Tennakoon, G. | Search bioRxiv

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

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↗