro-apoptotic effects of IFN-γ are ameliorated in DFT1.Tet/IFN-γ cultured with doxycycline. Cell proliferation and viability of DFT1.Tet/IFN-γ was compared against DFT1.WT cells cultured with or without 50 ng/ml IFN-γ for 10 days. (A) Differences in proliferation of DFT1.Tet/IFN-γ in the presence and absence of doxycycline for 1, 2, 5, and 10 days, assessed by WST-8 proliferation assay. The WST-8 assay uses absorbance at 450 nm as a proxy for the number of viable cells. Baseline absorbance was measured 2 days prior (t = −2) and change in absorbance was measured after 2 days (t = 0). Graph shows the average value of triplicates and a standard deviation error bar. (B) Cell viability of DFT1.Tet/IFN-γ in the presence and absence of doxycycline for 1, 2, 5, and 10 days. Cell death over 2 days (from t = −2 to t = 0) was quantitated by staining dead cells with DAPI and analysis by flow cytometry. The results shown are representative of n = 3 replicates/treatment. The percentages in the upper right and left quadrants were added to yield the total percentage of dead cells while the same was done for the lower quadrants to give the total percentage of viable cells.
While the model used here involved major histocompatibility mismatched allografts, there appears to be a striking similarity between our results and recent work on viral infections in large elastic arteries; increased transmural arterial inflammation occurs in GRKO mice infected with CMV (46) or γ-herpesvirus 68 (47), suggesting that the elastic media of great vessels may be an immune-privileged site, perhaps with privilege preserved in part by IFN-γ. The current study focused on Th1/Th2 cytokine milieu on AAA using allogeneic responses based on our previous find- ings of Th2-predominant cytokine expression in the human AAA lesions and did not explore the effects of atherosclerosis generally observed in human AAA lesions. It is possible that human ath- erosclerotic lesions need some additional (perhaps idiosyncratic) Th2-predominant inflammatory responses to develop aneurysm formation rather than Th1-dominated obstructive lesions. Fur- ther study remains to elucidate such triggers.
IL-2 is mainly produced by activated T-cell. The binding to the T-cell receptor stimulates IL-2 secretion and the expression of IL-2 receptor. Finally, the IL-2 receptor ligation activates effector T-cells and the establish- ment of T-cell memory. Millington et al. and Wang et al. have described that IL-2 has comparable sensitivity and specificity for active TB as well as IFN-γ and IP-10  . However, other recent studies suggest that IL-2 expression is lower in patients with active TB disease compared to latent TB infection (LTBI) and controls  . LTBI and infection control are dominated by central memory T-cells with potential IL-2 and option- ally IFN-γ co-secretion; where as active TB disease is characterized by loss of IL-2 production and T-cells with effector memory T-cell phenotype -. Mamishi et al. also described that IL-2 release stimulated by TB- specific antigens was significantly higher in individuals with LTBI compared to patients with active TB disease; therefore, IL-2 could be a potential biomarker for discriminating between active and latent TB infection  . On the other hand, concerning the position of TNF-α for IFN-γ and IL-2, Harari et al. evaluated CD4 T-cells producing IFN-γ, TNF-α and IL-2 by flow cytometry in patients with active TB disease and LTBI. As a result, the proportion of MTB-specific CD4+ TNF-α single –positive T-cells was predominantly found in pa- tients with active TB disease and that of polyfunctional MTB-specific CD4+T-cells producing TNF-α, IL-2 and IFN-γ was dominant in those with LTBI . Petruccioli et al. also described that bifunctional IFN-γ/TNF-α spe- cific cells and the effector memory phenotype were significantly associated with active TB disease compared to LTBI or cured TB . Although the reason for the lowest sensitivity and specificity of IL-2 compared to IFN-γ in this study may be explained by these previous reports, the lower sensitivity and specificity of TNF-α com- pared to IFN-γ was not compatible.
Direct proof of a cause and effect relationship between a Th1- like, proinflammatory circuit and the hepatobiliary injury has met remarkable experimental challenges as a result of the inability to study human samples prior to or at the time of bile duct obstruc- tion and the obvious ethical barriers to obtain normative data from livers of normal age-matched infants. Therefore, we used a mouse model of biliary atresia to test the hypothesis that IFN-γ plays a key regulatory role in the pathogenesis of bile duct injury and obstruction in biliary atresia. We found that hepatic lympho- cytes undergo Th1 commitment at the time of biliary injury and obstruction. More notably, loss of IFN-γ expression completely prevented the inflammatory and fibrosing obstruction of bile ducts, which resulted in resolution of symptoms and improved long-term survival.
Confirmatory testing and signature refinement across multiple cancer types demonstrates a common biology of responsiveness to PD-1 checkpoint blockade. Because activated Th1 and cytotoxic T cells are the likely direct effectors of checkpoint blockade–mediated tumor regression, we hypothesized that measures of this common biology would predict response to anti–PD-1 therapy regardless of tissue of origin. We therefore asked whether the biology of respon- siveness to PD-1 checkpoint blockade that was observed in mel- anoma would extend to unrelated tumor types. After confirming that the 2 preliminary signatures in melanoma showed statistical- ly significant associations with clinical outcome, both signatures were refined by removal of genes that did not reach a nominal 1-sided P value less than 0.05 for positive association with either objective response or PFS in the confirmatory set of 62 melano- ma tumors, yielding refined “IFN-γ (6-gene)” and “expanded immune (18-gene)” signatures. Subsequently, as data from the KEYNOTE-012 (NCT01848834) study became available, these signatures were tested in biopsy specimens from pembrolizum- ab-treated patients with HNSCC or gastric cancer. RNA expres- sion profiling data were available for tumors from 40 patients with HNSCC and were evaluable for objective response, with an objec- tive response rate of 22.5%. There were 33 such patients with gas- tric cancer, with 22.2% responding. The refined signatures (Table 2) both showed statistically significant associations with improved clinical outcome at P < 0.05 for objective response and PFS for HNSCC (19) and at P < 0.10 and P < 0.05 for objective response and PFS, respectively, in gastric cancer (Figure 3, A and B, and Table 3). Receiver operating characteristics (ROCs) for response status over the range of the signature scores demonstrated the high discriminatory ability of the signatures (Figure 3, C and D). Areas under the ROC curves and their 95% CIs were 0.80 (0.61–0.95) for HNSCC (19) and 0.66 (0.47–0.83) for gastric cancer. To illus- trate the potential clinical usefulness of these signatures to predict response to pembrolizumab, the Youden index–based cutoff val- ues (20) for the IFN-γ 6-gene signature were determined and were similar for the 2 cancer types (1.882 HNSCC; 1.856 gastric). For Table 2. IFN-γ and expanded immune gene signatures
Background: Rotavirus infection is one of the most common gastroenteritis in the world, and a million cases are registered to enter hospital every year. PMLs are IFN-up-regulated proteins, and one of their critical functions is working as antiviral proteins. Recently, PML-II has been depicted as an isoform responsible for the antiviral function. Methods: Rotavirus prevalence determination was achieved by PCR and Rapid Adeno/Rota Virus test, while the relative expression assay was carried out by real-time PCR technique. Blood and stool samples were collected from 34 children under five years admitted to the hospital with acute gastroenteritis showing signs of dehydration. RNA samples were extracted from blood specimens and converted to cDNA to be used in gene expression analysis of PML, PML-II, and IFN-γ in rotavirus positive or negative samples. Results: Rapid Adeno/Rota Virus Antigen Combo Test and PCR assay could detect the virus in stool samples in 45% and 17.6% of cases, respectively. PML in positive samples decreased to 10 4 fold less than the level in negative ones. The same trend was noticed in the level of IFN-γ and PML-II expression as their expression reduced to 10 4 or 13fold in rotavirus- infected samples compared to the control, respectively. Conclusion: Altogether, our data showed that the gene expression of PML, PML-II, and type II IFN considerably diminished in rotavirus-infected samples compared to the negative control. DOI: 10.29252/ibj.24.2.128
Reverse transcription (RT)-PCR analysis of IFN- γ inducible protein-10 (IP-10) mRNA expression induced by the interaction of synovial fluid (SF) leukocytes and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). SF monocytes (mono) or PMNs were layered onto RA FLS monolayers. Total RNA was isolated 12 hours later, after which RT-PCR was performed. (a) Representative expression of IP-10 mRNA; expression of β -actin mRNA served as an internal control. Lane M contains molecular weight markers (100 base pair [bp] ladder). (b) IP-10 mRNA expression was quantified and normalized to β -actin as the IP-10/ β -actin ratio. Data are expressed as means ± SEM for three independent experiments that were performed using three different RA fibroblasts and three different RA SF leukocytes.