NaCl enhances CD8+ T cell effector functions in cancer immunotherapy.

CD8+ T cells control tumors but inevitably become dysfunctional in the tumor microenvironment. Here, we show that sodium chloride (NaCl) counteracts T cell dysfunction to promote cancer regression. NaCl supplementation during CD8+ T cell culture induced effector differentiation, IFN-γ production and cytotoxicity while maintaining the gene networks responsible for stem-like plasticity. Accordingly, adoptive transfer of tumor-specific T cells resulted in superior anti-tumor immunity in a humanized mouse model. In mice, a high-salt diet reduced the growth of experimental tumors in a CD8+ T cell-dependent manner by inhibiting terminal differentiation and enhancing the effector potency of CD8+ T cells. Mechanistically, NaCl enhanced glutamine consumption, which was critical for transcriptional, epigenetic and functional reprogramming. In humans, CD8+ T cells undergoing antigen recognition in tumors and predicting favorable responses to checkpoint blockade immunotherapy resembled those induced by NaCl. Thus, NaCl metabolism is a regulator of CD8+ T cell effector function, with potential implications for cancer immunotherapy.

Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors.

Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4- CD8- unconventional αß T cells (UTCαß). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαß associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTCαß polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors.

Macrophage expression and prognostic significance of the long pentraxin PTX3 in COVID-19.

Long pentraxin 3 (PTX3) is an essential component of humoral innate immunity, involved in resistance to selected pathogens and in the regulation of inflammation1-3. The present study was designed to assess the presence and significance of PTX3 in Coronavirus Disease 2019 (COVID-19)4-7. RNA-sequencing analysis of peripheral blood mononuclear cells, single-cell bioinformatics analysis and immunohistochemistry of lung autopsy samples revealed that myelomonocytic cells and endothelial cells express high levels of PTX3 in patients with COVID-19. Increased plasma concentrations of PTX3 were detected in 96 patients with COVID-19. PTX3 emerged as a strong independent predictor of 28-d mortality in multivariable analysis, better than conventional markers of inflammation, in hospitalized patients with COVID-19. The prognostic significance of PTX3 abundance for mortality was confirmed in a second independent cohort (54 patients). Thus, circulating and lung myelomonocytic cells and endothelial cells are a major source of PTX3, and PTX3 plasma concentration can serve as an independent strong prognostic indicator of short-term mortality in COVID-19.

The complexity of neutrophils in health and disease: Focus on cancer.

Neutrophils are essential soldiers of the immune response and their role have long been restricted to their activities in defence against microbial infections and during the acute phase of the inflammatory response. However, increasing number of investigations showed that neutrophils are endowed with plasticity and can participate in the orchestration of both innate and adaptive immune responses. Neutrophils have an impact on a broad range of disorders, including infections, chronic inflammations, and cancer. Neutrophils are present in the tumour microenvironment and have been reported to mediate both pro-tumour and anti-tumour responses. Neutrophils can contribute to genetic instability, tumour cell proliferation, angiogenesis and suppression of the anti-tumour immune response. In contrast, neutrophils are reported to mediate anti-tumour resistance by direct killing of tumour cells or by engaging cooperative interactions with other immune cells. Here we discuss the current understandings of neutrophils biology and functions in health and diseases, with a specific focus on their role in cancer biology and their prognostic significance in human cancer.

Immune cell networking in solid tumors: focus on macrophages and neutrophils.

The tumor microenvironment is composed of tumor cells, stromal cells and leukocytes, including innate and adaptive immune cells, and represents an ecological niche that regulates tumor development and progression. In general, inflammatory cells are considered to contribute to tumor progression through various mechanisms, including the formation of an immunosuppressive microenvironment. Macrophages and neutrophils are important components of the tumor microenvironment and can act as a double-edged sword, promoting or inhibiting the development of the tumor. Targeting of the immune system is emerging as an important therapeutic strategy for cancer patients. However, the efficacy of the various immunotherapies available is still limited. Given the crucial importance of the crosstalk between macrophages and neutrophils and other immune cells in the formation of the anti-tumor immune response, targeting these interactions may represent a promising therapeutic approach against cancer. Here we will review the current knowledge of the role played by macrophages and neutrophils in cancer, focusing on their interaction with other immune cells.

BTN2A1 targeting reprograms M2-like macrophages and TAMs via SYK and MAPK signaling.

Tumor-associated macrophages (TAMs), often adopting an immunosuppressive M2-like phenotype, correlate with unfavorable cancer outcomes. Our investigation unveiled elevated expression of the butyrophilin (BTN)2A1 in M2-like TAMs across diverse cancer types. We developed anti-BTN2A1 monoclonal antibodies (mAbs), and notably, one clone demonstrated a robust inhibitory effect on M2-like macrophage differentiation, inducing a shift toward an M1-like phenotype both in vitro and ex vivo in TAMs from patients with cancer. Macrophages treated with this anti-BTN2A1 mAb exhibited enhanced support for T cell proliferation and interferon-gamma (IFNγ) secretion. Mechanistically, BTN2A1 engagement induced spleen tyrosine kinase (SYK) recruitment, leading to sequential SYK and extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of SYK or ERK phosphorylation abolished M2 reprogramming upon BTN2A1 engagement. Our findings, derived from an analysis of macrophages from healthy donors and human tumors, underscore the pivotal role of BTN2A1 in immunosuppressive macrophage differentiation and function, offering potential applications in cancer immunotherapy.

Neutrophils Mediate Protection Against Colitis and Carcinogenesis by Controlling Bacterial Invasion and IL22 Production by γδ T Cells.

Neutrophils are the most abundant leukocytes in human blood and play a primary role in resistance against invading microorganisms and in the acute inflammatory response. However, their role in colitis and colitis-associated colorectal cancer is still under debate. This study aims to dissect the role of neutrophils in these pathologic contexts by using a rigorous genetic approach. Neutrophil-deficient mice (Csf3r-/- mice) were used in classic models of colitis and colitis-associated colorectal cancer and the role of neutrophils was assessed by histologic, cellular, and molecular analyses coupled with adoptive cell transfer. We also performed correlative analyses using human datasets. Csf3r-/- mice showed increased susceptibility to colitis and colitis-associated colorectal cancer compared with control Csf3r+/+ mice and adoptive transfer of neutrophils in Csf3r-/- mice reverted the phenotype. In colitis, Csf3r-/- mice showed increased bacterial invasion and a reduced number of healing ulcers in the colon, indicating a compromised regenerative capacity of epithelial cells. Neutrophils were essential for γδ T-cell polarization and IL22 production. In patients with ulcerative colitis, expression of CSF3R was positively correlated with IL22 and IL23 expression. Moreover, gene signatures associated with epithelial-cell development, proliferation, and antimicrobial response were enriched in CSF3Rhigh patients. Our data support a model where neutrophils mediate protection against intestinal inflammation and colitis-associated colorectal cancer by controlling the intestinal microbiota and driving the activation of an IL22-dependent tissue repair pathway.

The long Pentraxin PTX3 serves as an early predictive biomarker of co-infections in COVID-19.

BACKGROUND: COVID-19 clinical course is highly variable and secondary infections contribute to COVID-19 complexity. Early detection of secondary infections is clinically relevant for patient outcome. Procalcitonin (PCT) and C-reactive protein (CRP) are the most used biomarkers of infections. Pentraxin 3 (PTX3) is an acute phase protein with promising performance as early biomarker in infections. In patients with COVID-19, PTX3 plasma concentrations at hospital admission are independent predictor of poor outcome. In this study, we assessed whether PTX3 contributes to early identification of co-infections during the course of COVID-19. METHODS: We analyzed PTX3 levels in patients affected by COVID-19 with (n = 101) or without (n = 179) community or hospital-acquired fungal or bacterial secondary infections (CAIs or HAIs). FINDINGS: PTX3 plasma concentrations at diagnosis of CAI or HAI were significantly higher than those in patients without secondary infections. Compared to PCT and CRP, the increase of PTX3 plasma levels was associated with the highest hazard ratio for CAIs and HAIs (aHR 11.68 and 24.90). In multivariable Cox regression analysis, PTX3 was also the most significant predictor of 28-days mortality or intensive care unit admission of patients with potential co-infections, faring more pronounced than CRP and PCT. INTERPRETATION: PTX3 is a promising predictive biomarker for early identification and risk stratification of patients with COVID-19 and co-infections. FUNDING: Dolce & Gabbana fashion house donation; Ministero della Salute for COVID-19; EU funding within the MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) and MUR PNRR Italian network of excellence for advanced diagnosis (Project no. PNC-E3-2022-23683266 PNC-HLS-DA); EU MSCA (project CORVOS 860044).

Neutrophil diversity in inflammation and cancer.

Neutrophils are the most abundant circulating leukocytes in humans and the first immune cells recruited at the site of inflammation. Classically perceived as short-lived effector cells with limited plasticity and diversity, neutrophils are now recognized as highly heterogenous immune cells, which can adapt to various environmental cues. In addition to playing a central role in the host defence, neutrophils are involved in pathological contexts such as inflammatory diseases and cancer. The prevalence of neutrophils in these conditions is usually associated with detrimental inflammatory responses and poor clinical outcomes. However, a beneficial role for neutrophils is emerging in several pathological contexts, including in cancer. Here we will review the current knowledge of neutrophil biology and heterogeneity in steady state and during inflammation, with a focus on the opposing roles of neutrophils in different pathological contexts.

Stromal and Immune Cell Dynamics in Tumor Associated Tertiary Lymphoid Structures and Anti-Tumor Immune Responses.

Tertiary lymphoid structures (TLS) are ectopic lymphoid organs that have been observed in chronic inflammatory conditions including cancer, where they are thought to exert a positive effect on prognosis. Both immune and non-immune cells participate in the genesis of TLS by establishing complex cross-talks requiring both soluble factors and cell-to-cell contact. Several immune cell types, including T follicular helper cells (Tfh), regulatory T cells (Tregs), and myeloid cells, may accumulate in TLS, possibly promoting or inhibiting their development. In this manuscript, we propose to review the available evidence regarding specific aspects of the TLS formation in solid cancers, including 1) the role of stromal cell composition and architecture in the recruitment of specific immune subpopulations and the formation of immune cell aggregates; 2) the contribution of the myeloid compartment (macrophages and neutrophils) to the development of antibody responses and the TLS formation; 3) the immunological and metabolic mechanisms dictating recruitment, expansion and plasticity of Tregs into T follicular regulatory cells, which are potentially sensitive to immunotherapeutic strategies directed to costimulatory receptors or checkpoint molecules.

Differential expression and regulation of MS4A family members in myeloid cells in physiological and pathological conditions.

The MS4A gene family encodes 18 tetraspanin-like proteins, most of which with unknown function. MS4A1 (CD20), MS4A2 (FcεRIß), MS4A3 (HTm4), and MS4A4A play important roles in immunity, whereas expression and function of other members of the family are unknown. The present investigation was designed to obtain an expression fingerprint of MS4A family members, using bioinformatics analysis of public databases, RT-PCR, and protein analysis when possible. MS4A3, MS4A4A, MS4A4E, MS4A6A, MS4A7, and MS4A14 were expressed by myeloid cells. MS4A6A and MS4A14 were expressed in circulating monocytes and decreased during monocyte-to-Mϕ differentiation in parallel with an increase in MS4A4A expression. Analysis of gene expression regulation revealed a strong induction of MS4A4A, MS4A6A, MS4A7, and MS4A4E by glucocorticoid hormones. Consistently with in vitro findings, MS4A4A and MS4A7 were expressed in tissue Mϕs from COVID-19 and rheumatoid arthritis patients. Interestingly, MS4A3, selectively expressed in myeloid precursors, was found to be a marker of immature circulating neutrophils, a cellular population associated to COVID-19 severe disease. The results reported here show that members of the MS4A family are differentially expressed and regulated during myelomonocytic differentiation, and call for assessment of their functional role and value as therapeutic targets.

Impact of RAS mutations on the immune infiltrate of colorectal liver metastases: A preliminary study.

Kirsten rat sarcoma viral oncogene homolog KRAS proto-oncogene is the most common altered gene in colorectal cancer (CRC). Determining its mutational status, which is associated with worse prognosis and resistance to anti-epidermal growth factor receptor (EGFR) inhibitors, is essential for managing patients with CRC and colon liver metastases (CLM). Emerging studies highlighted the relationship of KRAS-mutated cancers and tumor microenvironment components, mainly with T cells. The aim of this study was to analyze the relationship of CLM immune cell infiltrate with KRAS mutational status. We performed a retrospective study on paraffin-embedded CLM tissue sections from patients surgically resected at the Department of Hepatobiliary and General Surgery of Humanitas Clinical and Cancer Center. We studied the distribution of lymphocytes (CD3+ cells), macrophages (CD163+), and neutrophils (CD66b+) in CLM tumoral and peritumoral area. Percentage of positive cells was correlated with tumor macroscopic characteristic, clinical aspects, and KRAS mutation. We observed a significant increase in CD66b+ cells in the peritumoral area in patients KRAS-mutated compared to KRAS wild-type patients. Percentages of lymphocytes and macrophages did not show significant differences. Further, neutrophils were found to be significantly increased also in the bloodstream of KRAS-mutated patients, indicating increased mobilization of neutrophils and recruitment in the CLM site. In conclusion, this study reveals a new intriguing aspect of the peritumoral microenvironment, which could pave the way for new prognostic and predictive markers for patient stratification.

p38 MAPK differentially controls NK activating ligands at transcriptional and post-transcriptional level on multiple myeloma cells.

The mechanisms that regulate the expression of the NKG2D and DNAM-1 activating ligands are only partially known, but it is now widely established that their expression is finely regulated at transcriptional, post-transcriptional and post-translational level, and involve numerous stress pathways depending on the type of ligand, stressor, and cell context. We show that treatment of Multiple Myeloma (MM) cells with sub-lethal doses of Vincristine (VCR), an anticancer drug that inhibits the assembly of microtubules, stimulates the expression of NKG2D and DNAM-1 activating ligands, rendering these cells more susceptible to NK cell-mediated killing. Herein, we focused our attention on the identification of the signaling pathways leading to de novo surface expression of ULBP-1, and to MICA and PVR upregulation on VCR-treated MM cells, both at protein and mRNA levels. We found that p38MAPK differentially regulates drug-dependent ligand upregulation at transcriptional and post-transcriptional level. More specifically, we observed that ULBP-1 expression is attributable to both increased transcriptional activity mediated by ATM-dependent p53 activation, and enhanced mRNA stability; while the p38-activated E2F1 transcription factor regulates MICA and PVR mRNA expression. All together, our findings reveal a previously unrecognized activity of VCR as anticancer agent, and indicate that in addition to its established ability to arrest cell growth, VCR can also modulate the expression of NKG2D and DNAM-1 activating ligand on tumor cells and thus promoting NK cell-mediated immunosurveillance.

Hydrodynamic alterations during cyanobacteria (Arthrospira platensis) growth from low to high biomass concentration inside tubular photobioreactors.

The rheological behavior of an Arthrospira culture was studied from low to high biomass concentration. Two tubular undulating row photobioreactors (TURP-5r and TURP-10r), with a very short light path of 1.0 cm, were used during batch growth. In TURP-5r, the biomass concentration increased to 14.5 g(dw) L(-1), and alterations of the physical properties and hydrodynamic behavior occurred as a result. In the past, the rheological characteristics of photosynthetic-microbe cultures were rarely investigated because of the low biomass concentration attained in the systems. Developing closed photobioreactor technologies, the optimum biomass concentration rises and the viscosity, the generalized Reynolds number (N'(Re)), and the power required for culture recycling are also subject to alteration. Starting from a biomass concentration of 4.1 g(dw) L(-1), the Arthrospira culture already exhibits the characteristics of a non-Newtonian fluid. As a result of culture recycling from 2.0 to 20.5 g(dw) L(-1) and an available power of 1.67 W row(-1), we demonstrated that N'(Re) is reduced from 6265 to 1148. Our experimental results showed that N'(Re) of 2345 can be reached only at a cell concentration below 11.1 g(dw) L(-1), while at a cell concentration below 4.1 g(dw) L(-1) N'(Re) = 4080 was reached. The power consumption (P(c)) for culture recycling increased noticeably when the cell concentration rose; the highest P(c) increase attained was from 2.0 to 4.1 g(dw) L(-1). This is the range within which the Arthrospira culture changes from a Newtonian to a non-Newtonian fluid.