Biology subjects

Tanese de Souza, C.

Publications and source records attributed to Tanese de Souza, C..

4 recordsLinked to original sources

bioRxiv2024

LAG3 marks activated but hyporesponsive NK cells.

Natural Killer (NK) cells are critical for immunosurveillance yet become dysfunctional in contexts such as chronic stimulation by viral infections or cancer. This phenomenon is similar to T cell exhaustion but less well characterized, which limits therapeutic interventions. As shown for T cells, NK cells often display an increased expression of immune checkpoint proteins (ICP) following chronic stimulation, and ICP blockade therapies are currently being explored for several cancer types, which have remarkable patient benefits. Nevertheless, the nature of ICP expression in NK cells is still poorly documented. In this study, we aimed to identify the conditions that lead to and the phenotype of immune checkpoint LAG3 (Lymphocyte-activation gene 3) expressing NK cells. Using various experimental models, we found that LAG3 is expressed by murine NK cells upon activation in different contexts, including in response to cancer and acute viral infections. LAG3 marks a subset of immature, proliferating and activated cells, which, despite activation, have a reduced capacity to respond to a broad range of stimuli. Further characterization also revealed that LAG3+ NK cells exhibit a transcriptional signature similar to that of exhausted CD8+ T cells. Taken together, our results support the use of LAG3 as a marker of dysfunctional NK cells across diverse chronic and acute inflammatory conditions.

immunology↗

bioRxiv2024

Preventing surgery induced immune suppression and metastases by inhibiting PI3K-gamma signalling in Myeloid-Derived Suppressor Cells.

Myeloid derived suppressor cells (MDSCs) have a dominating presence in the postoperative period and mediate the suppression of Natural Killer (NK) cells and promotion of cancer metastases after surgery. However, their functional characteristics and effect on cellular immunity after surgery have not been comprehensively investigated. Here, we characterize the expansion of surgery-induced (sx) MDSCs via multi-colour flow cytometry, single-cell RNA sequencing, and functional ex vivo NK cell suppression assays. We then screened a small molecule library using our sx-MDSC:NK cell suppression assay to identify compounds that could inhibit sx-MDSCs. These studies provide evidence that PI3K-{gamma} signalling is upregulated in sx-MDSCs and blockade with PI3K-{gamma} specific inhibitors attenuates NK cell suppression in humans and mice and reduces postoperative metastases in murine models. Upregulated PI3K-{gamma} in sx-MDSCs is a potential pathway amenable to therapeutic targeting in the postoperative period. One Sentence SummaryThe suppressive mechanisms of surgery-induced myeloid derived suppressor cells use PI3K signalling and are amenable to PI3K-gamma specific inhibitors.

immunology↗

bioRxiv2020

An easily adopted murine model of distal pancreatectomy for investigating immunotherapy efficacy in resectable pancreatic adenocarcinoma

BackgroundAlthough surgery provides the greatest therapeutic benefit to eligible pancreatic ductal adenocarcinoma (PDAC) patients it does not significantly improve survival for the majority of patients. Unfortunately our understanding of the therapeutic benefit of combining surgery with different treatment modalities including promising immunotherapeutics is limited by the current lack of easily adopted surgical models. The purpose of this study was to develop a surgically resectable model of PDAC in immunocompetent mice for use in preclinical investigations. Materials and MethodsSurgically resectable orthotopic tumors were generated by injecting Pan02 cells into the tail of the pancreas. Fifteen days post implantation the primary tumors and tail of the pancreas were resected by laparotomy while preserving the spleen. Splenic function, tumor growth, immune phenotyping and survival were assessed following surgical resection of the primary tumor mass. ResultsAs expected orthotopic tumor implants recapitulated many of the major histological hallmarks of PDAC including disrupted lobular structure and vascular invasion. Preservation of splenic immune cell viability and function was not associated with improved survival following surgery alone. However, pre-operative vaccination with GVAX was associated with improved survival which was not impacted by surgery. ConclusionThis represents the first murine model of surgically resectable murine model of PDAC which recapitulates known pathological hallmarks of human disease in an immune competent model while allowing spleen preservation. This relatively simple and easily adopted approach provides an ideal platform to examine the efficacy of potential immunotherapy combinations for PDAC surgery patients.

cancer biology↗

bioRxiv2020

A Comprehensive Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood

The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artifacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 L of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 L of whole blood following co-incubation with interleukin (IL)-2/12 and the ECS mix for 20 minutes prior to cell fixation and RBC lysis and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFN{gamma}) was similarly assessed by incubating 1 mL of whole blood with PMA-ionomycin or IL-2/12 prior to incubation with ECS and subsequent ICS antibodies. In addition, plasma was collected from stimulated samples prior to ECS for quantification of secreted IFN{gamma} by ELISA. Results were consistent, despite inherent interpatient variability. Although we did not investigate an exhaustive list of targets, this approach enabled quantification of representative ECS surface markers including activating (NKG2D and DNAM-1) and inhibitory (NKG2D, PD-1, TIGIT, and TIM-3) receptors, cytokine receptors (CD25, CD122, CD132, and CD212) and ICS markers associated with NK cell activation following stimulation, including signaling protein phosphorylation (p-STAT4, p-STAT5, p-p38 MAPK, p-S6K) and IFN{gamma} in both healthy donors and cancer patients. In addition, we compared extracellular receptor expression using whole blood versus cryopreserved PBMCs and observed a significant difference in the expression of almost all receptors. The methods presented permit a relatively rapid parallel assessment of immune cell receptor expression, signaling protein activity, and cytokine production in a minimal volume of whole blood from both healthy donors and cancer patients.

immunology↗