Elucidating the Protective Role of Adaptive Natural Killer Cells in Malaria

Daniel Michelson1, Jules Sangala2, James Dalhvang2, Geoffrey Hart21University of Minnesota Medical School Twin Cities, 2Division of Infectious Disease and International Medicine, Department of Medicine, Minneapolis,

MinnesotaUniversity of Minnesota Center for Immunology

Autoimmunity*Cancer*Vaccines*Cures

University of Minnesota Center for Immunology

Autoimmunity*Cancer*Vaccines*Cures

Background:Malaria presents an ongoing global health crisis with 500 million cases and 500,000 deaths annually, and despite significant efforts there is currently no approved preventative malaria vaccine. However, the severity of disease varies across populations, with individuals in endemic regions experiencing a greater degree of natural immunity. Natural killer (NK) cells form an essential part to the body’s innate immune response to malaria, and recently the Hart lab has obtained data indicating NK cells may kill infected red blood cells (iRBCs) viaantibody-dependent cell-mediated cytotoxicity (ADCC). Although NK cells are commonlyassociated with the innate immune system, NK cell populations have been shown tophenotypically and functionally obtain characteristics of the adaptive immune response towardcytomegalovirus infection (CMV). Specifically, these NK cells tend to express CD57 and NKG2Cwhile underexpressing Fc receptor (FcR) γ chains and promyelocytic leukemia zing finger(PLZF) and are identified as “adaptive NK cells” 1,2,3. Surprisingly, FcRγ and PLZF negative NK cells increase with malaria exposure and are associated with reduced malaria risk and reducedparasitemia, but the mechanisms of these associations remain unclear. Preliminary data showsthat FcRγ negative NK cells exhibit enhanced ADCC function, which may be the reason for theirprotective association and increased RBC lysis, but the mechanism of this enhanced function is currently unknown.

Figure 1. Incidence of PLZF - and FcRγ - CD56dim NK cells is elevated in Malian cohort (malaria-endemic region) versus Sweden cohort.

Figure 2. FcRγ - NK cells correlate with decreased malaria susceptibility in Malian subjects. Increased numbers of

Aims: 1. Elucidate the mechanism of enhanced ADCC in FcR! negative NK cells2. Identify mechanism of NK lysis of infected red blood cells3. Identify antibodies responsible for the phenotypic change in adaptive NK cells

Future Directions:- Optimize primary NK culture and CRISPR-Cas9 protocol- Optimize guides for other pathway targets (Syk, Zap70, " chain, etc.)- Evaluate post-knockout NK ADCC function - Use luciferase assay to identify key monoclonal antibodies against Plasmodium proteins- Identify mechanism of RBC lysis (perforin, iNOS)- Induce adaptive NK cell phenotype in naive cells

Acknowledgements: Thanks to Branden Moriarity and the Moriarity lab, 2018 NIH T35 Medical Student Summer Research Program in Infection and Immunity, and Dr. Dan Mueller.

Conclusions:- Successful electroporation of primary NK cells requires cytokine stimulation prior to electroporation- CRISPR/Cas9 can be used to knockout FcRγ and perforin in primary NK cells - Luciferase ADCC assays utilizing Jurkat effector cells offer a convenient, scalable model for

studying antibody-mediated ADCC in NK cells

Results:

Methods:

Concepts:

PLZF - FcR γ - CD56 Bright

PLZF - FcR γ - CD56 Bright

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FcR γ neg

P value = 0.0028

Susceptible Resistant0

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PLZF neg

P value = 0.039

Susceptible Resistant0

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neg

(% o

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*

Conventional NK cell

g chain z chain

CD16(FcRgIIIa)

g and z chain signal

ADCC

GnegNK cell

z chain

CD16(FcRgIIIa)

z Chain only signal

ADCC

z chain

Figure 4. FcR ! chains contain two ITAMs, while " chains contain six ITAMs. We hypothesize that a reduction in ! chains causes an increase in " chain signaling, resulting in increased ADCC.

Figure 5. Outline of CRISPR/Cas9 protocol used to knock out selected genes in primary NK cells.

Figure 7. CRISPR/Cas9 knockout of FcR! in primary NK cells from a single subject visualized by flow cytometry. Stimulation was performed with either IL-15 or IL-2 for 4 days prior to electroporation. Two guide RNAs were used for all samples. Both populations showed increased proportions of FcR! - NK cells following electroporation (right).

Figure 9. Luciferase assay control data comparing cells with target antibodies versus background luminescence from isolated cells. Cell types and target antibodies used for the ADCC assay include P815 mouse mast cells (mouse monoclonal anti-CD16/32 antibody), red blood cells (rabbit polyclonal anti-RBC antibody target), and Plasmodium infected red blood cells (iRBCs) stimulated with US or Mali plasma.

Figure 8. CRISPR/Cas9 knockout of perforin (PRF1) in primary NK cells visualized by flow cytometry. Triplicate cells were stimulated with IL-15 at 2 ng/ml for 4 days prior to electroporation. Two guide RNAs were used for each sample.

Figure 10. Comparison of ADCC markers following exposure of Plasmodium infected RBCs to US or Mali plasma. A and B show increased percentages of primary NK cells positive for CD107a and IFNg on flow cytometry for the subjects exposed to iRBCs and Mali plasma versus US plasma. Cshows increased increased luminescence in CD16-expressing Jurkat cells exposed to iRBCs and Mali plasma versus US plasma. D illustrates the linear correlation between flow cytometry data for ADCC markers CD107a and IFNg and luciferase ADCC assay luminescence(R2 = 0.97, 0.96, respectively). Correlation between the luciferase ADCC assay and primary NK degranulation flow cytometry data were also collected for the P815, RBC, and iRBC/US plasma models, with R2

coefficients ranging from 0.88 to 0.96 for the P815 and RBC samples. The iRBC/US plasma model R2 values were 0.22 (CD107a) and 0.85 (IFNg).

Figure 3. CD16-mediated response pathways in NK cells leading to lysis of infected red blood cells through an unclear mechanism, likely involving perforin and/or nitric oxide (NO).

Figure 6 Concept diagram of luciferase ADCC assay. Antibodies on the target cell initiate a CD16 mediated response in the effector cell, resulting in an NFAT response element driven expression of luciferin.

Target cell

Antigen-antibody complex

CD16 Effector cell

NFAT-RE LucLuciferase

References: 1. Lopez-Verges, S., et al. “Expansion of a unique CD57(+)NKG2Chi natural killer cell subset during acute human cytomegalovirus infection.” Proceedings of the National Academy of Sciences 108 (2011): 14725-14732. 2. Guma, M. et al. “Expansionof CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts.” Blood 107 (2006): 3624-3631. 3. Tesi, B., et al. “Epigenetic regulation of adaptive NK cell diversification.” Trends in Immunology 37 (2016): 451-461.

0.00001 0.0001 0.001 0.010

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iRBC-US-Target

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CD56

FcRg

IL-2

IL-15

Perforin +39.1

Perforin -60.9

0 104

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107

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-103

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Perforin +39.4

Perforin -60.6

0 104

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Perforin +34.8

Perforin -65.2

0 104

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-103

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Perforin +97.8

Perforin -2.15

0 104

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-103

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Perforin +99.0

Perforin -0.52

0 104

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-103

103

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Perforin +99.3

Perforin -0.75

0 104

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107

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-103

103

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CD56

Perforin

Control

2 Perforin guides

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Mali/US CD107aMali CD107aUS CD107a

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ADCC After Plasma ExposureMali

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Mali/US IFNg

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Luminescence

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iRBC-Mali-ADCC

CD107a

IFNg

R squareCD107a0.9726

IFNg0.9635

A B

C D