A New Hope for CD56negCD16pos NK Cells as Unconventional Cytotoxic Mediators: An Adaptation to Chronic Diseases

Abstract

Natural Killer (NK) cells play an essential role in antiviral and anti-tumoral immune responses. In peripheral blood, NK cells are commonly classified into two major subsets: CD56^brightCD16^neg and CD56^dimCD16^pos despite the characterization of a CD56^negCD16^pos subset 25 years ago. Since then, several studies have described the prevalence of an CD56^negCD16^pos NK cell subset in viral non-controllers as the basis for their NK cell dysfunction. However, the mechanistic basis for their cytotoxic impairment is unclear. Recently, using a strict flow cytometry gating strategy to exclude monocytes, we reported an accumulation of CD56^negCD16^pos NK cells in Plasmodium falciparum malaria-exposed children and pediatric cancer patients diagnosed with endemic Burkitt lymphoma (eBL). Here, we use live-sorted cells, histological staining, bulk RNA-sequencing and flow cytometry to confirm that this CD56^negCD16^pos NK cell subset has the same morphological features as the other NK cell subsets and a similar transcriptional profile compared to CD56^dimCD16^pos NK cells with only 120 genes differentially expressed (fold change of 1.5, p < 0.01 and FDR<0.05) out of 9235 transcripts. CD56^negCD16^pos NK cells have a distinct profile with significantly higher expression of MPEG1 (perforin 2), FCGR3B (CD16b), FCGR2A, and FCGR2B (CD32A and B) as well as CD6, CD84, HLA-DR, LILRB1/2, and PDCD1 (PD-1), whereas Interleukin 18 (IL18) receptor genes (IL18RAP and IL18R1), cytotoxic genes such as KLRF1 (NKp80) and NCR1 (NKp46), and inhibitory HAVCR2 (TIM-3) are significantly down-regulated compared to CD56^dimCD16^pos NK cells. Together, these data confirm that CD56^negCD16^pos cells are legitimate NK cells, yet their transcriptional and protein expression profiles suggest their cytotoxic potential is mediated by pathways reliant on antibodies such as antibody-dependent cell cytotoxicity (ADCC), antibody-dependent respiratory burst (ADRB), and enhanced by complement receptor 3 (CR3) and FAS/FASL interaction. Our findings support the premise that chronic diseases induce NK cell modifications that circumvent proinflammatory mediators involved in direct cytotoxicity. Therefore, individuals with such altered NK cell profiles may respond differently to NK-mediated immunotherapies, infections or vaccines depending on which cytotoxic mechanisms are being engaged.

Keywords: CD56negCD16pos subset; endemic Burkitt lymphoma; epstein-barr virus; malaria; natural killer cells; transcription profile.

Figure 1

Gating strategy for FACS and HandE staining. PMBCs were gated on (A) lymphocytes size determined by SSC-A vs. FSC-A cytoplots, then (B) live, and (C) single cells were selected. (D) A Dump channel was used to eliminate CD3⁺, CD14⁺, and CD19⁺ cells. These cells were stained purple using hematoxylin and eosin (HandE), as shown in photo 1. Then (D) CD3⁻CD14⁻CD19⁻ cells were (E) sorted based on CD56 vs. CD16 expression and the three isolated NK cell subsets were stained with HandE: photo 2 CD56^brightCD16^neg, photo 3 CD56^dimCD16^pos, and photo 4 CD56^negCD16^pos.

Figure 2

Heatmap and MA plot of selected differentially expressed genes associated with NK cell function. (A) Heatmap of selected NK cell genes comparing CD56^negCD16^pos and CD56^dimCD16^pos NK cells. The id between the brackets are the protein name for that particulate gene, i.e., gene symbol (protein name), NCAM1 (CD56). (B) MA plot of differential expression analysis between CD56^negCD16^pos and CD56^dimCD16^pos NK cell subsets. The MA plot illustrates a log2 fold change of NK specific gene expression in CD56^negCD16^pos relative to CD56^dimCD16^pos cells and the average normalized expression counts of genes expressed by CD56^negCD16^pos cells. Protein names are used in the MA plot for ease of comparison to flow data even though these results are for gene expression. The Red dots indicate genes that have significantly higher expression in CD56^negCD16^pos relative to CD56^dimCD16^pos NK cells (with BH-adjusted p-value < 0.01 and FDR < 0.05), whereas blue dots indicate genes that have significantly lower expression (with BH-adjusted p-value < 0.01 and FDR < 0.05), and gray dots are genes that are similarly expressed. (C) Boxplot of gene expression profiles that define Innate Lymphoid Cells (ILCs): AHR, AREG, BCL11B, CSF2, GATA3, GFI1, IRGAE, RORC comparing CD56^negCD16^pos relative to CD56^dimCD16^pos NK cells.

Figure 3

Validation of RNA-sequencing data by flow cytometry. (A) Heatmap of markers expressed on both CD56^dimCD16^pos (in green) and CD56^negCD16^pos (in purple) NK cells from eBL (in turquoise) and healthy (in pink) children. (B) Violin plots showing the protein expression of IL18Ra, CD62L, DNAM1, TIM3, CD32, granzyme B and PD-1 from both CD56^dimCD16^pos (in green) and CD56^negCD16^pos (in purple) NK cells. *Represents a p-value < 0.05.

Figure 4

Manhattan plot of functional profiling of the list of upregulated genes from CD56^negCD16^pos relative to CD56^dimCD16^pos cells. Using the online tool gProfiler and the ordered g:GOSt query, we assessed which biological process (BP) will be linked to the list of 536 significantly differentially expressed genes from CD56^negCD16^pos relative to CD56^dimCD16^pos cells. The x-axis represents functional terms that are grouped and color-coded by data sources [molecular function (MF), biological process (BP), cell component (CC)]. The y-axis shows the adjusted enrichment p-values in negative log10 scale. Adjusted p-values g:GOSt used Bonferroni correction and a threshold of 0.01. On the table, adjusted p-values were color coded as yellow for insignificant findings to dark blue with highest significance.

Figure 5

Hypothetical model of CD56^dimCD16^pos and CD56^negCD16^pos NK cells cytotoxic pathways against both Plasmodium falciparum-infected red blood cells (Pf-iRBC) and endemic Burkitt lymphoma (eBL) tumor cells. (A) Proposed killing pathways used by CD56^dimCD16^pos NK cells against iRBCs and eBL tumors primarily mediated by natural direct cytotoxicity (direct killing) through the activation of cytotoxic receptors (NKp46, NKp30, NKp80, NKG2D…) but also antibody dependent cell cytotoxicity (ADCC) through CD16a activation. (B) Proposed killing pathways used by CD56^negCD16^pos NK cells against iRBC and eBL tumors. Due to the low expression of cytotoxic receptors, direct killing appears to be incapacitated however other killing pathways based on recognition of opsonized targets might be enhanced.