CD56 Bright CD16 Natural Killer Cells Research Articles

High co-expression of immune checkpoint receptors PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT on tumor-infiltrating lymphocytes in early-stage breast cancer

High expression of immune checkpoint receptors (ICRs) in the tumor microenvironment regulates the anti-tumor response. In this study, the differential expressions of ICRs on tumor-infiltrating lymphocytes (TILs) in patients with early-stage breast cancer were investigated.The study included 32 patients who underwent surgery with a diagnosis of early-stage breast cancer between September 2018 and March 2020. TIL isolation was performed using a MACS tumor separation device and tumor separation kit. PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT expression of cytotoxic T and natural killer (NK) cells on TILs and peripheral blood lymphocytes (PBLs) were determined by flow cytometry.Patients with a high Ki-67 index, high TIL density, and HER-2 positivity were more likely to have increased CD16+CD56dim NK cells on TILs. Patients with T2 tumors were more likely to have increased expression of PD-1, LAG-3, and TIGIT on tumor-infiltrating CD8+ cytotoxic T cells than those with T1 tumors. PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3 expression of CD8+ T and CD16-CD56bright NK cells in TILs showed significant positive correlations with each other. PD1+CD8+, TIGIT+CD16+, and CTLA-4+CD56+ cells in PBLs and TILs were found to be negatively correlated, whereas only TIM-3+ expression of CD8+ T and CD16+CD56dim cells in PBLs and TILs showed positive correlations.Our results suggest that CD16+CD56dim NK cells on TILs may play a major role in the immune response against HER2-positive or highly proliferating breast tumors in patients with early-stage breast cancer. Furthermore, various ICRs were found to be highly co-expressed with each other on TILs, including PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. These receptors may synergistically suppress the response to the tumor, which may trigger immune escape mechanisms in the early stage of carcinogenesis. However, ICR expressions other than TIM3 on PBLs were not found to accompany their counterparts on TILs.

Differential Expression of Novel Immune Checkpoint Receptors on Tumor Infiltrating Lymphocytes in Patients with Locally Advanced Breast Cancer after Neoadjuvant Chemotherapy.

Immune checkpoint receptors (ICRs) were recently found to modulate the anti-tumoral immune response. This study aimed to determine the clinical and pathological associations of ICRs expression on tumor-infiltrating lymphocytes (TILs) in patients with locally advanced breast cancer (LABC) treated with neoadjuvant chemotherapy (NAC). Expressions of ICRs including PD-1, LAG-3, TIM-3, TIGIT, and CTLA-4 on CD8+ T lymphocytes and Natural Killer (NK) cells on TILs were analyzed by flow cytometry. Patients <50 years were more likely to express CTLA-4 on CD8+ T lymphocytes compared to those ≥50 years (p=0.004). In addition, patients with ypT3-4 tumors were more likely to have increased LAG-3 expression on CD16-CD56bright NK cells (p=0.042) and PD-1 (p=0.014) and CTLA-4 (p=0.018) expressions on CD8+ T cells in regard to those with ypT1-T2, respectively. Contrarily, PD-1 expression on CD16-CD56bright NK cells was found to be decreased in patients with ypN+ compared to those with ypN- (p=0.022). Furthermore, patients with HER2+ tumors were more likely to have increased TIM-3 expression on CD8+ T cells (p=0.043), whereas patients with a better response to NAC were more likely to express TIGIT on CD8+ T (p=0.014) and CD16-CD56bright NK cells (p=0.003), respectively. The new generation ICRs, TIM-3, LAG-3, and TIGIT are highly expressed in LABC following NAC in patients with poor prognostic factors. Therefore, new evolving therapies using inhibitory mAbs directed to TIM-3, LAG-3, and TIGIT could be also be considered in locally advanced breast cancers expressing these ICRs.

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 μg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. N/A. Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.

CD3−CD16−CD56brightImmunoregulatory NK Cells are Increased in the Tumor Microenvironment and Inversely Correlate with Advanced Stages in Patients with Papillary Thyroid Cancer

The innate immune system is the first line of defense and plays a key role in thyroid cancer development. The role of the tumor-infiltrating natural killer (NK) cells is becoming increasingly important in research and potential cancer therapies. NK cell subpopulations, CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright), demonstrate a significant role in the tumor immuno-surveillance process. We investigated the distribution of CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK subpopulations in tissue and blood samples from patients with papillary thyroid cancer (PTC) and nodular goiter (NG). Twenty-eight patients with PTC, 13 patients with NG, and 50 healthy donors were included in the study. Tissue and blood samples from all patients and blood samples from healthy donors were analyzed for CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK cells by flow cytometry. A significant predominance of CD3(-)CD16(+)CD56(dim) cells compared to CD3(-)CD16(-)CD56(bright) NK cells was found in blood samples in all groups (p<0.0001 in PTC, NG, and healthy donors). Increased infiltration by CD3(-)CD16(-)CD56(bright) NK cells was observed in thyroid tissue of patients with PTC, as compared to CD3(-)CD16(+)CD56(dim) NK cells (p=0.046), while CD3(-)CD16(+)CD56(dim) NK cells demonstrated a higher infiltration of NG tissues. CD3(-)CD16(+)CD56(dim) NK cell tissue infiltration positively correlated with advanced stages of PTC. In contrast, the CD3(-)CD16(-)CD56(bright) NK cell population was negatively associated with tumor stage in patients with PTC. CD3(-)CD16(-)CD56(bright) NK cell infiltration seems to be associated with PTC progression. These findings contribute to a better understanding of the immune response in PTC and may lead to novel immunotherapeutic approaches in these patients.

Cytokine production by CD16-CD56bright natural killer cells in the human early pregnancy decidua.

Using a cell sorter, CD16-CD56bright natural killer (NK) cells were sorted from decidual mononuclear cells at an early stage of pregnancy. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) method for their expression of mRNA coding for the following 12 cytokines: IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and leukemia inhibitory factor (LIF). Although mRNA coding for every cytokine was detected in decidual mononuclear cells, mRNAs coding for only G-CSF, GM-CSF, M-CSF, TNF-alpha, IFN-gamma, and LIF were detected in CD16-CD56bright NK cells. Also, the supernatant of CD16-CD56bright NK cell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-alpha, IFN-gamma, and LIF. These findings indicate that CD16-CD56bright NK cells produce many different cytokines and that these cytokines may play an important role in a successful pregnancy.