DOI: 10.5061/dryad.nzs7h44xb
title: Antigen-specific memory NK cell responses against HIV and influenza utilize the NKG2/HLA-E axis

description: For over a decade, multiple studies have disputed the notion of natural
 killer (NK) cells as purely innate lymphocytes by demonstrating that they
 are capable of putative antigen-specific immunological memory against
 multiple infectious agents including two critical global health priorities
 – human immunodeficiency virus (HIV) and influenza. However, the
 mechanisms underlying antigen specificity remain unknown. Herein, we
 demonstrate that antigen-specific human NK cell memory develops upon
 exposure to both HIV and influenza, unified by a conserved and
 epitope-specific targetable mechanism largely dependent on the activating
 CD94/NKG2C receptor and its ligand HLA-E, and confirm these findings by
 three rigorous assays. We validated the permanent acquisition of
 antigen-specificity by individual memory NK cells by single-cell cloning.
 We identified biomarkers of antigen-specific NK cell memory through
 complex immunophenotyping by 30-parameter flow cytometry showing elevated
 expression of KLRG1, a4b7, and NKG2C. Finally, we show individual
 HLA-E-restricted peptides that may constitute the dominant NK cell
 response in HIV-1- and influenza-infected persons in vivo. Our findings
 clarify the mechanisms behind antigen-specific memory NK cell responses,
 and suggest they could be targeted for future vaccines, cure strategies,
 or other therapeutic interventions.
description: # Antigen-specific memory NK cell responses against HIV and influenza
 utilize the NKG2/HLA-E axis For over a decade, multiple studies have
 disputed the notion of natural killer (NK) cells as purely innate
 lymphocytes by demonstrating that they are capable of putative
 antigen-specific immunological memory against multiple infectious agents
 including human immunodeficiency virus (HIV) and influenza. However, the
 mechanisms underlying antigen specificity remain unknown. Altogether,
 these datasets were used to demonstrate that antigen-specific human NK
 cell memory develops upon exposure to both HIV and influenza, unified by a
 conserved and epitope-specific targetable mechanism largely dependent on
 the activating CD94/NKG2C receptor and its ligand HLA-E, and confirm these
 findings by three rigorous assays. We validated the permanent acquisition
 of antigen-specificity by individual memory NK cells by single-cell
 cloning. We identified biomarkers of antigen-specific NK cell memory
 through complex immunophenotyping by 30-parameter flow cytometry showing
 elevated expression of KLRG1, a4b7, and NKG2C. Finally, we show individual
 HLA-E-restricted peptides that may constitute the dominant NK cell
 response in HIV- and influenza-infected persons in vivo. Our findings
 clarify the mechanisms behind antigen-specific memory NK cell responses
 and suggest they could be targeted for future vaccines, cure strategies,
 or other therapeutic interventions. **Description of the data and file
 structure** The data used to generate figures for this manuscript are
 contained within the file adi3974_RawData.xlsx. The data are in tabular
 format with rows corresponding to samples and columns corresponding to
 features. Sheets in the file are named and their descriptions are detailed
 below. Unless otherwise denoted all numerical values represent percent
 expression for each sample. Data are reported as both manipulated
 (background subtracted) or unmanipulated (raw) values where applicable.
 For cytotoxicity assays, negative raw values were considered as “0” for
 background subtraction. All data were from human donors either as
 HIV-negative healthy controls or people living with HIV. The data
 represent human NK cells either as a bulk population purified from
 peripheral blood mononuclear cells or as single-cell clones generated per
 donor. Assays utilizing clonally expanded NK cells denote which specific
 clone was utilized. Empty cells mean the sample was not tested for a
 specific condition. **Abbreviations** PLWH: people living with HIV NKCL:
 NK cell clone ICS: intracellular cytokine staining CAM: Calcein
 acetoxymethyl BLCL: B-Lymphoblastoid Cell Line cART: combination
 antiretroviral therapy **Datasets included** 1.A S1.A: Antigen-specific
 responses against HIV by Bulk NK cells from PLWH or HIV-negative
 participants. Enriched NK cells from PLWH (N=7) or healthy donors (N=7)
 were co-cultured with autologous BLCL that had been pulsed with peptide
 pools derived from HIV Gag and NK cell responses assessed by ICS. NK cell
 responses for PLWH and healthy donors are displayed in different columns. 
 1.B: Antigen-specific responses against HIV by Bulk NK cells from PLWH or
 HIV-negative participants. Autologous BLCL were pulsed with a pool of HIV
 Gag overlapping peptides or with the CEF (CMV, EBV and influenza) control
 peptide pool and were labeled with the CellTrace Violet dye. Mock-pulsed
 BLCL serving as intra-well controls were labeled with the green dye CFSE.
 Purified NK cells from PLWH (N=12) or healthy donors (N=6) were
 co-cultured with BLCL at 5:1 E:T ratios (equal mixture of pulsed target
 BLCL and unpulsed control BLCL) for 16 hours, and specific lysis of BLCL
 was determined by flow cytometry. Killing of HLA-deficient K562 cells was
 used as additional positive control. CT, chronic treated; CU VIR, chronic
 untreated viremic; EC, elite controller; PC, previous controller. NK cell
 responses for PLWH and healthy donors are displayed in different columns. 
 \** ** 1.C S1.C: Antigen-specific responses against influenza by Bulk NK
 cells from HIV-negative healthy participants. Enriched NK cells from 11
 HIV-negative healthy donors were incubated overnight with peptide pools
 derived from influenza A/California/04/2009(H1N1) NP and
 A/California/08/2009(H1N1) MP1 and NK cell responses assessed by ICS. NP,
 nucleoprotein; MP1, matrix protein 1; PHA, Phytohaemagglutinin (positive
 control). \** ** 2.ABE S4.AB S5.ABC: Antigen-specific responses against
 HIV and CMV by NKCL from PLWH. Percentages of antigen-specific lysis by
 133 NKCL generated from 19 PLWH against HIV Env, HIV Gag and CMV pp65
 after subtracting background (mock-pulsed BLCL) or not (unmanipulated).
 CAM cytotoxicity assays were used to evaluate lysis after co-culture of
 NKCL with autologous BLCL pulsed with indicated peptide pools.
 Non-specific lysis was assessed by measuring killing of mock-pulsed
 autologous BLCL, self-peptides-pulsed BLCL (negative control) and
 HLA-E-deficient K562 cells (positive control). The percent-specific lysis
 was calculated as follows: (test release – spontaneous release)/(maximum
 release - spontaneous release) x 100. \** ** 2.CD S4.CD: Antigen-specific
 responses against influenza by NKCL from HIV-negative donors. Percentages
 of antigen-specific lysis by 28 NKCL generated from 10 healthy donors
 against influenza H1N1 NP after subtracting background (mock-pulsed BLCL)
 or not (unmanipulated). CAM cytotoxicity assays were used to evaluate
 lysis after co-culture of NKCL with autologous BLCL pulsed with indicated
 peptide pools. Non-specific lysis was assessed by measuring killing of
 mock-pulsed autologous BLCL, self-peptides-pulsed BLCL (negative control)
 and HLA-E-deficient K562 cells (positive control). The percent-specific
 lysis was calculated as follows: (test release – spontaneous
 release)/(maximum release - spontaneous release) x 100. 3.B S9.A:
 Antigen-specific responses against HIV by NKCL from PLWH in the presence
 of control antibodies or antibodies blocking NK cell receptors.
 Percentages of antigen-specific killing of HIV Env-pulsed BLCL by 8 NKCL
 in the presence of isotype control or NKG2C, NKp44 or NKp30 blocking
 antibodies after subtracting background (mock-pulsed BLCL). CAM
 cytotoxicity assays were used to evaluate lysis after co-culture of NKCL
 with autologous BLCL pulsed with HIV Env peptide pools. \** ** 3.DE:
 Antigen-specific responses against HIV and influenza by NKCL following
 nucleofection with siRNA control or siRNA targeting NKG2C. Top 3 rows:
 percentages of lysis by 3 NKG2C+ NKCL from 2 PLWH reacting against HIV Env
 48 hours following nucleofection with siRNA control or siRNA targeting
 NKG2C. Bottom 2 rows: percentages of lysis by 2 NKG2C+ NKCL reacting
 against influenza NP (A/AnnArbor/6/1960(H2N2)) 48 hours following
 nucleofection with siRNA control or siRNA targeting NKG2C. MFI of NKG2C on
 each NKCL 48 hours following nucleofection with siRNA control or siRNA
 targeting NKG2C is indicated in the last 2 columns. \** ** 3.FG S9.CDEF:
 Positive correlation between NKG2C expression and antigen-specific killing
 by NKCL. Percentages of specific killing of BLCL pulsed with HIV Env (73
 NKCL from 14 PLWH) or influenza NP, MP1, or HA (31 NKCL from 10 donors)
 and frequencies of NKG2C+, NKG2A+ and MFI of NKG2C for each NKCL. \** **
 4A: Identification of peptides derived from HIV and influenza that
 stabilize HLA-E. K562 cells stably expressing HLA-E*0101 were either left
 mock pulsed or pulsed with nonameric peptides derived from HIV consensus B
 Gag or Env or from A/California/04/2009(H1N1) NP. Controls included CMV
 pp65-derived NLVPMVATV that do not stabilize HLA-E (NEG), and VMAPRTLFL, a
 CMV/HLA-G leader sequence-derived peptide that stabilizes HLA-E (POS).
 HLA-E surface stabilization was assessed by flow cytometry. Each row
 represents an independent experiment. Data was obtained by calculating the
 relative mean fluorescence intensity of HLA-E on K562 HLA-E*01:01 cells
 pulsed with indicated individual peptides (in each column) compared to
 HLA-E expression in the absence of peptide (No Peptide). \** ** 4.BC
 S12.ABC: Identification of NKCL from PLWH that react to single HIV-derived
 HLA-E stabilizing peptides. Percentages of peptide-specific lysis by 15
 NKCL from 5 untreated viremic PLWH after subtracting non-specific lysis
 (killing of mock pulsed autologous BLCL) or not (unmanipulated). CAM
 cytotoxicity assays were used to evaluate lysis after co-culture of NKCL
 with autologous BLCL pulsed with peptide pools encompassing the whole HIV
 Env or HIV Gag sequence or with indicated single HLA-E-binding nonameric
 peptides derived from CMV UL40, HIV Gag or HIV Env. 4.DE S12.DE:
 Identification of NKCL from healthy donors that react to single
 influenza-derived HLA-E stabilizing peptides. Percentages of
 peptide-specific lysis by 14 NKCL from 5 healthy donors after subtracting
 non-specific lysis (killing of mock pulsed autologous BLCL) or not
 (unmanipulated). CAM cytotoxicity assays were used to evaluate lysis after
 co-culture of NKCL with autologous BLCL pulsed with peptide pools
 encompassing the whole H1N1 NP sequence or with indicated single
 HLA-E-binding nonameric peptides derived from H1N1 NP. \** ** 4.F S5.D:
 NKCL from HIV-negative donors do not react to HIV-derived peptides.
 Percentages of peptide-specific lysis by 27 NKCL from 7 HIV-negative
 healthy donors after subtracting non-specific lysis (killing of mock
 pulsed autologous BLCL). CAM cytotoxicity assays were used to evaluate
 lysis after co-culture of NKCL with autologous BLCL pulsed with indicated
 peptide pools or single HLA-E stabilizing peptides derived from HIV or
 H1N1 NP or with the CEF (CMV, EBV and influenza) control peptide pool.
 \** ** 5.AB S13AB: Antigen-specific responses against HIV-derived
 HLA-E-stabilizing single peptides by Bulk NK cells from PLWH. PBMCs
 samples from 12 PLWH (8 cART-treated and 4 untreated viremic) were
 incubated for 16h with pools of peptides spanning HIV Env or single HIV
 Env- and CMV UL40-derived nonamers in the presence of CD107a antibodies.
 GolgiStop and GolgiPlug were added for the last 2 hours incubation prior
 to surface and intracellular cytokine staining to measure CD107a
 upregulation and IFN-y production by ICS. \** ** 5.CDE S14A:
 Antigen-specific responses against influenza-derived HLA-E-stabilizing
 single peptides by Bulk NK cells from healthy donors. Enriched NK cells
 from 19 healthy donors were incubated for 6 hours with pools of peptides
 spanning H1N1 NP or indicated single H1N1 NP-derived nonamers in the
 presence of CD107a antibodies, GolgiStop and GolgiPlug to evaluate CD107a
 upregulation and IFN-y production by ICS. For a subset of donors, NK cell
 responses to H1N1 NP-derived TMDSNTLEL was evaluated in the presence of
 control or anti-NKG2C antibodies. \** ** S6 S7: Phenotypic analysis of
 NKCL reacting or not to HIV by flow cytometry. Proportions of NKCL
 expressing indicated surface markers evaluated by flow cytometry and
 comparing 5 NKCL from 2 healthy donors with 14 NKCL from one untreated
 viremic PLWH, including 7 HIV Gag/Env-specific NKCL and 7 NKCL that did
 not react to HIV Gag/Env. Each row represents data for the same NKCL.
 \** ** S10.BCDEFG: Stabilization of HLA-E*01:01 by HIV Env-, HIV Gag- and
 influenza NP-derived nonameric peptides. K562 cells stably expressing
 HLA-E*01:01 were either left mock pulsed or pulsed for 16h with 40uM of
 nonameric peptides derived from A/California/04/2009(H1N1) NP (18 total),
 HIV consensus B Env (84 total) and HIV consensus B Gag (60 total) with
 strong binding prediction scores based on the NetMHC pan 4.0 and IEDB
 epitope prediction tools. Controls included CMV pp65-derived NLVPMVATV
 that do not stabilize HLA-E (NEG CTRL), and VMAPRTLFL, a CMV/HLA-G leader
 sequence-derived peptide that stabilizes HLA-E (POS CTRL). HLA-E surface
 stabilization was assessed by flow cytometry using anti-HLA-E antibody
 (3D12). Values represent the relative mean fluorescence intensity (RFI) of
 HLA-E on K562 cells as compared to HLA-E expression in the absence of
 peptide (No Peptide) for all tested peptides. Percentages of HLA-E+ K562
 cells are also included for all tested peptides. Each row represent data
 for one single peptide. Columns represent independent experiments. S11
 ABD: HIV Env, HIV Gag and influenza NP encode for HLA-E-stabilizing
 peptides. S11.A box: Relative mean fluorescence intensity (RFI) of HLA-E
 on K562 HLA-E*01:01 cells as compared to HLA-E expression in the absence
 of peptide after pulsing with increasing concentrations of indicated
 peptides. Exp #1 and #2 represent independent experiments. S11.B box:
 Competition assay for binding to HLA-E. K562-HLA-E*0101 cells were
 incubated overnight at 26°C with 10uM of FITC-conjugated VMAPRTLFL CMV
 peptide and increasing concentrations of the indicated unlabeled
 competitor peptides. Data represent the relative mean fluorescence
 intensity (RFI) of HLA-E-bound FITC-labelled VMAPRTLFL peptide pooled from
 3 independent experiments as compared to bound FITC-labelled VMAPRTLFL in
 the presence of 10uM of competitor peptide. S11.D box**:** Data represent
 the relative mean fluorescence intensity (RFI) of HLA-E on BLCL targets as
 compared to HLA-E expression in the absence of peptide after pulsing with
 indicated individual peptides at 40μM for 2 hours in R10 medium. NLVPMVATV
 (NEG CTRL); VMAPRTLIL (POS CTRL). S13 CDEF: Antigen-specific responses
 against HIV-derived HLA-E-stabilizing single peptides by bulk NK cells
 from PLWH. Proportions of CD107a+ or IFN-y+ NK cells following stimulation
 of enriched NK cells from 5 PLWH with indicated single HLA-E-stabilizing
 peptides derived from HIV Env or CMV UL40 using ICS. S14CD: Influenza
 NP-derived HLA-E-binding peptides elicit primary NKG2C+ NK cell responses.
 Enriched NK cells from 19 healthy donors were stimulated with indicated
 single HLA-E-stabilizing peptides derived from influenza NP and
 proportions of CD107a+ and IFN-y+ NK cells evaluated using ICS.
 Proportions of NKG2C+, NKG2A+, and NKG2C-NKG2A- degranulating CD107a+ NK
 cells, as well as NKG2C+, NKG2A+ and NKG2C-NKG2A- IFN-y producing NK cells
 are indicated after subtracting proportions of CD107a+ and IFN-y+
 unstimulated NK cells, respectively. **Sharing/Access information** All
 data, metadata, and methods used to generate figures and findings are
 contained within the repository and manuscript methods section. Raw flow
 cytometry files are available upon reasonable request to the corresponding
 author. **Code/Software** This manuscript generated no new code or
 programs. All data were generated using BD FACSDiva software with BD FACS
 Symphony flow cytometer. Flow cytometry analysis was performed using
 FlowJo software and exported data were statistically analyzed in GraphPad
 Prism software.