Memory-like NK Cells Exploit Innate Priming and Alternative Signaling Mechanisms to Enhance Function in HIV/SIV infection R. Keith Reeves, Ph.D. Center for Virology and Vaccine Research Harvard Medical School/BIDMC

‘Memory-like’ FcR∆g NK cells x FcR signaling Lack gamma signaling chain and adaptor Syk Still require antibodies to grant antigen-specificity Rapid mobilization and proliferation upon secondary antigen exposure Increased cytokine production More potent ADCC functions Previously only described in human blood Massively expanded by CMV (70% seropositivity) CMV differences % of NK cells Lee et al., Immunity, 2015

FcR∆g NK cells in rhesus macaques Traditional NK CD56 CD56 FcR∆g NK Phenotypically analogous in humans and macaques No clear differences in NK cell subpopulations (by CD56 and CD16) FcR∆g NK expand with rhCMV Nearly all adult captive rhesus are rhCMV+, unless from SPF colonies FcR∆g NK expand further in SIV NKG2a/c R = 0.570 p < 0.0001 All these expanded cells, where are they going?

Trafficking patterns of FcR∆g memory-like NK cells *** *** Circulating FcR∆g NK cells express lower levels of LN-homing molecules CCR7 and CD62L ** Circulating FcR∆g NK cells express higher levels of gut-homing a4b7 Further increased in SIV infection May suggest preferential mucosal homing which is consistent with cytotoxic NK cells ***

FcR∆g NK cells are systemically distributed (by flow) ** * FcR∆g NK cells are found systemically Increased in tissues during SIV infection

FcR∆g memory-like NK function Binding of fluorescent HIVJRFL immune complexes Functional activation after CD16 cross-linking CD107a Human Rhesus CD16 Binding of HIV immune complexes does not differ from bulk NK cells Stimulation via CD16 demonstrates more robust cytotoxic function in FcR∆g compared to traditional NK CD16 density upregulates on FcR∆g NK cells following exposure to CMV Memory-like FcR∆g may require innate priming to achieve functional potential

What mechanisms underlie memory-like FcR∆g NK cells enhanced potency for antibody-mediated functions?

FcR∆g memory-like NK are disparately transcriptionally regulated Syk Helios FcgR Eomes Lower Syk, Helios, and Eomes – results in a broader functional repertoire Altered transcriptional profile suggests different developmental program (liver) OR epigenetic modifications Fits in well with other ‘memory’ NK cell subsets *** In addition to lack of gamma chain and syk, also have lower helios and eomoes ***

FcR∆g memory-like NK don’t signal through γ-chain and Syk adaptor pathway PhosphoFlow DG NK Phospho- Syk CD16 Lack gamma chain Low basal Syk expression pSyk absent in FcR∆g following stimulation Syk, if present, is generally inactive FcgR

Alternate signaling pathways Paul J Leibson Signal Transduction during Natural Killer Cell Activation: Inside the Mind of a Killer null, Volume 6, Issue 6, 1997, 655–661

FcR∆g memory-like NK cell functional signaling CD3 zeta Most NK cells express CD3ζ Typical signaling for non antibody-dependent killing, but also for ADCC Hypothesized alternate pathway Increased expression in Syk-deficient FcR∆g NK cells FcR∆g NK cells have higher pCD3ζ indicating an active pathway PhosphoFlow Phospho- CD3 zeta FcgR Zap70 CD3ζ uses either Syk or Zap70 as adaptor Zap70 higher in FcR∆g NK cells

Summary & Conclusions Memory-like FcR∆g NK cells are functionally more potent than other FcR-bearing NK cells and recall rapidly after functional priming Memory-like FcR∆g NK cells are systemic and mobilize in the mucosae during SIV infection Functional priming of FcR∆g NK cells depends on prior CMV status and uses CD3zeta-Zap70 signaling as an alternative to gamma chain/Syk to enhance function

Acknowledgements Reeves Lab/ Immunology Group — Center for Virology and Vaccine Research Spandan Shah Cordelia Manickam Valerie Varner Tristan Evans Estelle Autissier Daniel Ram Megan Powell Amanda Jimenez George Tweet Vaishnavi Parthasarathy Scott Smith Michelle Lifton Stephanie Jost Mary Laughridge Haiying Li New England Primate Research Center Jackie Gillis Michelle Connole Harvard Medical School Tulane Primate Center Miti Kaur Pyone Aye Marion Ratterree Uli von Andrian Yerkes NPRC Beth Israel Deaconess Dan Barouch Paul Johnson Premi Rajakumar Duke University Biomere Georgia Tomaras Guido Ferrari Sallie Permar Institute of Human Virology Mark Nedelman Angela Carville Christian Grant George Lewis Tony DeVico FUNDING R01 DE026327 R01 DE026014 P01 AI120756 R21 AI118468 UM1 AI124377 UM1 AI068614 108547-53-RGRL P30 AI060354

FcR∆g signaling Mouse Human Human FcR∆g ?? Zap70 Zap70 Zap70 Syk Syk In the absence of intact gamma chain, FcR∆g NK cells could signal through homodimeric CD3ζ and Zap70