Innate Immunity Research Toolkit

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What Is It?

Natural Killer (NK) cells are part of the innate immune system with inherent cancer cell-killing capabilities, providing an exciting alternative to costly custom T cell immunotherapies. However, engineering NK cells is difficult due to their heterogeneity and plasticity, requiring further research into realizing their use as cell therapies. Here we present a starter toolkit for engineering NK cells in support of fundamental research towards cell therapies.

The NK cell research toolkit product contains three classes of genes: ligands, surface receptors, and transcription factors. Using these genes, researchers can manipulate NK cells, cancer cells, and “feeder” cells (i.e. cells used to trigger NK cell proliferation). More specifically:

The ligands contained within this FreeGenes product can be transduced or transfected into:

non-stimulatory feeder cells, cancer cells or NK cell lines to isolate their effects
stimulatory feeder cells to enhance their activating effects on NK cells

The transcription factors can be transduced or transfected into NK cells to alter their behavior or phenotype.
The activating or inhibitory receptors can similarly be transduced or transfected into NK cells to perturb their behavior.

What Can the NK Cell Toolkit Be Used For?

The genes listed here can be expressed on NK cells, feeder cells, or tumor cells to study individual NK cell signaling pathways, strengths of activation of different receptor-ligand interactions, and whether certain NK cells are more or less resistant to inhibitory cues from cancer cells. The provided genes can be used individually or combined to assess synergistic effects.

The combination of these ligands, receptors, and transcription factors represents a powerful toolbox with which to intelligently explore a myriad of iterations of feeder cell-NK cell-tumor cell pipelines. The NK Cell Research Toolkit enables researchers to examine the effects of distinct molecules on NK cells’ targeting and eradication of discrete tumor cell types, and to design novel chimeric antigen receptors to modify endogenous signaling pathways. Research into NK cells may advance the development of effective off-the-shelf cell therapies, which will drastically lower the cost of immunotherapy and make these life-saving treatments available to more patients.

Shipped as purified and dried down plasmid DNA stained with Cresol Red in one 96-well plate. Cresol Red will not impact plasmid transformation. Plate map can be found here.

Note: The genes KIR2DL4IB (BBF10K_000506), KIR2DL4IC (BBF10K_000507) and NR4A2 (BBF10K_000531) are not currently included in this collection.

Sample Workflow

PCR amplify the gene of interest out of the supplied backbone
Clone the gene into a mammalian expression vector of your choice

Note that these genes do not come with a promoter sequence, so one must be present on your new backbone in order for the gene to be expressed

Transfect or transduce NK cells, feeder cells, or tumor cells to express the corresponding protein

Note that if you are transducing, you will need to produce virus - see here: https://www.addgene.org/protocols/lentivirus-production/

Select the NK cells, feeder cells, or tumor cells that have successfully been transduced or transfected using a fluorescent molecule or selection agent such as blasticidin

Note that the selection agents or fluorescent proteins are not included in the gene sequences, but the provided sequences all end with the beginning of the P2A self-cleaving peptide sequence so that a blasticidin resistance gene, GFP, or other gene can be incorporated into your vector without altering the function of the protein of interest - see here for more information about P2A: https://en.wikipedia.org/wiki/2A_self-cleaving_peptides and see here for information about blasticidin resistance: https://en.wikipedia.org/wiki/Blasticidin_S

Test the expansion of NK cells after culture with modified feeder cells and compare to the unmodified feeder cells, or test the cytotoxic activity of engineered versus non-engineered NK cells against engineered or non-engineered tumor cells

Where Can I Find More Information?

https://en.wikipedia.org/wiki/Cancer_immunotherapy

https://en.wikipedia.org/wiki/Chimeric_antigen_receptor_T_cell

https://en.wikipedia.org/wiki/Natural_killer_cell

Designed By: Nina Horowitz, Stanford University

Genes

Gene	Name	NCBI ID	Freegenes ID
IL15P1	Interleukin 15 Precursor 1	NP_000576.1	BBF10K_000502
IL15P2	Interleukin 15 Precursor 2	NP_751915.1	BBF10K_000503
IL2	Interleukin 2	NP_000577.2	BBF10K_000504
KIR2DL4IA	killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 isoform A	NP_002246.5	BBF10K_000505
KIR2DL4IB	killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 isoform B	NP_001074241.1	BBF10K_000506
KIR2DL4IC	killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 isoform C	NP_001074239.1	BBF10K_000507
HLAG	Homo sapiens major histocompatibility complex, class I, G (HLA-G), transcript variant 1	NP_001350496.1	BBF10K_000508
HLAG1	Homo sapiens major histocompatibility complex, class I, G (HLA-G), transcript variant 1	NP_001371219.1	BBF10K_000509
NKG2D	KLRK1 killer cell lectin like receptor K1	NP_031386.2	BBF10K_000510
ULBP1I1	UL16 binding protein 1 isoform 1	NP_079494.1	BBF10K_000511
ULBP1I2	UL16 binding protein 1 isoform 2	NP_001304018.1	BBF10K_000512
ULBP2	UL16 binding protein 2	NP_079493.1	BBF10K_000513
CD28H	TMIGD2 transmembrane and immunoglobulin domain containing 2	NP_653216.2	BBF10K_000514
B7H7	HHLA2 HERV–H LTR-associating protein 2	NP_009003.1	BBF10K_000515
CD40	cluster of differentiation 40	NP_001241.1	BBF10K_000516
CD40L	CD154 cluster of differentiation 154	NP_000065.1	BBF10K_000517
TGFb	transforming growth factor beta 1	NP_000651.3	BBF10K_000518
PDGF-DDtv1	platelet-derived growth factor D transcription variant 1	NP_079484.1	BBF10K_000519
PDGF-DDtv2	platelet-derived growth factor D transcription variant 2	NP_149126.1	BBF10K_000520
SDC2	HSPG syndecan 2	NP_002989.2	BBF10K_000521
2B4	Homo sapiens CD244 molecule	NP_057466.1	BBF10K_000522
NKp44	NCR2 natural cytotoxicity triggering receptor 2	NP_004819.2	BBF10K_000523
StrepCAR-CD28z	anti-streptavidin chimeric antigen receptor, intracellular CD28z	N/A	BBF10K_000524
StrepCAR-41BBz	anti-streptavidin chimeric antigen receptor, intracellular 4-1BBz	N/A	BBF10K_000525
StrepCAR-2B4	anti-streptavidin chimeric antigen receptor, intracellular 2B4	N/A	BBF10K_000526
CD19CAR-CD28z	anti-CD19 chimeric antigen receptor, intracellular CD28z	N/A	BBF10K_000527
CD19CAR-2B4	anti-CD19 chimeric antigen receptor, intracellular 2B4	N/A	BBF10K_000528
CD19CAR-41BBz	anti-CD19 chimeric antigen receptor, intracellular 4-1BBz	N/A	BBF10K_000529
NR4A1	Nuclear Receptor Subfamily 4 Group A Member 1	NP_001189162.1	BBF10K_000530
NR4A2	Nuclear Receptor Subfamily 4 Group A Member 2	NP_006177.1	BBF10K_000531
CRTAM	Cytotoxic and regulatory T cell molecule	NP_062550.2	BBF10K_000532
CD96	TACTILE T cell activation, increased late expression	NP_937839.1	BBF10K_000533
CLEC12A	C-lectin type domain family 12 member A	NP_612210.4	BBF10K_000534
CXCR6	c-x-c motif chemokine receptor 6	NP_006555.1	BBF10K_000535
RGS1	regulator of G protein signaling 1	NP_002913.3	BBF10K_000536
Hobit-i1	Homolog of Blimp-1 in T cells isoform 1	NP_001108231.1	BBF10K_000537
Hobit-i2	Homolog of Blimp-1 in T cells isoform 2	NP_001294854.1	BBF10K_000538
ID2	inhibitor of DNA binding 2	NP_002157.2	BBF10K_000539
GPR183	G-protein coupled receptor 183	NP_004942.1	BBF10K_000540
CD49a	ITGA1 Integrin subunit alpha 1	NP_852478.1	BBF10K_000541
CD103	ITGAE integrin subunit alpha E	NP_002199.3	BBF10K_000542

Download all of this information as a CSV from our GitHub.

Bionet

The bionet enables open peer-peer exchange of functional biomaterials and associated data. This product may also be available from bionet nodes that are more convenient to you. Here are other bionet nodes who may be willing to provide you this specific product.

Name	Contact	Country
David Truong	www.truonglab.com	United States
Alexandr Dorif	dorif11 {at} gmail {dot} com	Moldova, Republic of
Jordan Gonzalez	jgonzalez {at} thecitizensciencelab {dot} org	United States

Download all of this information as a CSV from our GitHub.

MTA

Unilateral OpenMTA

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