Tim 3 f38 2e2

Human PBMC were stained using directly conjugated mAb against human CD3 (BW264/56), CD56 (REA196), CD57 (TB03), from Miltenyi Biotec (San Diego, CA, USA), Tim-3 (F38-2E2) from BioLegend and NKG2C (134591) from R&D Systems. Cells were stimulated with 1 μg anti-CD16 mAb (3G8; BioLegend) per 10⁶ PBMC and prepared for intracellular staining by adding brefeldin A (Sigma-Aldrich) 1 h after the start of incubation to a final concentration of 10 μg/mL and continuing the incubation for an additional 4 h. NK cell degranulation was detected by introducing directly conjugated anti-CD107a mAb (H4A3; BioLegend) at a 0.25 μg per 10⁶ PBMC at the time of brefeldin A addition. Cells were fixed and permeabilized after 5 h incubation using the Inside Stain Kit (Miltenyi Biotec) as per manufacturer's instructions and then stained with directly conjugated polyclonal Ab against human FcRγ from MilliporeSigma (Burlington, MA, USA) and anti-human IFN-γ mAb (4S.B3) from eBioscience (San Diego, CA, USA). Non-viable cells were excluded by fixable live/dead stain (Invitrogen) as per manufacturer's instructions. Data were acquired using a MoFlo Astrios EQ flow cytometer and data analyses and illustration performed using Kaluza software (both Beckman Coulter, Brea, CA, USA).

For flow cytometry and FACS sorting experiments, fluorochrome-conjugated antibodies against the following antigens were used: CD45 (clone HI30; conjugated to BV421), CD3 (OKT3; BV605 & AF700), CD56 (HCD56; PE/Cy7), CD8 (SK1; BV510 & APC/Cy7), CD4 (SK3; FITC), CD19 (HIB19, PerCP/Cy5.5), CD14 (M5E2; APC), HLA-DR (LN3; BV711), PD-L1 (29E.2A3; BV605), LAG-3 (11C3C65; AF647), TIM-3 (F38-2E2; FITC & BV785), HLA-ABC (W6/32; PE/Cy7), Isotype (eBMG2b; PE) (MOPC-173; PE/Cy7) (all Biolegend); CD4 (SK3; UV395) and PD-L2 (MIH18; BV711 & BV785) (all BD Biosciences); CTLA-4 (14D3; PE/Cy7), PD-L1 (MIH1; APC), PD-1 (MIH4; FITC) (all ThermoFIsher, Waltham, MA); VZV gE (MilliporeSigma, Burlington, MA) conjugated in house to R-PE as previously described [31 (link)]. For immunofluorescence analyses experiments, unconjugated antibodies against the following antigens were used: VZV gB (Mouse: Abcam, Cambridge, MA) and VZV ORF63 (Rabbit) that was previously described [46 (link)]. Secondary antibodies consisted of Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 594 donkey anti-mouse IgG (MilliporeSigma). Tetramers of HLA A*0201 and VZV ORF34 and ORF18 proteins were obtained from the Fred Hutchinson Cancer Research Center Immune Monitoring Core.

The following fluorochrome conjugated monoclonal antibodies reactive with macaque cells were used for flow cytometry studies: CD3 (SP34-2, BD Biosciences [BD]), CD4 (L200, BD), CD8 (SK1, BD), CD95 (DX2, BD), CD28 (CD28.2, BioLegend), CCR5 (3A9, BD), CXCR5 (MU5UBEE, Invitrogen), PD-1 (EH12.2H7, BioLegend), ICOS (C398.4A, BioLegend), CCR7 (150503, BD), α4β7 (A4B7, NHP Reagent Resource), LAG3 (3DS223H, eBioscience), Tim-3 (F38-2E2, BioLegend), TIGIT (MBSA43, Invitrogen), CD20 (2H7, BioLegend), CD19 (J3-119, Beckman Coulter), HLA-DR (L243, BioLegend), CD10 (HI10A, BioLegend), CD21 (B-ly4, BD), CD27 (O323, BioLegend), IgD (IADB6, Southern Biotech), IgG (G18-145, BD), IL-21R (2G1-K12, BioLegend), Ki-67 (B56, BD), BCL6 (IG191E/A8, BioLegend), CD80 (2D10, BioLegend), CD56 (B159, BD), CD45 (D058-1283, BD), CD14 (MoP9, BD), CD16 (3G8, BD), CCR2 (48607, R&D Systems), CD11b (ICRF44, BioLegend), CD11c (3.9, Invitrogen), PD-L1 (29E.2A3, BioLegend), PD-L2 (24F.10C12, BioLegend), CD80 (2D10, BioLegend), CD86 (FUN-1, BD), CD141 (1A4, BD), CD163 (GHI/61, BioLegend), and CD123 (7G3, BD).