Interferon-γ couples CD8+ T cell avidity and differentiation during infection.

Effective responses to intracellular pathogens are characterized by T cell clones with a broad affinity range for their cognate peptide and diverse functional phenotypes. How T cell clones are selected throughout the response to retain a breadth of avidities remains unclear. Here, we demonstrate that direct sensing of the cytokine IFN-γ by CD8+ T cells coordinates avidity and differentiation during infection. IFN-γ promotes the expansion of low-avidity T cells, allowing them to overcome the selective advantage of high-avidity T cells, whilst reinforcing high-avidity T cell entry into the memory pool, thus reducing the average avidity of the primary response and increasing that of the memory response. IFN-γ in this context is mainly provided by virtual memory T cells, an antigen-inexperienced subset with memory features. Overall, we propose that IFN-γ and virtual memory T cells fulfil a critical immunoregulatory role by enabling the coordination of T cell avidity and fate.

IFNγ signaling in cytotoxic T cells restricts anti-tumor responses by inhibiting the maintenance and diversity of intra-tumoral stem-like T cells.

IFNγ is an immune mediator with concomitant pro- and anti-tumor functions. Here, we provide evidence that IFNγ directly acts on intra-tumoral CD8 T cells to restrict anti-tumor responses. We report that expression of the IFNγ receptor ß chain (IFNγR2) in CD8 T cells negatively correlates with clinical responsiveness to checkpoint blockade in metastatic melanoma patients, suggesting that the loss of sensitivity to IFNγ contributes to successful antitumor immunity. Indeed, specific deletion of IFNγR in CD8 T cells promotes tumor control in a mouse model of melanoma. Chronic IFNγ inhibits the maintenance, clonal diversity and proliferation of stem-like T cells. This leads to decreased generation of T cells with intermediate expression of exhaustion markers, previously associated with beneficial anti-tumor responses. This study provides evidence of a negative feedback loop whereby IFNγ depletes stem-like T cells to restrict anti-tumor immunity. Targeting this pathway might represent an alternative strategy to enhance T cell-based therapies.

New Paradigms to Assess Consequences of Long-Term, Low-Dose Curcumin Exposure in Lung Cancer Cells.

Curcumin has been investigated extensively for cancer prevention, but it has been proposed that long-term treatments may promote clonal evolution and gain of cellular resistance, potentially rendering cancer cells less sensitive to future therapeutic interventions. Here, we used long-term, low-dose treatments to determine the potential for adverse effects in non-small cell lung cancer (NSCLC) cells. IC50s for curcumin, cisplatin, and pemetrexed in A549, PC9, and PC9ER NSCLC cells were evaluated using growth curves. IC50s were subsequently re-assessed following long-term, low-dose curcumin treatment and a three-month treatment withdrawal period, with a concurrent assessment of oncology-related protein expression. Doublet cisplatin/pemetrexed-resistant cell lines were created and the IC50 for curcumin was determined. Organotypic NSCLC-fibroblast co-culture models were used to assess the effects of curcumin on invasive capacity. Following long-term treatment/treatment withdrawal, there was no significant change in IC50s for the chemotherapy drugs, with chemotherapy-resistant cell lines exhibiting similar sensitivity to curcumin as their non-resistant counterparts. Curcumin (0.25-0.5 µM) was able to inhibit the invasion of both native and chemo-resistant NSCLC cells in the organotypic co-culture model. In summary, long-term curcumin treatment in models of NSCLC neither resulted in the acquisition of pro-carcinogenic phenotypes nor caused resistance to chemotherapy agents.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Paracrine costimulation of IFN-γ signaling by integrins modulates CD8 T cell differentiation.

The cytokine IFN-γ is a critical regulator of immune system development and function. Almost all leukocytes express the receptor for IFN-γ, yet each cell type elicits a different response to this cytokine. Cell type-specific effects of IFN-γ make it difficult to predict the outcomes of the systemic IFN-γ blockade and limit its clinical application, despite many years of research. To better understand the cell-cell interactions and cofactors that specify IFN-γ functions, we focused on the function of IFN-γ on CD8 T cell differentiation. We demonstrated that during bacterial infection, IFN-γ is a dominant paracrine trigger that skews CD8 T cell differentiation toward memory. This skewing is preferentially driven by contact-dependent T cell-T cell (T-T) interactions and the localized IFN-γ secretion among activated CD8 T cells in a unique splenic microenvironment, and is less sensitive to concurrent IFN-γ production by other immune cell populations such as natural killer (NK) cells. Modulation of CD8 T cell differentiation by IFN-γ relies on a nonconventional IFN-γ outcome that occurs specifically within 24 hours following infection. This is driven by IFN-γ costimulation by integrins at T-T synapses, and leads to synergistic phosphorylation of the proximal STAT1 molecule and accelerated IL-2 receptor down-regulation. This study provides evidence of the importance of context-dependent cytokine signaling and gives another example of how cell clusters and the microenvironment drive unique biology.

Detection of Plasma Curcuminoids from Dietary Intake of Turmeric-Containing Food in Human Volunteers.

SCOPE: Curcumin (from turmeric), has been extensively investigated for potential beneficial properties in numerous diseases. Most work has focused on supra-dietary concentrations/doses that would necessitate curcumin supplementation. However, much evidence instigating curcumin research is underpinned by epidemiological data based on low dietary intake via turmeric consumption. METHODS AND RESULTS: Here, a novel, highly sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) method for detection of curcuminoids is described. Assay sensitivity is demonstrated in a pilot pharmacokinetic volunteer study following ingestion of foodstuffs containing a standardized mass of turmeric, representative of daily consumption by certain South Asian populations. Free parent curcumin was detectable in plasma from one individual, reaching maximal plasma concentrations (Cmax ) of 3.2 nm. Curcumin conjugates were detected in all volunteers; Cmax for curcumin glucuronide is 47.6 ± 28.5 nm 30 min post-food, while Cmax for demethoxycurcumin glucuronide and curcumin sulfate is ≈2 nm. Curcumin and its major metabolites persist in plasma for at least 8 h. CONCLUSION: Despite poor absorption and rapid conjugation, dietary intake of standard culinary turmeric within complex food matrices furnished human plasma with detectable levels of curcuminoids. Whether sustained low systemic concentrations of these non-nutritive, biologically active, dietary components may have pharmacological activity for human health benefit, warrants further research.

An HPLC-UV method for the simultaneous quantification of curcumin and its metabolites in plasma and lung tissue: Potential for preclinical applications.

Curcumin, derived from turmeric, has been extensively investigated for its broad spectrum of biological activities. Previously reported HPLC-UV methods have focussed on analysis of the parent compound. Here, a sensitive HPLC-UV method was developed and partially validated, then used for the simultaneous determination of curcumin and its glucuronide and sulfate metabolites in plasma and lung tissue from mice. The assay was applied to an in vivo pharmacokinetic study comparing formulated curcumin (Meriva™) with standard curcumin. Plasma levels of glucuronide and sulfate metabolites were 5- and 2-fold higher after Meriva™ administration compared with standard curcumin. In lung tissue, free curcumin was 4-fold higher following Meriva™ administration vs standard curcumin. This assay represents a rapid, cheap method for simultaneous detection of curcumin and its major metabolites that has applicability in pre-clinical settings.

The role of stromal fibroblasts in lung carcinogenesis: A target for chemoprevention?

The tumour microenvironment plays an essential role in the development and spread of cancers. Tumour cells interact with the surrounding extracellular matrix (ECM), embedded within which, are a variety of non-cancer cells including cells of the vasculature, immune system and fibroblasts. The essential role of fibroblasts in the cultivation and maintenance of an environment in which tumour cells are able to maintain their aggressive phenotypic traits is becoming increasingly well documented. Cancer-associated fibroblasts are able to secrete a vast array of ECM-modulating factors, meaning that they have potential for a functional role in every step of the carcinogenic process. In particular, they are likely to have a role in early tumour-initiating inflammatory events, and so may provide a potential target for chemopreventive intervention. This review summarises the known interactions between lung tumour cells and surrounding reactive fibroblasts, highlighting the need to further investigate cancer-associated fibroblasts as therapeutic targets in lung cancer chemoprevention strategies.

Translating curcumin to the clinic for lung cancer prevention: evaluation of the preclinical evidence for its utility in primary, secondary, and tertiary prevention strategies.

Lung cancer is responsible for over one million deaths worldwide each year. Smoking cessation for lung cancer prevention remains key, but it is increasingly acknowledged that prevention strategies also need to focus on high-risk groups, including ex-smokers, and patients who have undergone resection of a primary tumor. Models for chemoprevention of lung cancer often present conflicting results, making rational design of lung cancer chemoprevention trials challenging. There has been much focus on use of dietary bioactive compounds in lung cancer prevention strategies, primarily due to their favorable toxicity profile and long history of use within the human populace. One such compound is curcumin, derived from the spice turmeric. This review summarizes and stratifies preclinical evidence for chemopreventive efficacy of curcumin in models of lung cancer, and adjudges the weight of evidence for use of curcumin in lung cancer chemoprevention strategies.