Soluble IFN-γ Research Articles

T lymphocyte-derived IFN-γ facilitates breast cancer cells to pass the blood-brain barrier: An in vitro study corroborating translational data

The appearance of brain metastasis is the most serious complication of breast cancer with mostly fatal outcomes. To reach the brain, tumor cells need to pass the blood-brain barrier (BBB). The molecular mechanisms underlying penetration of the BBB are largely unknown. Previously we found that tumor-infiltrating T lymphocytes enhance the development of brain metastasis of estrogen receptor-negative (ER-) breast cancer. In the current study, we investigate the contribution of T lymphocytes and the IFN-γ pathway in enabling breast cancer cells to pass the in vitro BBB. CD8+ cells display the strongest stimulatory effect on breast cancer cell passage. We show that inhibition of the IFN-γ receptor in MDA-MB-231 breast cancer cells, or neutralization of soluble IFN-γ, impairs the in vitro trespassing of breast cancer cells. Importantly, we validated our findings using gene expression data of breast cancer patients. The CXCL-9,-10,-11/CXCR3 axis, dependent on IFN-γ signaling activity, was overexpressed in primary breast cancer samples of patients who developed brain metastasis. The data support a role for T-lymphocytes and the IFN-γ pathway in the formation of brain metastasis of ER-breast cancer, and offer targets to design future therapies for preventing breast cancer cells to cross the BBB.

Abstract 2810: Increasing the efficacy of KRASG12Cinhibitor therapy through the adjuvant use of FAP targeted radiotherapy in a murine model of lung cancer

Abstract The purpose of this study was to evaluate the combined effects of KRAS targeted therapy and FAP targeted radiotherapy compared to either therapy alone in an animal model of lung cancer. KRAS is a protein that acts as a molecular switch regulating cell proliferation mediated through binding of GTP versus GDP. When mutated, the switch remains “on” and cells undergo unrestrained proliferation. KRAS mutations are frequently found in lung cancer and it appeared for years as if they could not be inhibited. Recently KRASG12C inhibitors have been developed by targeting a site adjacent to the binding pocket that induces a conformational change in the protein preventing GTP binding thus inhibiting unrestrained cell cycling. While a promising advance, KRAS targeted therapy does not confer a durable response and therefore it is essential to develop adjuvant therapies that enhance efficacy of KRAS targeting agents. FAP targeted radiotherapy is a new anticancer therapeutic that delivers a radioactive payload directly to tumors and has been shown to increase efficacy when used with immunomodulatory therapy. We studied the effect of KRAS targeted therapy alone and in combination with FAP targeted radiotherapy. Animals were implanted with KRASG12C heterozygote LLC1 lung cancer cells and treated according to the following groups: untreated control, sotorasib treatment, 177Lu-FAPI-04 treatment, and combined treatment. Tumor growth was evaluated every 2-3 days and animals were euthanized at 21 days and tumors extracted for analysis. anti-Ki67 and anti-caspase-3 immunofluorescence assessed cell cycling and apoptotic cell death, respectively. Immunoassays were performed to quantify soluble analytes, including IL-12p70, TNF-α, TGF-β1, and IFN-γ. Tumor growth in dual treated animals was significantly reduced compared to either therapy alone. Ki67 immunofluorescence demonstrated a significantly decreased number of cells in the combined therapy group than either therapy alone (p=0.01 and 0.02 when compared to sotorasib alone and radiotherapy alone, respectively). Anti-caspase-3 immunofluorescence demonstrated a significant increase in positive cells in the combined group compared to animals treated with sotorasib alone (p=0.003). Immunoassays assessing expression of TNF-α, TGF-β1, and IFN-γ from tumors from animals in each treatment group demonstrated statistically significant differences (p=0.006, p=0.03, p=0.02, respectively). Combined KRAS targeted therapy and FAP targeted radiotherapy results in decreased tumor growth compared to either therapy alone. Effects are mediated through decreased cell cycling, increased apoptotic cell death, and changes in key cell signaling pathways. Future translational studies will assess this therapy combination in patients to evaluate its ability to increase response to therapy. Citation Format: Kathleen M. Capaccione, Mikhail Doubrovin, Brian Braumuller, Sophia Huang, Nikunj Bhatt, Fatemeh Momen-Heravi, Andrei Molotkov, Michael Kissner, Alessandra Ali, Akiva Mintz. Increasing the efficacy of KRASG12Cinhibitor therapy through the adjuvant use of FAP targeted radiotherapy in a murine model of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2810.

Delivery of Immobilized IFN-γ With PCN-333 and Its Effect on Human Mesenchymal Stem Cells

Interferon-gamma (IFN-γ) plays a vital role in modulating the immunosuppressive properties of human mesenchymal stem/stromal cells (hMSCs) used in cell therapies. However, IFN-γ suffers from low bioavailability and degrades in media, creating a challenge when using IFN-γ during the manufacturing of hMSCs. Metal-organic frameworks (MOFs), with their porous interiors, biocompatibility, high loading capacity, and ability to be functionalized for targeting, have become an increasingly suitable platform for protein delivery. In this work, we synthesize the MOF PCN-333(Fe) and show that it can be utilized to immobilize and deliver IFN-γ to the local extracellular environment of hMSCs. In doing so, the cells proliferate and differentiate appropriately with no observed side effects. We demonstrate that PCN-333(Fe) MOFs containing IFN-γ are not cytotoxic to hMSCs, can promote the expression of proteins that play a role in immune response, and are capable of inducing indoleamine 2,3-dioxygenase (IDO) production similar to that of soluble IFN-γ at lower concentrations. Overall, using MOFs to deliver IFN-γ may be leveraged in the future in the manufacturing of therapeutically relevant hMSCs.

Serum biomarker panel diagnostics in pancreatic ductal adenocarcinoma: the clinical utility of soluble interleukins, IFN-γ, TNF-α and PD-1/PD-L1 in comparison to established serum tumor markers

PurposeNovel biomarkers to better predict outcome and select the best therapeutic strategy for the individual patient are necessary for pancreatic ductal adenocarcinoma (PDAC).MethodsUsing a panel assay, multiple biomarkers (IFN-γ, IL-10, IL-6, IL-8, TNF-α, CEA, CA 19–9, CYFRA 21–1, HE4, PD-1 and PD-L1 levels) were measured in serum samples of 162 patients with resected, locally advanced and metastatic PDAC in this retrospective single-center study. Optimal cut-off values to differentiate prognostic subgroups with significantly different overall survival (OS) were determined by receiver operator characteristics and Youden Index analysis. Marker levels were assessed before the start of chemotherapy and correlated with OS by univariate and multivariate Cox analysis.ResultsMedian OS for resected patients was 28.2 months, for locally advanced patients 17.9 months and for patients with metastatic disease 8.6 months. CYFRA 21–1 and IL-8 discriminated metastatic from locally advanced patients best (AUC 0.85 and AUC 0.81, respectively). In univariate analyses, multiple markers showed prognostic relevance in the various subgroups. However, multivariate Cox models comprised only CYFRA 21–1 in the resected group (HR 1.37, p = 0.015), IL-10 in locally advanced PDAC (HR 10.01, p = 0.014), as well as CYFRA 21–1 and CA 19–9 in metastatic PDAC (p = 0.008 and p = 0.010) as an independent prognostic marker for overall survival.ConclusionIL-10 levels may have independent prognostic value in locally advanced PDAC, whereas CYFRA 21–1 levels are prognostic after PDAC surgery. CYFRA 21–1 and IL-8 have been identified to best discriminate metastatic from locally advanced patients.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for cell therapies due to their immunosuppressive capacity that can be enhanced in the presence of interferon-gamma (IFN-γ). In this study, multilayers of heparin (HEP) and collagen (COL) (HEP/COL) were used as a bioactive surface to enhance the immunomodulatory activity of hMSCs using soluble IFN-γ. Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. We evaluated the viability, adhesion, real-time growth, differentiation, and immunomodulatory activity of hMSCs on (HEP/COL) multilayers. HMSCs viability, adhesion, and growth were superior when cultured on (HEP/COL) multilayers compared to tissue culture plastic. We also confirmed that hMSCs osteogenic and adipogenic differentiation remained unaffected when cultured in (HEP/COL) multilayers in the presence of IFN-γ. We measured the immunomodulatory activity of hMSCs by measuring the level of indoleamine 2,3-dioxygenase (IDO) expression. IDO expression was higher on (HEP/COL) multilayers treated with IFN-γ. Lastly, we evaluated the suppression of peripheral blood mononuclear cell (PBMC) proliferation when co-cultured with hMSCs on (HEP/COL) multilayers with IFN-γ. hMSCs cultured in (HEP/COL) multilayers in the presence of soluble IFN-γ have a greater capacity to suppress PBMC proliferation. Altogether, (HEP/COL) multilayers with IFN-γ in culture medium provides a potent means of enhancing and sustaining immunomodulatory activity to control hMSCs immunomodulation.

Increased Circulating Levels of Inflammatory Markers in A Cohort of Adults with Youth-Onset Type 2 Diabetes Mellitus

Background: Young-onset type 2 diabetes (T2DMY) is considered to have a more aggressive disease phenotype, unfavourable long-term outcomes, and a close association with low-grade chronic inflammation. The aim of this study was to explore the circulating inflammatory profile in a cohort of adults with T2DMY and assess its potential associations. Methods: T2DMY adults (n=65) and non-diabetic controls (n=40) underwent detailed anthropometric and biochemical characterisation. Serum levels of six pro-inflammatory biomarkers (soluble E-selectin, IL-1β, IFN-γ, IL-6, IL-8 and angiopoietin like protein-4) were measured by Magnetic Luminex Assays and were compared between study groups. Potential associations of these biomarkers with anthropometric/biochemical parameters and microvascular complications were also tested in the T2DMY group. Results: T2DMY patients had significantly higher circulating levels of all measured inflammatory markers compared to controls, even after adjusting for age and gender (E-selectin: 65.9 ± 4.5 vs. 37.9 ± 8.2 ng/ml; IFN-γ: 220.9 ± 7.4 vs. 160.6 ± 13.5 pg/ml; IL1-β: 50.1 ± 1.8 vs. 38.0 ± 3.3 pg/ml; IL-6: 17.5 ± 0.7 vs. 11.4 ± 1.1 pg/ml; IL-8: 18.1 ± 1.5 vs. 11.7 ± 2.4 pg/ ml; ANGPTL-4: 162.8 ± 9.8 vs. 109.0 ± 15.6 ng/ml; all p-values<0.05). Significant positive correlations were found between fat mass and IL-6 and ANGPTL-4; waist-to-hip ratio and E-selectin, IFN-γ, IL-1β and IL-6; and HbA1c and E-selectin, IFN-γ, IL-1β and IL-6. Within the T2DMY group, E-selectin was significantly elevated in patients with combined albuminuria and retinopathy compared with those without these complications (88.97 ± 43.59 vs 59.38 ± 25.28 ng/ml; p=0.003). Conclusions: T2DMY patients exhibit an adverse profile of key circulating pro-inflammatory biomarkers, which appears associated with worse anthropometric/biochemical parameters and microvascular complications. The role of these biomarkers as predictors of T2DMY and disease progression should be further elucidated.

Metabolomic and Signaling Programs Induced by Immobilized versus Soluble IFN γ in Neural Stem Cells.

Neural stem cells (NSCs) provide a strategy to replace damaged neurons following traumatic central nervous system injuries. A major hurdle to translation of this therapy is that direct application of NSCs to CNS injury does not support sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply physical and biochemical signals to instruct the encapsulated NSCs to be neurogenic. However, the immobilization of factors, including IFN-γ, versus soluble delivery of the same factor, has been incompletely characterized especially with respect to activation of signaling and metabolism in cells over longer time points. In this study, protein and metabolite changes in NSCs induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs compared to immobilized IFN-γ, indicating that immobilization may not sustain signaling or has altered ligand/receptor interaction and integrity. However, both IFN-γ delivery types supported increased βIII tubulin expression in parallel with canonical and noncanonical receptor-signaling compared to no IFN-γ. Global metabolomics and pathway analysis revealed that soluble and immobilized IFN-γ altered metabolic pathway activities including energy, lipid, and amino acid synthesis, with soluble IFN-γ having the greatest metabolic impact overall. Finally, soluble and immobilized IFN-γ support mitochondrial voltage-dependent anion channel (VDAC) expression that correlates to differentiated NSCs. This work utilizes new methods to evaluate cell responses to protein delivery and provides insight into mode of action that can be harnessed to improve regenerative medicine-based strategies.

Persistent Immune Activation in HIV-1-Infected Ex Vivo Model Tissues Subjected to Antiretroviral Therapy: Soluble and Extracellular Vesicle-Associated Cytokines.

Residual immune activation after successful antiretroviral therapy (ART) in HIV-1-infected patients is associated with the increased risk of complications. Cytokines, both soluble and extracellular vesicle (EV)-associated, may play an important role in this immune activation. Ex vivo tissues were infected with X4LAI04 or R5SF162 HIV-1. Virus replicated for 16 days, or tissues were treated with the anti-retroviral drug ritonavir. Viral replication and production of 33 cytokines in soluble and EV-associated forms were measured with multiplexed bead-based assays. Both variants of HIV-1 efficiently replicated in tissues and triggered upregulation of soluble cytokines, including IL-1β, IL-7, IL-18, IFN-γ, MIP-1α, MIP-1β, and RANTES. A similar pattern was observed in EV-associated cytokine release by HIV-infected tissues. In addition, TNF-α and RANTES demonstrated a significant shift to a more soluble form compared with EV-associated cytokines. Ritonavir treatment efficiently suppressed viral replication; however, both soluble and EV-associated cytokines remained largely upregulated after 13 days of treatment. EV-associated cytokines were more likely to remain elevated after ART. Treatment of uninfected tissues with ritonavir itself did not affect cytokine release. We demonstrated that HIV-1 infection of ex vivo lymphoid tissues resulted in their immune activation as evaluated by upregulation of various cytokines, both soluble and EV-associated. This upregulation persisted despite inhibition of viral replication by ART. Thus, similar to in vivo, HIV-1-infected human tissues ex vivo continue to be immune-activated after viral suppression, providing a new laboratory model to study this phenomenon.

Covalently Immobilizing Interferon-γ Drives Filopodia Production through Specific Receptor-Ligand Interactions Independently of Canonical Downstream Signaling.

Immobilizing a signaling protein to guide cell behavior has been employed in a wide variety of studies. This approach draws inspiration from biology, where specific, affinity-based interactions between membrane receptors and immobilized proteins in the extracellular matrix guide many developmental and homeostatic processes. Synthetic immobilization approaches, however, do not necessarily recapitulate the in vivo signaling system and potentially lead to artificial receptor-ligand interactions. To investigate the effects of one example of engineered receptor-ligand interactions, we focus on the immobilization of interferon-γ (IFN-γ), which has been used to drive differentiation of neural stem cells (NSCs). To isolate the effect of ligand immobilization, we transfected Cos-7 cells with only interferon-γ receptor 1 (IFNγR1), not IFNγR2, so that the cells could bind IFN-γ but were incapable of canonical signal transduction. We then exposed the cells to surfaces containing covalently immobilized IFN-γ and studied membrane morphology, receptor-ligand dynamics, and receptor activation. We found that exposing cells to immobilized but not soluble IFN-γ drove the formation of filopodia in both NSCs and Cos-7, showing that covalently immobilizing IFN-γ is enough to affect cell behavior, independently of canonical downstream signaling. Overall, this work suggests that synthetic growth factor immobilization can influence cell morphology beyond enhancing canonical cell responses through the prolonged signaling duration or spatial patterning enabled by protein immobilization. This suggests that differentiation of NSCs could be driven by canonical and non-canonical pathways when IFN-γ is covalently immobilized. This finding has broad implications for bioengineering approaches to guide cell behavior, as one ligand has the potential to impact multiple pathways even when cells lack the canonical signal transduction machinery.

P.P476S mutation of RBPJL inhibits the efficacy of anti‐PD‐1 therapy in oesophageal squamous cell carcinoma by blunting T‐cell responses

ObjectivesAnti‐PD‐1 immune checkpoint blockade represents the onset of a new era in cancer immunotherapy. However, robust predictors are necessary for screening patients with immune checkpoint‐responsive oesophageal squamous cell carcinoma (ESCC).MethodsWe obtained biopsy samples from an ESCC patient with mixed responses. The expression of CD4, CD8, CD68, PD‐L1, RBPJL and IL‐16 was analysed by immunohistochemistry, and the correlation with prognostic value was obtained from the GEPIA portal. T‐cell functions were examined by flow cytometry, MTS and transwell assays. The secreted cytokines were identified using an Inflammation Array Kit. The concentration of soluble IFN‐γ was measured by enzyme‐linked immunosorbent assay. The clinical benefit of RBPJL was examined in a PBMC xenograft mouse model.ResultsThe patient had an exceptional clinical response with shrinkage of the primary oesophageal and lung metastatic lesions as well as enlargement of liver metastatic lesions after toripalimab monotherapy. Four liver‐specific gene mutations were identified. RBPJL showed better response to toripalimab in the PBMC cell‐derived xenograft (CDX) ESCC model. Conditional medium from RBPJL overexpression induced chemotaxis and proliferation of T lymphocytes, as well as Th2/Th1 differentiation through the RBPJL‐NF‐κB‐IL‐16 axis in vitro. These functions were all inhibited by the p.P476S mutation of RBPJL (RBPJL (p.P476S)).ConclusionsWe report for the first time that RBPJL (p.P476S) promotes tumor growth in ESCC and inhibits the efficacy of anti‐PD‐1 therapy through blunting T‐cell responses. Our findings provide a potential new predictor for evaluating the efficacy of anti‐PD‐1 therapy in ESCC patients.

P11.10 The IFNγ pathway mediates brain metastasis formation of breast cancer

Abstract BACKGROUND In previous work we showed the prominence of the T-cell response in the formation of brain metastases of primary ER negative breast cancers (Mustafa et al, Acta Neuropathol 2018). We also showed that breast cancer cells co-cultured with stimulated T lymphocytes overexpress Guanylate-binding protein 1 (GBP1) accompanying increased trespassing ability through an in vitro blood-brain barrier (BBB) model. In addition, we demonstrated a predilection for metastasizing to brain of breast cancer cells that were co-cultured with activated T cells in a mouse model. We now scrutinize the importance of the IFNγ pathway for tresspassing of the tumor cells through the BBB following T cell contact. MATERIAL AND METHODS Anti-hIFN-γ-IgA antibodies were used to neutralize the IFNγ effects on the tumor cells. The effects on the tumor cells is only due to native IFNγ produced by activated T cells, not by recombinant IFNγ. Since the IFNγ expression itself enhances its expression by the T cells, we blocked IFNγ receptors prior to adding CD3+ T cell conditioned media to the breast cancer cells. The receptor blocking was achieved by antibodies to the IFNγα and IFNγβ subunits. Activation of the STAT1 pathway was monitored by GBP1 expression. For functional read-out the in vitro BBB model was used. RESULTS The presence of T-lymphocyte-secreted IFNγ in the primary breast cancer microenvironment activates the STAT1-dependent IFNγ pathway in breast cancer cells, endowing them with an increased ability to trespass the in vitro BBB. Moreover, direct inhibition of soluble IFNγ, or blocking of the IFNγ-specific receptor in breast cancer cells significantly decreases their ability to cross the BBB. CONCLUSION The results illustrate the specific action of T lymphocytes in the formation of cerebral metastasis involves the IFNγ signaling pathway as one of the crucial entangled pathways Subsequent studies should aim at the interference with the IFNγ pathway to develop preventive strategies against the formation of cerebral metastases of breast cancer.

BSCI-25. THE ROLE OF THE IFNγ PATHWAY IN BREAST CANCER BRAIN METASTASIS FORMATION

In previous work, we showed the prominence of the T cell response in the formation of brain metastases of primary ER-negative breast cancers. We also showed that prior co-cultured breast cancer cells with stimulated T lymphocytes bear an overexpression of Guanylate-binding protein 1 (GBP1) and possess an increased trespassing ability through an in vitro blood-brain barrier (BBB) model. In addition, we demonstrated a predilection for metastasizing to the brain of breast cancer cells that were co-cultured with activated T cells in a mouse model. In the present work, we show that activated CD8+ cytotoxic T lymphocytes, rather than CD4+ lymphocytes, are the main cause of increasing the ability of breast cancer cells to cross the BBB. While synthetic IFNγ does not change the ability of breast cancer cells to cross the BBB, this study shows that the T lymphocyte-secreted IFNγ activates the STAT1-dependent IFNγ pathway in breast cancer cells, enabling them to cross the in vitro BBB. Direct inhibition of soluble IFNγ or blocking of the IFNγ-specific receptor in breast cancer cells significantly decreases their ability to cross the BBB. The results illustrate that IFNγ signaling pathway is one of the crucial pathways in the formation of brain metastasis of ER- breast cancer. The interference with the IFNγ pathway will develop preventive strategies against the formation of brain metastases of breast cancer.

SOLUBLE ST2 AND CD163 AS POTENTIALBIOMARKERS TO DIFFERENTIATE PRIMARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS FROM MACROPHAGE ACTIVATION SYNDROME

The differentiation of primary hemophagocytic lymphohistiocytosis (pHLH) and macrophage activation syndrome (MAS) poses a challenge to hematologists. The aim of this study was (1) to compare the levels of soluble ST2 (sST2), sCD163, IFN-γ, IL-10, IL-18, TNF-α and Serum soluble interleukin-2 receptor (sCD25) in patients with pHLH and MAS and (2) to investigate whether they can help differentiate the two diseases. A total of 52 participants were recruited in this study, including 12 pHLH patients, 20 MAS patients, and 20 healthy subjects. We measured the levels of sST2, sCD163 and sCD25 in serum by ELISA. The serum levels of IFN-γ, IL-10, IL-18, and TNF-α were detected using a Luminex 200 instrument. The serum levels of sST2 and sCD163 in MAS patients were markedly higher than that in pHLH patients (363.13 ± 307.24 ng/ml vs 80.75 ± 87.04 ng/ml, P = 0.004; 3532.72 ± 2479.68 ng/ml vs 1731.96 ± 1262.07 ng/ml, P = 0.046). There was no significant difference in the expression of IFN-γ (306.89 ± 281.60 pg/ml vs 562.43 ± 399.86 pg/ml), IL-10 (20.40 ± 30.49 pg/ml vs 8.3 ± 13.14 pg/ml), IL-18 (463.33 ± 597.04 pg/ml vs 1247.82 ± 1318.58 pg/ml), TNF-α (61.48 ± 84.69 pg/ml vs 106.10 ± 77.21 pg/ml), and sCD25 (21062.1 ± 18515.26 pg/ml vs 11074.78 ± 11149.96 pg/ml) between pHLH and MAS. Patients with pHLH and MAS show some differences in cytokine profiles. The elevated levels of IFN-γ, IL-10, TNF-α, IL-18, and sCD25 can contribute to the diagnosis of HLH, but may not discriminate pHLH from MAS. Levels of sST2 and sCD163 may serve as markers to distinguish pHLH from MAS.

STAT4 and T-bet control follicular helper T cell development in viral infections.

Follicular helper T (Tfh) cells promote germinal center (GC) B cell survival and proliferation and guide their differentiation and immunoglobulin isotype switching by delivering contact-dependent and soluble factors, including IL-21, IL-4, IL-9, and IFN-γ. IL-21 and IFN-γ are coexpressed by Tfh cells during viral infections, but transcriptional regulation of these cytokines is not completely understood. In this study, we show that the T helper type 1 cell (Th1 cell) transcriptional regulators T-bet and STAT4 are coexpressed with Bcl6 in Tfh cells after acute viral infection, with a temporal decline in T-bet in the waning response. T-bet is important for Tfh cell production of IFN-γ, but not IL-21, and for a robust GC reaction. STAT4, phosphorylated in Tfh cells upon infection, is required for expression of T-bet and Bcl6 and for IFN-γ and IL-21. These data indicate that T-bet is expressed with Bcl6 in Tfh cells and is required alongside STAT4 to coordinate Tfh cell IL-21 and IFN-γ production and for promotion of the GC response after acute viral challenge.

The expression of triggering receptor expressed on myeloid cells receptor-1 in Aspergillus infected mice

Objective: To investigate the expression of triggering receptor expressed on myeloid cells receptor-1 (TREM-1) in plasma and bronchoalveolar lavage fluid (BALF) and its correlation with Galactomannan, IFNγ, IL-6 and IL-10 in Aspergillus infected mice. Methods: Cyclophosphamide(CTX) was intraperitoneally injected and fumigatus spore suspension was inhaled by nose to establish the immunocompromised invasive pulmonary aspergillosis(IPA) mouse model.Healthy controls, immunocompromised only and IPA only groups were also established. Each group had 6 mice. After inoculation, mice were sacrificed. Lung tissue specimens, BALF, and plasma samples were collected. Plasma and BALF soluble TREM-1 (sTREM-1), Galactomannan, IFNγ, IL-6, and IL-10 were detected by ELISA. Results: Positive Aspergillus fumigatus was found by tissue culture in the lung. Infiltration of inflammatory cells, blood congestion and interstitial lung tissue injury were observed in histological sections of both IPA and immunocompromised IPA mice. Compared to IPA group [(453.78±74.18) ng/L, P<0.001; (10.21±1.46) ng/L, P<0.001] and control group [(245.16±65.85) ng/L, P<0.001; (6.60±3.74) ng/L, P<0.001], the plasma and BALF sTREM-1 significantly increased in immunocompromised IPA group [(1 537.64±359.52) ng/L; (20.12±2.72) ng/L]. Compared to control group, both the BALF sTREM-1 in IPA group (P=0.041) and the plasma and BALF Galactomannan, IFNγ, IL-6, and IL-10 levels in IPA and immunocompromised IPA groups were significantly higher (P<0.01). Pearson correlation analysis showed that plasma and BALF sTREM-1 were significantly correlated with Galactomannan (r=0.83, P<0.001; r=0.82, P<0.001), IFNγ (r=0.79, P<0.001; r=0.61, P<0.01), IL-6 (r=0.81, P<0.001; r=0.66, P<0.01), and IL-10 (r=0.70, P=0.001; r=0.54, P=0.02). Conclusions: Plasma and BALF sTREM-1 appears highly expressed in Aspergillus infected mice. sTREM-1 in mice plasma and BALF is closely correlated with Galactomannan, IFNγ, IL-6, and IL-10 levels, which suggests that sTREM-1 has great diagnostic value during invasive fungal infection.

Establishing the reference intervals of NK cell functions in healthy adults

Natural killer (NK) cells play a key role in host defense against microbial pathogens. Establishing the reference intervals (RIs) of NK cell functions would be valuable in assessing the immune status of hosts. We evaluated the NK cell activity in healthy adults. We further established and validated the RIs of representative NK cell functions. Flow cytometry was used to evaluate the cytokine production and CD107a degranulation of NK cells. Levels of soluble IFN-γ in the culture supernatants were evaluated by ELISA. Our results demonstrated that the intracellular IFN-γ production of NK cells was positively correlated with CD107a expression and soluble IFN-γ levels. There were no significant differences in NK cell functions between different age and gender groups. The mean values and RIs of representative NK cell functions are as following: IFN-γ+ NK cells (%): 28.09 (11.3–51.95); CD107a+ NK cells (%): 17.90 (9.852–27.56); soluble IFN-γ (pg/ml): 330.4 (41.38–717.8). In addition, the intracellular IFN-γ production and degranulation activity of NK cells in patients with colorectal cancer were significantly lower than that in healthy adults. Our study has established the RIs of NK cell functions in healthy adults, which might be used for monitoring the immune status of the hosts.

IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability and survival by activating Erk1/2 and S6K1 pathways in neoplastic B-lymphoid cells

B-cell activating factor of the TNF family (BAFF) has been documented to act as a critical factor in the development of aggressive B lymphocytes and autoimmune diseases. However, the effect of various cytokines on BAFF-elicited neoplastic B-lymphoid cells is not known. In this study, we exhibited that administration of human soluble BAFF (hsBAFF), IL-2, IL-4, IFN-γ, or TNF-α alone increased cell viability and survival in Raji cells concentration-dependently, yet a more robust viability/survival was seen in the cells co-treatment of IL-2, IL-4, IFN-γ, or TNF-α with hsBAFF, respectively. Further research revealed that both Erk1/2 and S6K1 signaling pathways were essential for IL-2, IL-4, IFN-γ, or TNF-α enhancement of the viability/survival in the hsBAFF-stimulated cells, as inhibition of Erk1/2 with U0126 or down-regulation of Erk1/2, or blockage of S6K1 with rapamycin or silencing S6K1, or silencing S6K1/Erk1/2, respectively, reduced the cell viability/survival in the cells treated with/without hsBAFF±IL-2, IL-4, IFN-γ, or TNF-α. These findings indicate that IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability/survival by activating Erk1/2 and S6K1 signaling in neoplastic B-lymphoid cells. Our data suggest that modulation of IL-2, IL-4, IFN-γ and/or TNF-α levels, or inhibitors of Erk1/2 or S6K1 may be a new approach to prevent BAFF-induced aggressive B-cell malignancies.

Dysregulation of T helper-type cytokines appears prior to and concurrent with appearance of autoantibody specificities during early SLE pathogenesis and contributes to prognosis of transition to classified disease

Abstract Autoantibodies and immune dysregulation are associated with systemic lupus erythematosus (SLE), but how these factors influence disease development is unknown. This study evaluates timing and changes in SLE-linked autoantibodies and soluble mediator pathways in the years preceding SLE classification. Serial sera from 84 SLE cases spanning pre- and post-SLE classification (average time = 5.98 years) and matched healthy controls (HC) were obtained from the Department of Defense Serum Repository. Adjusting for multiple comparison, a number of soluble mediators, including IL-5 (q=4.35×10−6), IL-6 (q=8.26×10−6), and IFN-γ (q=0.037), were significantly elevated in cases vs. HC &amp;gt;3.5 years pre-classification, prior to (IL-5 and IL-6) and concurrent with (IFN-γ) the earliest SLE-specific autoantibody specificity, anti-Ro/SSA. Th1-type, Th17-type, and TNF superfamily soluble mediators increased longitudinally in cases approaching SLE classification, but not in HC (q&amp;lt;0.05). In particular, serum levels of BLyS and APRIL were comparable in cases and HC until &amp;lt;10 months pre-classification (q=0.003 and q=0.019, respectively). Random forest models incorporating IL-5, IL-6, and IFN-γ levels (79–82% accuracy) distinguished future SLE patients better than models with ANA alone (58% accuracy) &amp;gt;3.5 years before classification. These data indicate that pre-clinical immune system perturbations allow for the accumulation of autoantibodies preceding clinical symptoms and SLE classification.

The Aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNFα, and IFNγ cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNFα- and IFNγ-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes.

T lymphocyte-derived TNF and IFN-γ repress HFE expression in cancer cells

The immune system and tumors are closely intertwined initially upon tumor development. During this period, tumors evolve to promote self-survival through immune escape, including by targeting crucial components involved in the presentation of antigens to the immune system in order to avoid recognition. Accordingly, components involved in MHC I presentation of tumor antigens are often mutated and down-regulated targets in tumors. On the other hand, the immune system has been shown to influence tumors through production of immunosuppressive cytokines, recruitment and polarization of cells favoring or impeding tumor escape or through production of anti-tumor cytokines promoting tumor rejection. We previously discovered that the hemochromatosis protein HFE, a negative regulator of iron absorption, dampens classical MHC I antigen presentation. In this study, we evaluated the impact of activated T lymphocytes purified from peripheral blood mononuclear cells (PBMC) on HFE expression in tumor cell lines. We co-cultured tumor cell lines from melanoma, lung, and kidney cancers with anti-CD3-activated PBMC and established that HFE expression is increased in tumor cell lines compared to healthy tissues, whilst being down-regulated significantly upon exposure to activated PBMC. HFE down-regulation was mediated by both CD4 and CD8 T lymphocytes, through production of soluble mediators, namely TNF and IFN-γ. These results suggest that the immune system may modulate tumor HFE expression in inflammatory conditions in order to regulate MHC I antigen presentation and promote tumor clearance.