Antigen-specific memory NK cell responses against HIV and influenza use the NKG2/HLA-E axis

Abstract

Multiple studies have broadened the roles of natural killer (NK) cells functioning as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including HIV-1 and influenza. However, the mechanisms underlying antigen specificity remain unknown. Here, 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. We validated the permanent acquisition of antigen specificity by individual memory NK cells by single-cell cloning. We identified elevated expression of KLRG1, α4β7, and NKG2C as biomarkers of antigen-specific NK cell memory through complex immunophenotyping. Last, we uncovered 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 contributing to antigen-specific memory NK cell responses and suggest that they could be potentially targeted therapeutically for vaccines or other therapeutic interventions.

Fig. 1.. Human NK cells mediate antigen-specific responses against HIV and influenza.

(A) Enriched NK cells from PLWH (N=7) or healthy donors (N=7) were co-cultured with autologous BLCL that had been pulsed with 2ug/mL peptide pools derived from HIV Gag (HIV-1 Consensus B; provided by the NIH AIDS Reagent Program) and NK cell responses assessed by ICS. Dead cells were excluded. Dot plots show proportions of IFN-γ⁺ and CD107a⁺ NK cells after subtracting background (unstimulated). (B) 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. (C) Enriched NK cells from 11 HIV-negative healthy donors were incubated overnight with 2ug/mL 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. Dead cells were excluded. Dot plots show proportions of IFN-γ⁺ and CD107a⁺ NK cells after subtracting background (unstimulated). Full circle, positive for IgG antibodies against influenza A by ELISA. Empty circle, not tested for the presence of IgG antibodies against influenza A. Asterisks, significant differences compared to unstimulated controls. Data are represented as median and individual data points (A and C) or mean ± SEM and individual data points (B). Statistical significance was tested using Mann-Whitney U test for comparisons between PLWH (HIV+) and HIV-negative donors (HIV-) (A and B), or Wilcoxon signed-rank test for comparisons between unstimulated and stimulated NK cells (C). * p<0.05.

Fig. 2.. Single-cell cloning of human antigen-specific NK cells.

Percentages of antigen-specific lysis by 133 NKCL generated from 19 PLWH against HIV Env, HIV Gag and CMV pp65 (A) after subtracting background (mock-pulsed BLCL) and (B) compared to non-specific killing of mock-pulsed BLCL for each NKCL. Percentages of antigen-specific lysis by 28 NKCL generated from 10 healthy donors against influenza H1N1 NP (C) after subtracting background (mock-pulsed BLCL) and (D) compared to non-specific killing of mock-pulsed BLCL for each NKCL. Full circle, positive for IgG antibodies against influenza A. Empty circle, not tested for the presence of IgG antibodies against influenza A. 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). (E) Heatmap of antigen-specific lysis after subtracting background for 16 NKCL from 8 PLWH with the highest specific killing against HIV Gag and/or ENV. NKCL generated from the same PLWH are displayed in a unique color. NKCL displaying over 15% specific killing against both HIV Env and HIV Gag or HIV Env and CMV pp65 are indicated in bold font. Statistical significance was tested using Wilcoxon signed-rank test (B and D). ns, not significant.

Fig. 3.. Human antigen-specific NK cell responses are associated with NKG2C expression.

(A) Non-linear t-SNE (bh-SNE) plots of 14 NKCL from one untreated viremic PLWH, showing distinct clustering for 7 HIV reactive and 7 non-HIV-reactive NKCL, with HIV-reactive NKCL being partly associated with cell clusters expressing high levels of the inhibitory NKG2A or high levels of activating NKG2C and/or inhibitory KIR3DL1. (B) Antigen-specific killing of HIV Env-pulsed BLCL by 6 NKCL in the presence of isotype control or NKG2C blocking antibodies. Bars represent mean+SEM. Statistical significance was tested using Wilcoxon signed-rank test. (C) Representative flow cytometry plots showing NKG2C expression on one NKCL 48 hours after nucleofection with a scramble siRNA control (Ctrl siRNA) or with siRNA targeting NKG2C. (D) Percentages of lysis by 3 NKG2C+ NKCL reacting against HIV Env (left panel) and MFI of NKG2C on the respective NKCL (right panel) from 2 PLWH 48 hours following nucleofection with siRNA control or siRNA targeting NKG2C. (E) Percentages of lysis by 2 NKG2C+ NKCL reacting against influenza NP (A/AnnArbor/6/1960(H2N2)) (left panel) and MFI of NKG2C on the respective NKCL (right panel) from 1 healthy donor 48 hours following nucleofection with siRNA control or siRNA targeting NKG2C. Percentages of NKG2C down-modulation are indicated for each NKCL. Only NKCL displaying responses over 5% specific killing and at least 1.5-fold above the background (unpulsed) after nucleofection with Ctrl siRNA were included in the analysis. (F), (G) Spearman correlation analysis between frequencies of NKG2C⁺ NKCL and specific killing of BLCL pulsed with HIV Env (F) or influenza NP, MP1, or HA (G). Frequencies of NKG2C⁺ NKCL were determined as the proportion of NKCL beyond an arbitrary cut-off line set by at the right-hand side border of the NKG2C histogram in FMO controls (See Fig. S9).

Fig. 4.. Human antigen-specific memory NK cell responses are mediated via recognition of HLA-E-binding viral peptides.

(A) K562 cells stably expressing HLA-E*0101 were either left mock pulsed or pulsed for 16h with 40nm of 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. Bars represent the relative mean fluorescence intensity (RFI) + SEM of HLA-E on K562 HLA-E*01:01 cells pulsed with indicated individual peptides found to stabilize HLA-E and pooled from at least 2 distinct experiments as compared to HLA-E expression in the absence of peptide (No Peptide). The dotted line marks the cut off for increased HLA-E expression, defined as the mean value plus 2 standard deviations of non-stabilizing peptides. Asterisks, significant differences compared to K562 HLA-E*01:01 pulsed with non-binding peptides (NEG). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Percentages of peptide-specific lysis by 17 NKCL from 5 untreated viremic PLWH (B) after subtracting non-specific lysis (killing of mock pulsed autologous BLCL) and (C), compared to non-specific killing of mock-pulsed BLCL for each NKCL. Percentages of peptide-specific lysis by 14 NKCL from 5 healthy donors (D) after subtracting non-specific lysis (killing of mock pulsed autologous BLCL) and (E), compared to non-specific killing of mock-pulsed BLCL for each NKCL. 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, HIV Gag or H1N1 NP sequence or with indicated single HLA-E-binding nonameric peptides derived from CMV UL40, HIV Gag, HIV Env, or H1N1 NP. (F) 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 single HLA-E-binding nonameric peptides derived from HIV Env or H1N1 NP. NKG2C⁺ NKCL are indicated in red. Statistical significance was tested using Kruskal Wallis test with Dunn’s multiple comparison test (A) or Wilcoxon signed-rank test (C, E and F).

Fig. 5.. HIV Env- and H1N1 NP-derived HLA-E-binding peptides elicit primary NK cell responses.

(A)(B) PBMCs samples from 12 PLWH (8 cART-treated and 4 untreated viremic) were incubated for 16h with 2μg/mL of 1 pool of 15 amino acid (aa) peptides overlapping by 11 aa and 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 (A) and IFN-y production (B). Enriched NK cells from 19 healthy donors were incubated for 6 hours with 2μg/mL of 1 pool of 15 aa peptides overlapping by 11 aa and spanning H1N1 NP or 40uM of indicated single H1N1 NP-derived nonamers in the presence of CD107a antibodies, GolgiStop and GolgiPlug to evaluate CD107a upregulation (C) and IFN-γ production (D) by ICS. NK cell responses to H1N1 NP-derived TMDSNTLEL was evaluated in the presence of control or anti-NKG2C antibodies. Dot plots represent proportions of actively degranulating, cytotoxic NK cells, as judged by cell surface expression of CD107a (A, C left panel) or IFN-y producing NK cells (B, D) after subtracting proportions of CD107a⁺ and IFN-γ⁺ unstimulated NK cells, respectively. Bars represent the median. Paired plots compare CD107a expression between unstimulated and single-peptide-stimulated NK cells (C, right panels) or between NK cells stimulated with H1N1 NP-derived TMDSNTLEL in the presence of isotype control or anti-NKG2C antibodies after subtracting proportions of CD107a⁺ unstimulated NK cells (E). NK cells from all donors expressed NKG2C (PLWH: median 15.6%, range 2%-75%; healthy donors: median 10.3%, range 2%-48%). Statistical significance was tested using Wilcoxon signed-rank test (C and E).