Natural killer (NK) cells, cytotoxic lymphocytes critical to the immune system, are similar to T cells and play a key role in the immune system’s anti-tumor and anti-viral responses. NK cells express a wide range of receptors that upon engagement by ligands on target tumor cells, regulate their anti-tumor activity. However, unlike T cells, NK cells do not require activation to lyse cancerous or infected cells.
When activated, NK cells kill their target cells in a multistep process which involves synapse of NK cells and target cells, expression of FAS ligand, and secretion of pro-inflammatory cytokines and cytotoxic granules that induce cell death.
These cytotoxic lymphocytes are of great interest in the field of cancer immunotherapies given that they do not present the risk of graft versus host disease and can be used in an allogeneic donor setting, which is a unique advantage over T cell therapies.
In a recently published paper, scientists from Case Western University, discovered a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expression and control of NK cell cytotoxic function.
Cytotoxic Activity of NK Cells
The cytotoxic activity of NK cells can be regulated through the expression of activating and inhibitory receptors on the cell surface. Activating receptors recognize ligands expressed by unhealthy or infected cells and interact with signaling adaptors, resulting in the formation of an immunological synapse between the NK cell and target cell.
The balance in the expression of activating and inhibitory receptors determines whether or not NK cells will exert their cytotoxic effects. For example, tumors often have downregulated expression of inhibitory ligands, increasing the chances that an activating immunological synapse will be formed between the NK and tumor cell, promoting cytotoxicity.1
NK cells use a variety of mechanisms to kill tumor and infected cells. One method is through NK cell expression of Fas ligand (FasL) and TRAIL, which induces target cell death through death receptor-mediated apoptosis. Binding of FasL on NK cells to Fas on tumor cells starts the caspase enzymatic cascade and results in tumor cell death. And the binding of TRAIL on NK cells to DR4 or DR5 on tumors cells induces apoptosis.
An additional way NK cells kill tumor cells is the granule exocytosis pathway, which is commonly used by NK cells to exert cytotoxic effects. Once secreted from the NK cells, perforin will create holes in the membrane of the target cell so that granzyme B can enter and lyse the cell.
Consequences of NK Cell Dysfunction
Phenotypic alterations can occur to NK cells in the tumor microenvironment (TME), decreasing the expression of activating receptors important for NK cell functionality and increasing the expression of inhibitory receptors
Prf1 and gzmb, the genes for perforin and granzyme B, are often downregulated in diseases such as cancer, resulting in NK cells with a reduced cytotoxic capacity. This compromises cytotoxic function and leads to NK cell exhaustion. This is why it is critical to identify the key factors that regulate cytotoxic function in NK cells.
NK cells in the TME have a reduced expression of the effector molecules IFNγ, CD107a, FasL, granzyme B, TRAIL, and perforin. In addition, the TME disrupts the metabolism of NK cells, resulting in decreased cell health and cytotoxic capacity. Understanding the mechanisms behind the development of NK cell dysfunction provides avenues to therapeutically target the cells and restore normal functionality.
Uncovering a Potential Mechanism for NK Cell Dysfunction
The nuclear factor-κB (NFκB) proteins are a family of transcription factors (p50, p52, RelB, p65, c-Rel) that play a central role in many biological processes such as stress, inflammation, and innate immune responses. c-Rel is primarily expressed in B cells and T cells and its target genes are involved in lymphoid growth and survival and plays a key role in processes related to B and T cell growth, proliferation, and survival.
In previous studies, c-Rel deficient animals have shown that reduced expression of this transcription factor results in impaired antibody production by B cells and impaired cytokine secretion by T cells2,3
While it is known that c-Rel plays a part in T and B cell activation, its role in NK cells was unknown. In this study, researchers used IsoPlexis’ single-cell proteomics technology to reveal that when c-Rel was inhibited, NK cells had reduced polyfunctionality and a less heterogenous cytokine secretion profile, including a reduction in granzyme B secretion (Figure 1). This reduced polyfunctionality was also associated with reduced cytotoxicity in c-Rel deficient NK cells.
The results suggest that c-Rel regulates the anti-tumor functionality of NK cells and that therapeutically targeting c-Rel could potentially restore the cytotoxic capacity of dysfunctional NK cells.
The data from this paper indicates that c-Rel plays an essential role in regulating NK cell anti-tumor cytotoxicity by regulating the expression of prf1 and gzmb. In cancer, these genes are often downregulated causing NK cells to have reduced cytotoxicity and leads to NK cell exhaustion. The team was able uncover a previously unknown regulator of NK cell cytotoxicity, helped by insights uncovered using IsoPlexis’ single-cell technology.
Figure 1. An IsoPlexis single-cell cytokine analysis using control and PTXF treated human primary NK cells was performed to verify results. Data showed that activation with PMA/Ionomycin (PI) in control NK cells treated with water delivers the expected high Polyfunctional Strength Index (PSI), while treatment with the PTXF leads to lower PSI values (top). The bottom figure shows a heatmap visualization demonstrating that the positive PI stimulation of control NK cells delivers the expected heterogenous heatmap visualization, with a higher frequency of polyfunctional cell subsets, while the PTXF treatment leads to a less heterogenous secretion profile, with a lower frequency of polyfunctional cell subsets.
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- Frontiers | Human NK cell lytic granules and regulation of their exocytosis | Immunology (frontiersin.org)
- Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression – PubMed (nih.gov)
- B-cell survival and development controlled by the coordination of NF-κB family members RelB and cRel – PubMed (nih.gov)
- Frontiers | NF-κB c-Rel Is Dispensable for the Development but Is Required for the Cytotoxic Function of NK Cells (frontiersin.org)