CRISPR-MEDIATED DELETION OF FLI1 IN NK CELLS
20250092373 ยท 2025-03-20
Assignee
Inventors
- Timothy E. O'Sullivan (Los Angeles, CA, US)
- Luke Riggan (Los Angeles, CA, US)
- Joey H. Li (Los Angeles, CA, US)
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12N2501/125
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C12M35/02
CHEMISTRY; METALLURGY
C12N2501/22
CHEMISTRY; METALLURGY
A01K2207/12
HUMAN NECESSITIES
International classification
C12N9/22
CHEMISTRY; METALLURGY
C12M1/42
CHEMISTRY; METALLURGY
Abstract
Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus (MCMV) infection. Ly6C memory precursor (MP) NK cells displayed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. Our studies show that a NK cell-intrinsic checkpoint is controlled by the transcription factor Fli1 which limits MP NK formation by regulating early effector NK cell fitness during viral infection. Building upon this discovery, we have designed methods and materials for modulating the molecular mechanisms that regulate memory cell fate in NK cells, such as genetically modified NK cells having a deletion in the gene for the transcription factor Fli1.
Claims
1. A method of decreasing levels of Bcl-2-like protein 11 (BIM) observed in early effector NK cells following viral infection, the method comprising perturbing a Friend leukemia integration 1 transcription factor (FLI1) gene in the early effector NK cells such that levels of BIM in the early effector NK cells are observed to be decreased following viral infection as compared to early effector NK cells not having a perturbation in the FLI1 gene.
2. The method of claim 1, wherein perturbing the FLI1 gene comprises making a deletion in the FLI1 gene made using a clustered regularly interspaced short palindromic repeats (CRISPR) process comprising: (a) combining a CRISPR ribonucleoprotein complex with the early effector NK cells; (b) electroporating the combination of (a) under conditions comprising: a voltage between 1700-2000 volts; and a pulse width of width of about 120-30 milliseconds; such that the CRISPR ribonucleoprotein complex is electroporated into the early effector NK cells.
3. The method of claim 1, wherein the early effector NK cell is selected to be a mature naive NK cell.
4. The method of claim 1, wherein the early effector NK cell is disposed within a culture media comprising one or more cytokines, wherein the one or more cytokines is selected from: IL-2, IL-3, IL-4, IL-15, the notch ligand DLL1, stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), GM-CSF, and M-CSF.
5. The method of claim 1, wherein the early effector NK cell comprises a further modification to genomic DNA comprising a further deletion of one or more genes in the early effector NK cell and/or the addition of one or more genes in the early effector NK cell.
6. An isolated NK cell having a deletion in a Friend leukemia integration 1 transcription factor (FLI1) gene such that levels of Bcl-2-like protein 11 (BIM) in the NK cell are decreased.
7. The NK cell of claim 6, wherein the NK cell is a mature naive NK cell.
8. The NK cell of claim 6, wherein the cell is disposed within a culture media comprising one or more cytokines, wherein the one or more cytokines is selected from: IL-2, IL-3, IL-4, IL-15, the notch ligand DLL1, stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), GM-CSF, and M-CSF.
9. The NK cell of claim 6, wherein the cell comprises a further modification to genomic DNA comprising a further deletion of one or more genes in the NK cell and/or the addition of one or more genes in the NK cell.
10. A method of making a genetically modified NK cell, the methods comprising making a perturbation in a Friend leukemia integration 1 transcription factor (FLI1) gene of an NK cell so that the genetically modified NK cell is made.
11. The method of claim 10, wherein the perturbation in the FLI1 gene is made using a clustered regularly interspaced short palindromic repeats (CRISPR) process.
12. The method of claim 11, wherein the CRISPR process comprises: (a) combining a CRISPR ribonucleoprotein complex with the NK cells; (b) electroporating the combination of (a) under conditions comprising: a voltage between 1700-2000 volts; and a pulse width of width of about 120-30 milliseconds; such that the CRISPR ribonucleoprotein complex is electroporated into the NK cells.
13. The method of claim 10, wherein the NK cells are combined with one or more cytokines following collection and prior to electroporation; wherein the one or more cytokines is selected from: IL-2, IL-3, IL-4, IL-15, the notch ligand DLL1, stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), GM-CSF, and M-CSF.
14. The method of claim 10, wherein the method comprises deleting the FLI1 gene in the early effector NK cells such that levels of the pro-apoptotic factor Bim in the early effector NK cells are decreased.
15. The method of claim 10, wherein the NK cell is a primary cell obtained from the peripheral blood leukocytes of an individual.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0018] In the description of embodiments, reference may be made to the associated figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention.
[0019] As discussed below, we have discovered strategies for modulating molecular mechanisms that regulate memory cell fate in NK cells by perturbing (e.g. deleting) the gene for the transcription factor Fli1 in such cells. Typically such strategies involve CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of the NK cells. Surprisingly, such methods can be used to modulate the physiology of early effector NK cells, for example so that levels of the pro-apoptotic factor Bim in these cells are decreased following viral infection. Aspects and embodiments of this invention are described below and also found in Riggan et al., Nature Immunology volume 23, pages 556-567 (2022) (this specific publication is referred to herein as Riggan et al.).
[0020] The invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, an isolated NK cell having a perturbation (e.g. a deletion) in a FLI1 gene such that levels or functional activities of the pro-apoptotic factor Bim in the NK cell are decreased (e.g. as compared to a control NK cell not having a perturbation in a FLI1 gene). Briefly, as is known in the art, Friend leukemia integration 1 transcription factor (FLI1) is a transcription factor also known as transcription factor ERGB. Seem for example, NCBI Reference Sequence: NM_001271012.1. The FLI1 gene maps to 128,686,535-128,813,267 in GRCh38 coordinates. BIM, also referred to as BCL2, is a mammalian regulator of cell death. See, for example, NCBI Reference Sequence: NM_001204106 4836 bp mRNA linear PRI 22 Jul. 2018; ACCESSION: NM_001204106, VERSION NM_001204106.1. the BCL2L11/Bim gene is located on 2q13.
[0021] Embodiments of the invention include methods of making a genetically modified NK cell, the methods comprising making a perturbation in the FLI1 gene of an NK cell (e.g. a primary NK cell obtained from the peripheral blood leukocytes of an individual) so that the genetically modified NK cell is made. In illustrative working embodiments of the invention, the methods comprise deleting the FLI1 gene in the early effector NK cells such that levels of the pro-apoptotic factor Bim in the early effector NK cells are decreased (as compared to early effector NK cells not having a perturbation in the FLI1 gene). Typically in these methods, the perturbation in the FLI1 gene is made using a clustered regularly interspaced short palindromic repeats (CRISPR) process. In certain of these embodiments, the CRISPR process comprises: combining a CRISPR ribonucleoprotein complex with the NK cells; and then electroporating this combination under conditions comprising: a voltage between 1700-2000 volts; and a pulse width of width of about 120-30 milliseconds; such that the CRISPR ribonucleoprotein complex is electroporated into the NK cells. In certain embodiments of these methods, the NK cells are combined with one or more cytokines following collection and prior to electroporation; wherein the one or more cytokines is selected from: IL-2, IL-3, IL-4, IL-15, the notch ligand DLL1, stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), GM-CSF, and M-CSF.
[0022] Certain embodiments of the invention include using such methods to modulate memory cell fate in NK cells, for example by modulating levels of the pro-apoptotic factor Bim in NK cells. Illustrative embodiments of the invention include methods of decreasing levels/activities of the pro-apoptotic factor Bim in NK cells such as the levels of Bim that are observed in early effector NK cells following viral infection, the methods comprising perturbing a FLI1 gene in the early effector NK cells such that levels/activities of the pro-apoptotic factor Bim in the early effector NK cells are observed to be decreased following viral infection as compared to early effector NK cells not having a perturbation in the FLI1 gene. In certain embodiments of these methods, perturbing the FLI1 gene comprises making a deletion in the FLI1 gene made using a clustered regularly interspaced short palindromic repeats (CRISPR) process comprising: combining a CRISPR ribonucleoprotein complex with the early effector NK cells; and then electroporating the combination of (a) under conditions comprising: a voltage between 1700-2000 volts; and a pulse width of width of about 120-30 milliseconds; such that the CRISPR ribonucleoprotein complex is electroporated into the early effector NK cells. In some embodiments of the invention, the early effector NK cell is a mature naive NK cell. Optionally in these methods, an early effector NK cell is disposed within a culture media comprising one or more cytokines, wherein the one or more cytokines is selected from: IL-2, IL-3, IL-4, IL-15, the notch ligand DLL1, stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), GM-CSF, and M-CSF. In some embodiments of these methods, the early effector NK cell comprises a further modification to genomic DNA comprising a further deletion of one or more genes in the early effector NK cell and/or the addition of one or more genes in the early effector NK cell.
[0023] As discussed in detail below, embodiments of the invention include, for example, methods of electroporating a CRISPR ribonucleoprotein complex into mammalian NK cells. As used herein, the phrase CRISPR ribonucleoprotein complex refers to a ribonucleoprotein complex having CRISPR-associated endonuclease activity. Exemplary CRISPR ribonucleoprotein complexes include CRISPR/Cas9 CRISPR-associated endonuclease activity and CRISPR/Cpf1 CRISPR-associated endonuclease activity. CRISPR/Cas9 gene targeting requires a custom single-lead RNA (sgRNA) consisting of a targeted sequence (crRNA sequence) and a Cas9 nucleic acid recruitment sequence (tracrRNA). The crRNA region is a sequence of about 20 nucleotides, homologous to one of the regions of the gene you are interested in, that will guide the activity of the Cas9 nuclease. As used herein, the phrase CRISPR-associated RNA refers to an RNA component that, when combined with a CRISPR-associated protein, results in an CRISPR ribonucleoprotein complex. Exemplary CRISPR ribonucleoprotein complexes include ribonucleoprotein complexes having an CRISPR-associated protein, such as CRISPR/Cas9 protein or CRISPR/Cpf1 protein. An exemplary CRISPR-associated RNA includes a gRNA, including a crRNA and tracrRNA, for CRISPR/Cas9 protein that forms the CRISPR/Cas9 endonuclease system. Another exemplary CRISPR-associated RNA includes a crRNA for CRISPR/Cpf1 protein that forms the CRISPR/Cpf1 endonuclease system. Examples of these CRISPR ribonucleoprotein complexes, the CRISPR-associated RNA and protein components, and CRISPR-associated endonuclease systems are disclosed in the following references: Collingwood, M. A., Jacobi, A. M., Rettig, G. R., Schubert, M. S., and Behlke, M. A., CRISPR-BASED COMPOSITIONS AND METHOD OF USE, U.S. patent application Ser. No. 14/975,709, filed Dec. 18, 2015, published now as U.S. Patent Application Publication No. US2016/0177304A1 on Jun. 23, 2016 and issued as U.S. Pat. No. 9,840,702 on Dec. 12, 2017; and Behlke, M. A. et al. CRISPR/CPF1 SYSTEMS AND METHODS, U.S. patent application Ser. No. 15/821,736, filed Nov. 22, 2017, and U.S. Patent Application Publication No. 20190032131, the contents of which are hereby incorporated by reference herein in their entirety.
[0024] As is known in the art, primary cells (e.g. NK Cells) are those directly removed from an individual (e.g. a cancer patient), as compared to cell lines which are permanently established cell cultures. Typically, the methods of the invention comprise combining the CRISPR ribonucleoprotein complex with NK cells; and then electroporating this combination under conditions comprising: a voltage between 1700-2000 volts; and a pulse width of width of about 120-30 milliseconds; such that the CRISPR ribonucleoprotein complex is electroporated into the leukocytes. Typically in these methods, the CRISPR ribonucleoprotein complex comprises from 40 to 100 pmol Cas9 complexed with from 120 to 300 pmol sgRNA. In this context, a gRNA is comprised of a tracrRNA and crRNA. In particular, the crRNA and tracrRNA can be fused into a single chimeric nucleic acid (a single-guide RNA, or sgRNA) or they can be separate nucleic acids. In certain embodiments of the invention, these methods comprise not more than 1, 2 or 3 individual electroporations. Typically in these methods, the electroporation of the CRISPR ribonucleoprotein complex into the primary leukocytes results in modulation of expression of a gene in the NK cells targeted by the sgRNA. Electroporation methods, materials and devices that can be used with embodiments of the invention are disclosed, for example in US Patent Application Publication Nos.: 20200332276, 20200171303, 20200131500, 20200115668, 20200048600, 20200048599, 20190382792, 20190292510, 20190284579, 20190125165, 20190100721, 20190093125, 20180340186, 20180179485, 20180155688, 20180066222, 20180064073, 20170348525, 20170298390, 20170218355, 20160215297, and 20160129246, the contents of which are incorporated herein by reference.
[0025] As discussed in detail below, using single cell RNA sequencing (scRNAseq), we identified an MP-like transcriptional state of NK.sub.eff cells following MCMV-driven expansion in vivo. We provide further evidence that Ly6C.sup. NK.sub.eff cells preferentially survive the contraction phase to generate memory NK cells in a Bcl2-dependent manner. MP NK cells displayed distinct transcriptional and epigenetic signatures compared to Ly6C.sup.+ NK.sub.eff cells, with an overlapping epigenetic signature shared with MP CD8.sup.+ T cells enriched in ETS1 and Fli1 transcription factor binding motifs. Fli1 regulated MP NK cell formation by promoting Bim levels in early effector NK cells to decrease the fitness of expanding NK cells during viral infection. These results provide evidence that memory NK cells, similar to memory T cells, are generated by a subset of epigenetically distinct MP cells that preferentially survive during the contraction phase of the response to viral infection and identify Fli1 as a critical regulator of MP NK cell formation. Aspects and embodiments of the invention are discussed in the following sections.
Time-Resolved scRNA-Seq Analysis Reveals an MP-Like NK Cell State
[0026] To determine whether transcriptional heterogeneity exists within NK.sub.eff cells following viral infection, we adoptively transferred splenic Ly49H.sup.+ NK cells into recipient Klra8.sup./ (hereafter referred to as Ly49H.sup./) mice and infected with MCMV. On day (D) 7 post-infection (PI), adoptively transferred NK.sub.eff cells from the spleen were sorted and then profiled using 10 Genomics Chromium droplet scRNA-seq (Extended Data
[0027] To test this hypothesis in silico, we utilized RNA velocity analysis.sup.26 to determine the time-resolved transcriptional fates of D7 PI NK.sub.eff cells (Extended
Memory Ly6C.sup.+ NK Cells are Derived from Ly6C.sup. MP NK Cells
[0028] To determine whether a distinct MP NK cell subset could be identified in vivo, we analyzed cell surface expression of several genes predicted by our scRNA-seq dataset to be differentially expressed at D7 PI. While several cell surface proteins identified (CD44, Scal, NKG2D, CD16) did not display bimodal expression in effector NK cells, there were clear positive and negative populations of Ly6C and CX.sub.3CR1 NK.sub.eff cells on D7 PI (
[0029] Previous studies have suggested >95% of MCMV-induced memory NK cells are Ly6C.sup.+ on D28 PI.sup.30, 31. However, our adoptive transfer data suggested that Ly6C.sup. NK.sub.eff give rise to the majority of memory NK cells present 12 days following transfer, suggesting that Ly6C.sup.+ NK.sub.eff likely do not represent MP NK cells (
Bcl2 is Required for the Survival of NK.SUB.eff .Cells During Contraction
[0030] To determine the mechanisms of MP NK cell persistence following MCMV infection, we adoptively transferred CD45.1.sup.+Ly49H.sup.+ NK cells into Ly49H.sup./ mice, infected with MCMV, sorted Ly6C.sup.+ and Ly6C.sup. Ly49H.sup.+ NK cells from the spleens of the Ly49H.sup./ mice on D7 PI and performed RNA sequencing (RNA-seq). RNA-seq analysis identified 43 differentially expressed genes (DEGs) between Ly6C.sup.+ NK.sub.eff and MP NK cells, with MP NK cells displaying increased expression of Bcl2 and the transcription factor Zbtb33 (
MP NK Cells Display a Distinct Epigenetic Signature
[0031] To determine the mechanisms of MP NK cell formation following MCMV infection, we adoptively transferred CD45.1.sup.+Ly49H.sup.+NK cells into Ly49H.sup./ mice, infected with MCMV, sorted Ly6C.sup.+ and Ly6C.sup. Ly49H.sup.+ NK cells from the spleen of the Ly49H.sup./ mice on D7 PI and performed ATAC sequencing (ATAC-seq). ATAC-seq analysis identified 811 significantly differentially accessible (DA) peaks between Ly6C.sup.+ NK.sub.eff and MP NK cells (
[0032] Our ATAC-seq analysis suggested that Fli1 may be a critical transcriptional regulator of MP NK cells. Therefore, we examined whether mature NK cells express Fli1 during MCMV infection. RNA-seq analysis demonstrated that Fli1 was repressed early during viral infection, but subsequently increased during the expansion phase of the response on D4 PI (
Fli1 Increases BIM Levels in Early NK.SUB.eff .to Limit MP NK Cell Formation
[0033] To determine the significance of Fli1 in regulating MP NK cells, we ablated Fli1 in mature NK cells using CRISPR cRNPs (
Discussion
[0034] We utilized single cell sequencing to identify a MP-like NK cell state following mouse cytomegalovirus (MCMV) infection. Ly6C.sup. NK.sub.eff cells displayed enhanced survival during the contraction phase and were determined to be the main precursors of Ly6C.sup.+ memory NK cells. MP NK cells displayed distinct transcriptional and epigenetic signatures compared to Ly6C.sup.+ NK.sub.eff cells, with increased protein expression of Bcl2. While Bcl2 was required for the survival of Ly49H.sup.+ NK cells during the contraction phase, STAT5 signaling likely induced Fli1 in early effector NK cells to increase Bim levels and restrict the formation of MP NK cells.
[0035] Time-resolved trajectory analysis of our scRNA-seq data suggested that a subset of D7 PI NK.sub.eff cells transition to a cell state enriched in the MP CD8.sup.+ T cell transcriptional signature. Although bulk RNA-seq analysis demonstrated that MP CD8.sup.+ T cells and MP NK cells did not display overlapping gene signatures, they both display higher expression of Bcl2 transcript and protein, which is required for effector T cells to preferentially survive during the contraction phase of the anti-viral response.sup.36, 37. These data suggest that Mcl1 expression, which is required for NK cell survival during homeostasis.sup.33, 38, is not sufficient to inhibit apoptosis in NK.sub.eff cells due to the decreased levels of Bcl2 observed on D7 PI. The discrepancy between our bulk RNA-seq and scRNA-seq data may be explained by the possibility that D7 PI Ly6C.sup. NK.sub.eff cells contain a mixture of transitional NK.sub.eff and MP NK cells rather than a homogenous MP cell state identified by scRNA-seq. Furthermore, because a small frequency of adoptively transferred Ly6C.sup.+ NK.sub.eff persist during the contraction phase, it is possible that D7 PI Ly6C.sup.+ NK.sub.eff contain a small fraction of transitional NK.sub.eff that survive to generate memory NK cells on D14 PI. Irrespective of these points, our adoptive transfer data suggest that the majority of memory NK cells present on D19 PI are derived from D7 PI Ly6C.sup. NK.sub.eff cells, making this a functional MP population.
[0036] RNA velocity analysis suggested that MP-like NK cells undergo continuous differentiation to a terminally differentiated state on D14 PI, which could explain the constant decay of memory NK cell numbers observed in all studies to date following the expansion phase in response to MCMV infection.sup.39. Indeed, monocle trajectory analysis identified Zeb2 expression increasing towards the terminally differentiated memory NK cell state on D14 PI, and Zeb2 has been implicated in the terminal maturation of naive NK cells during homeostasis as well as the terminal differentiation of effector CD8.sup.+ T cells following viral infection.sup.27, 40, 41. However, our results indicate that Zeb2 is required for the expansion of NK.sub.eff cells, but not the terminal differentiation of MP-like NK cells (data not shown). Similarly, Id2 has been shown to maintain the terminal differentiation of effector CD8.sup.+ T cells through sustained repression of central memory-associated transcriptional programs in addition to regulating NK cell development and epigenetic regulation of effector functionality.sup.42-45. While we observed higher expression of Id2 in the D7 PI MP-like NK cell cluster from our scRNA-seq analysis, we did not observe an increase in MP NK cells following inducible deletion of Id2 during the contraction phase following MCMV infection (data not shown). Together, these findings suggest that there may be important epigenetic differences between effector NK and CD8.sup.+ T cells that dictate lineage specific terminal differentiation programs, although future studies will be necessary to support this hypothesis.
[0037] Our ATAC-seq dataset revealed that Ly6C.sup.+ NK.sub.eff and TE CD8.sup.+ T cells share common enrichment in Runx1, Tbet, and Runx2 binding motifs, with Ly6C.sup. NK.sub.eff and MP CD8.sup.+ T cells show enrichment for Fli1 and ETS1 binding motifs. While Tbet and Runx1 are important for NK cell survival during homeostasis and MCMV infection.sup.46,47, the precise roles of these transcription factors during NK.sub.eff terminal differentiation will need to be studied in further detail. In mouse and human NK cells, ETS-1 has been shown the be required for mature NK cell development and regulation of genes involved in apoptosis, and can be induced by IL-2/IL-15 signaling in human NK cells.sup.48-50. Similarly, we found that STAT5 signaling induced expression of Fli1 in mature mouse NK cells in response to IL-2 and IL-15 ex vivo. Interestingly, CRISPR-mediated deletion of Fli1 in mature naive NK cells leads to decreased Bim levels and a greater persistence of early effector NK cells following MCMV infection, with a larger proportion of MP NK cells persisting through the contraction phase as a result. This finding suggests that Fli1 acts as a repressor of MP NK cell formation, likely to limit bystander immunopathology of expanded Ly49H.sup.+ NK cells following clonal proliferation. Similarly, Fli1-deficient CD8.sup.+ T cells accumulate more effector cells following LCMV infection.sup.51, implicating Fli1 as a critical intrinsic checkpoint regulator of effector lymphocyte formation. While future studies will be needed to determine the epigenetic and/or transcriptional mechanisms by which Fli1 represses early effector lymphocyte fitness, these results identify an important regulator of effector lymphocyte formation. Thus, understanding the transcriptional and epigenetic pathways that induce MP states in NK cells could inform strategies aimed at enhancing adaptive NK cell adoptive immunotherapies.
Method Details
Mice
[0038] Mice were bred at UCLA in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). The following mouse strains were used this study: C57BL/6 (CD45.2) (Jackson Labs, #000664), B6.SJL (CD45.1) (Jackson Labs, #002114), Klra8.sup./ (Ly49H-deficient). Experiments were conducted using 6-8 week old age- and gender-matched mice in accordance with approved institutional protocols.
MCMV Infection
[0039] MCMV (Smith) was serially passaged through BALB/c hosts three times, and then salivary gland viral stocks were prepared with a dounce homogenizer for dissociating the salivary glands of infected mice 3 weeks after infection. Experimental mice in studies were infected with MCMV by i.p. injection of 7.510.sup.3 plaque-forming units (PFU) in 0.5 mL of PBS. In other experiments, Ly49H.sup./ mice were intravenously injected with 25,000 sort purified D7 PI Ly49H.sup.+ NK cells or PBS as control. 7 days later, recipient mice were infected with MCMV by i.p. injection of 7.510.sup.3 plaque-forming units (PFU) in 0.5 mL of PBS. Mice were monitored and weighed daily and sacrificed when body weight decreased 10% from initial weight.
Isolation and Enrichment of Mouse NK Cells
[0040] Mouse spleens, livers, lungs, lymph nodes, and blood were harvested and prepared into single cell suspensions as described previously.sup.11. Splenic single cell suspensions were lysed in red blood cell lysis buffer and resuspended in EasySep buffer (Stemcell). To avoid depleting Ly6C.sup.+ NK cells we developed a custom antibody cocktail as follows: splenocytes were labeled with 10 g per spleen of biotin conjugated antibodies against CD3 (17A2), CD19 (6D5), CD8 (53-6.7), CD88 (20/70), Ly6G (1A8), SiglecF (S17007L), TCR (H57-597), CD20 (SA275A11), CD172a (P84) and magnetically depleted from total splenocyte suspensions with the use of anti-biotin coupled magnetic beads (Biolegend).
Cell Sorting and Adoptive Transfer Experiments
[0041] Isolated splenic NK cells were sorted using Aria-H Cytometer. NK cells were sorted to >95% purity. Approximately 210.sup.5 enriched NK cells were injected intravenously into mice. In adoptive co-transfer experiments, equal numbers of Ly49H.sup.+ NK cells from each population (CD45.1.sup.+ and CD45.2.sup.+) were injected into recipients 16 hours prior to MCMV infection. In other experiments, TCR.sup.CD3.sup. NK1.1.sup.+Ly49H.sup.+KLRG1.sup.+CD45.1.sup.+CX.sub.3CR1.sup.+ and TCR.sup.CD3.sup.NK1.1.sup.+Ly49H.sup.+KLRG1.sup.+CD45.2.sup.+CX.sub.3CR1.sup. or TCR.sup.CD3.sup.NK1.1.sup.+Ly49H.sup.+KLRG1.sup.+Ly6C.sup.+ and TCR.sup.CD3.sup.NK1.1.sup.+Ly49H.sup.+KLRG1.sup.+Ly6C.sup. D7 PI NK.sub.eff were sorted and then transferred at a 1:1 ratio into naive or D7 PI Ly49H.sup./ hosts that had received CD45.1CD45.2 Ly49H.sup.+ NK cells 7 days prior to MCMV infection. Adoptively transferred cells were recovered by harvesting recipient mouse splenic NK cells, preforming magnetic enrichment, and analysis by flow cytometry at various time points post-transfer.
Guide RNA Design
[0042] Synthetic gRNAs were purchased from SYNTHEGO. gRNA sequences were derived from a mouse whole genome CRISPR library described previously.sup.52. 10 gRNA sequences from this dataset were ranked according to predicted indel percentage and low off-target score using the inDelphi machine-learning algorithm for each gene target.sup.53. The top 3-7 guides were validated for high indel percentages by Sanger sequencing and protein knockdown by western blot before utilization in experiments.
Electroporation and cRNP Complex Formation
[0043] gRNAs (Synthego) were diluted to 100 M (100 pMol/L) in 1 TE buffer (Sythego). 1.2 L (120 pMol) of gRNA, 0.9 L of 100 M Alt-R Cas9 Electroporation Enhancer (IDT) and 3.9 L water were added to a 1.5 mL tube per sample for a total of 6 L. 1 L of recombinant Cas9 (20 pMol) (Synthego) was added to 5 L water in a separate 1.5 mL tube. 6 L of diluted Cas9 was added to 6 L of gRNA-enhancer mixture for a total of 12 L cRNP complex at a 1:3 molar ratio. The cRNP complex was allowed to incubate for at least 10 minutes at room temperature (RT) and electroporated using the Neon Transfection system (Thermo-Fisher) as described previously.sup.34, 54. Cells were then incubated at 37 C. for either 10 minutes before adoptive transfer or 90 minutes before centrifugation and resuspension in complete media supplemented with 50 ng rmIL-15 for culturing in vitro. Cells were cultured in vitro for 3 days following electroporation prior to reading out gene editing efficiency by flow cytometry or sanger sequencing.
Ex Vivo Stimulation of Lymphocytes
[0044] For plate-bound antibody stimulation experiments, 510.sup.5 isolated NK cells were stimulated with 4 mg/mL precoated antibody against NK1.1 (PK136) for 4 hours in complete media containing Brefeldin A (1:1000; BioLegend) and Monensin (2 uM; BioLegend). Cells were cultured in media alone as a negative control. In cytokine stimulation experiments, isolated NK cells were incubated with various concentrations of mouse IL-15 or IL-2, in the presence or absence of 100 M CAS 285986-31-4 STAT5 inhibitor (Millipore Sigma).
Adoptive Transfer cRNP Experiments
[0045] Adoptive NK cell co-transfer studies were performed by injecting a total of 110.sup.6 NK cells; Rosa26 cRNP-edited WT, and gene x cRNP-edited WT NK cells purified from spleens of congenically distinct WT mice (CD45.1, CD45.1.2 or CD45.2) into Ly49H/.sup./ mice 16 hours prior to MCMV infection.
Proliferation Assays
[0046] CellTrace Violet (CTV) stock solution was prepared per the manufacturers' instructions (Thermo) and diluted at 1:1000 in 37 C. PBS. Isolated NK cells were incubated in 0.5 mL of diluted CTVsolution for 10 minutes at 37 C. The solution was quenched with 10 the volume of CR-10 media. Cells were then washed and injected i.v. Division, proliferation and expansion indexes were quantified using FlowJo V10 software using the following calculations. Division Index: Total Number of Divisions/The number of cells at start of culture. Proliferation Index: Total Number of Divisions/Cells that went into division. Division Index: Total Number of Divisions/The number of cells at start of culture.
Flow Cytometry
[0047] Cells were analyzed for cell-surface markers using fluorophore-conjugated antibodies (BioLegend, eBioscience). Cell surface staining was performed in 1 PBS and intracellular staining was performed by fixing and permeabilizing using the eBioscience Foxp3/Transcription Factor kit for intranuclear proteins or BD Cytofix/Cytoperm kits for cytokines. Flow cytometry was performed using the Attune NT Acoustic Focusing cytometer and data were analyzed with FlowJo software (BD). Cell surface and intracellular staining was performed using the following fluorophore-conjugated antibodies: CD45.1 (A20), CD45.2 (104), NK1.1 (PK136), KLRG1 (2F1), TCR (H57-597), CD3 (17A2), Ly49H (3D10), IFN- (XMG1.2), Ly6C (HK1.4), CD44 (IM7), CD16 (93), Sca-1 (E13-161.7), CX3CR1 (SA011F11), NKG2D (CX5), BCL2 (BCL/10C4), CD11b (M1/70), CD27 (LG.3A10), Bim (C34C5), Ki-67 (16A8), Cytochrome-C (6H2.B4). For mitochondrial dyes, NK cells were enriched from spleens as described above, stained with cell-surface antibodies, and then incubated with various dyes in Hank's balanced salt solution plus Mg and Ca as follows: 100 nM Mitotracker Green (Life Technologies) for 30 min at 37 C. to measure mitochondrial mass or 100 nM TMRE (Thermofisher) for 30 min at 37 C. to measure mitochondrial membrane potential. BH3 profiling was performed as previously described.sup.55. Briefly, purified NK cells were resuspended in MEB buffer (150 mM Mannitol 10 mM HEPES-KOH, 50 mM KCl, 0.02 mM EGTA, 0.02 mM EDTA, 0.1% BSA, 5 mM Succinate). 50 l of cell suspension (110.sup.5 cells/well) were plated in wells holding 50 L MEB buffer containing 0.002% digitonin and BCL2 inhibitor ABT-199. Plates were then incubated at 25 C. for 50 min. Cells were then fixed with 4% paraformaldehyde for 10 min, followed by neutralization with N2 buffer (1.7M Tris, 1.25M Glycine pH 9.1) for 5 min. Samples were stained for 1 hour with 20 L of staining solution (10% BSA, 2% Tween 20 in PBS) containing anti-cytochrome c (BioLegend). Immediately afterwards, cytochrome c release was quantified using Attune Flow Cytometer.
Single Cell Metabolism Assay (SCENITH)
[0048] To profile single cell metabolic responses, 95 uL of purified NK cells in complete media were plated at 0.10.510.sup.6 cells/mL in v-bottom 96-well plates. Experimental triplicates were performed in all conditions. Wells were then treated with Control (DMSO), 2-Deoxy-D-Glucose (DG) final concentration 100 mM, Oligomycin (O) final concentration 1 mM, or a sequential combination of DG and O at the final concentrations mentioned. As negative control, the translation initiation inhibitor Harringtonine was added (Harringtonine, 2 mg/mL). Puromycin (final concentration 10 mg/mL) is added immediately after the metabolic inhibitor treatment. After puromycin treatment, cells were washed in cold PBS and stained for surface markers. Intracellular staining of puromycin was achieved using a custom anti-puromycin antibody.sup.27 was performed by incubating cells during 1 h at 4 C. diluted in permeabilization buffer.
PCR and Sanger Sequencing
[0049] DNA from NK cells was isolated using DNeasy Blood and Tissue kits (Qiagen). DNA concentration was measured using the NanoDrop OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific) and then diluted to 50 ng/l in water before PCR amplification of cRNP-targeted genomic regions of approximately 500-1000 base pairs. PCR samples from WT and cRNP-edited cells were submitted for Sanger sequencing (GENEWIZ) and then indel percentage was calculated using ICE analysis (SYNTHEGO).
10 Library Preparation, Sequencing, and Alignment
[0050] Single cell RNA-sequencing libraries were generated with the Chromium Single Cell 3 v2 (Day 7) and v3 (Day 14) assay (10 Genomics). Libraries were sequenced using the HighSeq 4000 platform (Illumina) to a depth of approximately 300 million reads per library with 250 read length. Raw reads were aligned to mouse genome (mm10) and cells were called using cellranger count (v3.0.2).
scRNA-Seq Cell Clustering
[0051] The R package Seurat (v3.1.).sup.56 was used to cluster the cells. Cells with less than 100 genes detected or more than 10% mitochondrial gene expression were first filtered out as low-quality cells. The gene counts for each cell were divided by the total gene counts for the cell and multiplied by a scale factor 10,000, then natural-log transformation was applied to the counts. The FindVariableFeatures function was used to select variable genes with default parameters. The ScaleData function was used to scale and center the counts in the dataset. Principal component analysis (PCA) was performed on the variable genes, and 20 principal components were used for cell clustering (resolution=0.5) and uniform manifold approximation and projection (UMAP) dimensional reduction. The cluster markers were found using the FindAllMarkers function. Module scores were calculated using the AddModuleScore function with default parameters. In the comparison between D7 and D14 PI NK cells, the FindIntegration Anchors and IntegrateData functions were used to find anchors and integrate the D7 and D14 PI datasets, and the other steps were the same as described above.
RNA Velocity Analysis
[0052] To estimate the RNA velocities of single cells, velocyto.sup.26 was used to distinguish unspliced and spliced mRNAs in each sample. The python package scVelo.sup.57 was then used to recover the directed dynamic information by leveraging RNA splicing information. Specifically, the data was first normalized using the filter_and_normalize function. The first- and second-order moments were computed for velocity estimation using the moments function. The velocity vectors were obtained using the velocity function. The velocities were projected into a lower-dimensional embedding using the velocity_graph function. Finally, the velocities were visualized in the UMAP embedding using the velocity_embedding_stream function. All scVelo functions were used with default parameters.
Pseudo-Time Trajectory Construction
[0053] Pseudo-time trajectories were constructed using the R package Monocle.sup.58 (version 2.10.1). The raw counts for cells in the intended cell types were extracted and normalized by the estimateSizeFactors and estimateDispersions functions with the default parameters. Genes with average expression larger than 0.5 and detected in more than 10 cells were retained for further analysis. Variable genes were determined by the differentialGeneTest function with a model against the cell type identities. The top 2,000 variable genes with the lowest adjusted p value were used to order the cells. The orders were determined by the orderCells function, and the trajectory was constructed by the reduceDimension function with default parameters.
Bulk RNA Sequencing
[0054] RNA was isolated from the cells using RNeasy Mini kit (Qiagen) and used to generate RNA-seq libraries followed by sequencing using Illumina HighSeq 4000 platform (single end, 50 bp). The reads were mapped with STAR.sup.59 (version 2.5.3.a) to the mouse genome (mm10). The counts for each gene were obtained by usingquantMode GeneCounts commands in STAR, and the other parameters during alignment were set to default. Differential expression analyses were carried out using DESeq2.sup.60 (version 1.24.0) with default parameters. Genes with adjusted p value <0.05 were considered significantly differentially expressed. Sequencing depth normalized counts were used to plot the expression values for individual genes. The T cell bulk RNA-seq datasets were downloaded from GEO (GSE111902).sup.61, and the same DESeq2 procedure was applied. Genes with the absolute log2 fold change >0.5 and adjusted p value <0.05 in both NK cell and T cell datasets were plotted in
ATAC-Seq Analysis
[0055] For ATAC-Seq, 50,000 cells per sample were lysed to collect nuclei and treated with Tn5 transposase (Illumina) for 30 minutes at 37 C. with gentle agitation. The DNA was isolated with DNA Clean & Concentrator Kit (Zymo) and PCR amplified and barcoded with NEBNext High-Fidelity PCR Mix (New England Biolabs) and unique dual indexes (Illumina). The ATAC-Seq library amplification was confirmed by real-time PCR, and additional barcoding PCR cycles were added as necessary while avoiding overamplification. Amplified ATAC-Seq libraries were purified with DNA Clean & Concentrator Kit (Zymo). The purified libraries were quantified with Kapa Library Quant Kit (KAPA Biosystems) and quality assessed on 4200 TapeStation System (Agilent). The libraries were pooled based on molar concentrations and sequenced on an Illumina HighSeq 4000 platform (paired end, 100 bp).
[0056] ATAC-seq fastq files were trimmed to remove low-quality reads and adapters using Cutadapt.sup.62 (version 2.3). The reads were aligned to the reference mouse genome (mm10) with bowtie2.sup.63 (version 2.2.9). Peak calling was performed with MACS2.sup.64 (version 2.1.1). The peaks from all samples were merged into a single bed file, peaks from different samples that were closer than 10 bp were merged into a single peak. HTseq.sup.65 (version 0.9.1) was used to count the number of reads that overlap each peak per sample. The peak counts were analyzed with DESeq2.sup.60 (version 1.24.0) to identify differentially accessible genomic regions. Peaks with adjusted p value <0.15 were considered significantly differentially accessible. The peak counts were visualized with IGV, version 2.5.0. The differentially accessible peaks were analyzed using the findMotifsGenome.pl function from homer.sup.66 (version 4.9.1) to identify enriched cis-regulatory motifs of transcription factors. The T cell ATAC-seq datasets were downloaded from GEO (GSE111902), the same pipeline described above were used to analyze the datasets. Cytokine stimulated NK cell ATAC-seq datasets were downloaded from GEO (GSE140044) and visualized using the same pipeline described above. Peaks in the T cell ATAC-seq datasets with adjusted p value <0.05 were considered significantly differentially accessible. Significant peaks with the same change directions in the NK and T cell datasets were plotted in
ChIP-Seq Analysis
[0057] STAT5 ChIP-seq datasets derived from IL-15/IL-2 stimulated splenic NK cells were downloaded from GEO (GSE140044).sup.35 and visualized using IGV, version 2.5.0.
Western Blot
[0058] Protein was extracted from enriched primary splenic NK cells using Pierce RIPA buffer (Thermo-Fisher) with Halt protease inhibitor cocktail (Thermo-Fisher) and protein concentration was quantified using the Pierce BCA Protein Assay kit (Thermo-Fisher). Samples were electrophoresed on NuPage Novex 4-12% Bis-Tris Protein Gels, transferred to PVDF membranes, and blocked for one hour at room temperature with 5% w/v nonfat milk in 1 TBS and 0.1% Tween-20. Immunoblots were performed using rabbit anti-Fli1 (Abcam ab133485), rabbit anti--actin (Cell Signaling CST4970), and rabbit anti-GAPDH (Millipore Sigma G9545). Proteins were detected using the SuperSignal West Pico PLUS ECL kit (Thermo-Fisher) and visualized using the Azure Biosystems c280 imager. Band density was quantified using ImageJ version 1.53.
Statistical Analyses
[0059] For graphs, data are shown as meanSEM, and unless otherwise indicated, statistical differences were evaluated using a Student's t test with Welch's correction to assume a non-normal variance in our data distribution. Samples were compared using the Log-rank (Mantel-Cox) test with correction for testing multiple hypotheses. p <0.05 was considered significant. Graphs were produced and statistical analyses were performed using GraphPad Prism.
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[0126] Additional electroporation methods, materials and devices that can be used with embodiments of the invention are disclosed, for example in US Patent Application Publication Nos.: 20200362355, 20200048606, 20200000851 and 20190388469, as well as literature references such as: Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. Schumann et al. Nat Immunol (2020); CRISPR screen in regulatory T cells reveals modulators of Foxp3; Cortez et al., Nature 2020, 29 Apr.; Pooled Knockin Targeting for Genome Engineering of Cellular
[0127] Immunotherapies. Roth et al., Cell 2020 Apr. 30; Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nguyen et al., Nat Biotechnol. 2019 Dec. 9; Landscape of stimulation-responsive chromatin across diverse human immune cells; Calderon et al., Nat Genet. 2019 Sep. 30; Large dataset enables prediction of repair after CRISPR-Cas9 editing in primary T cells; Leenay et al., Nat Biotechnol. 2019 September;37(9):1034-1037; A large CRISPR-induced bystander mutation causes immune dysregulation; Simeonov et al., Commun Biol. 2019 Feb. 18;2:70; Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Shifrut et al., Cell 2018 December; 175:1-14; Reprogramming human T cell function and specificity with non-viral genome targeting; Roth et al., Nature. 2018 July;559(7714):405-409; T-bet-ing on autoimmunity variants. Nguyen et al., PLOS Genetics. 13(9); e1006924 (2017); Discovery of stimulation-responsive immune enhancers with CRISPR activation; Simeonov et al., Nature. 549; 111-115 (2017); A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human Cells. Hulquist et al., Cell Reports. 17; 138-1452 (2016); and Generation of Knock-in Primary Human T Cells Using Cas9 Ribonucleoproteins Schumamn et al., PNAS. (2015), the contents of all of which are incorporated herein by reference.
[0128] All publications mentioned herein such as Riggan et al., Nature Immunology volume 23, pages 556-567 (2022) (this publication is also referred to herein as Riggan et al.); PCT Publication Serial No. PCT/US21/62365, and those identified above are incorporated by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications.
[0129] Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.