The NK Receptor NKp30 Mediates Direct Fungal Recognition and Killing and Is Diminished

Transcription

1 S1 Cell Host & Microbe, Volume 14 Supplemental Information The NK Receptor NKp30 Mediates Direct Fungal Recognition and Killing and Is Diminished in NK Cells from HIV-Infected Patients Shu Shun Li, Stephen K. Kyei, Martina Timm-McCann, Henry Ogbomo, Gareth J. Jones, Meiqing Shi, Richard F. Xiang, Paul Oykhman, Shaunna M. Huston, Anowara Islam, M. John Gill, Stephen M. Robbins, and Christopher H. Mody

2 S2 Figure S1, related to Figure 1 Identification of the molecule recognized by the monoclonal antibody 1C01. A, Selection criteria of hybridoma supernatants. Supernatants containing mab from the hybridomas (2680 clones) were used to label YT cells and the fluorescent intensity was determined by flow cytometry. Anti-CD18: positive control; clones A, B and C are representative clones showing weakly positive, positive and strongly positive immune fluorescence. B, Screening of hybridoma supernatants for the ability to block anticryptococcal activity. YT cells were co-cultured overnight with C. neoformans in the presence of mab (selected from strong positive binding in supplementary figure

3 S3 1), or control IgG. YT cells were lysed and serial dilutions were placed onto Sabouraud dextrose agar and, incubated at 32 C for hours. 1C01: 50 μl in hybridoma supernatant (25 μg/ml); Control: mouse IgG in 50 μl conditioned medium. C&D, mab 1C01 recognizes a protein of kda in YT cells. C, Immunoblot analysis of equal amounts of YT whole cell lysate. YT lysate was evenly loaded and the membrane was cut into strips between each lane. Each strip of the membrane was blotted with different amounts of mab 1C01 as indicated. D, Immunoblot analysis of different amounts of YT whole cell lysate. Increasing amounts of YT lysate as indicated was loaded, and the whole membrane was blotted with mab 1C01. β-actin was used as a loading control.

4 S4 Figure S2, related to Figure 4 Perforin in culture media of YT cells after NKp30 knock down. Perforin in the conditioned culture media from YT cells that had NKp30 knocked down cultured in the presence or absence of C. neoformans (strain B3501) was determined by ELISA. Experiments were repeated twice with similar results. sirnac: control sirna;, sirna4: target specific sequences of sirna for human NKp30. *, p<0.05.

5 S5 Figure S3, related to Figure 5 CFU and MTS determination of YT cell killing. The anti-candida activity (CFU) was confirmed using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2h-tetrazolium] measurement. Different effector to target ratios were used in the killing assay including Candida alone (0:1) and YT alone (200:0). CFU (left vertical axis), and MTS determination (right vertical axis) were performed on the same wells.

6 S6 Figure S4, related to Figure 5 mab 1C01 blocked NK cell killing of C. neoformans (strain B3501) that had been grown in capsule-inducing conditions. Strain B3501 was cultured in the presence or absence of desferoxamine (final concentration 80 μm) at 32 C for 24h before being visualized by India ink (A, arrowhead indicates capsule). The anti-cryptococcal activity was determined by assessing CFU from desferoxamine-treated and untreated organisms in the presence of mab 1C01 or isotypematched control antibody (B). * P < 0.05.

7 S7 Supplemental Experimental Procedures Antibody generation from mice The mouse immunization protocol was adapted from Phillips and Babcock. Briefly, BALB/c mice were injected via the intraperitoneal route on day 1 with YT cells and boosted two weeks later with YT cells. After another two weeks, mouse serum was checked for the presence of anti-yt cell antibodies. Spleens in mice with high titer of anti-yt cell antibodies were isolated and fusion of splenocytes with the SP2/MIL6 myeloma cell line was performed to make hybridoma cells. Selected hybridomas were sub-cloned and expanded to make monoclonal antibodies (mab). Flow cytometry for hybridoma screening, competitive binding and conjugate formation Prior to use, hybridoma supernatants were centrifuged at 1,500 rpm at 4 C for 5 min to remove contaminating hybridoma cells or debris. YT cells were mixed with supernatants followed by incubation at 4 C for 1 hour. Labeling was revealed with a PE-conjugated goat anti-mouse IgG (Invitrogen, Cat No. P852). To assess the competitive binding of the ligand from a preparation of cryptococcal cell wall/membrane (CCW/M) with mab 1C01 to YT cells, YT cells were pre-treated with various amounts of CCW/M (stock suspension containing protein of μg/ml) at 37 C overnight. The cells were washed with cold PBS and labeled with mab 1C01. Conjugate formation of NK cells with Cryptococcus was adapted from the protocol of Barber et al (2003). Briefly, YT cells were labeled with 1.5 μm TRITC (tetramethylrhodamine-5-(and 6)-isothiocyanate) (Invitrogen, T-490) and C. neoformans with 0.1 μm of FITC (Sigma, ). The TRITC-labeled YT cells ( ) or FITC-labeled C. neoformans ( ) were co-cultured in the presence of 50 μl of

8 S8 conditioned medium containing mab 1C01 (25 μg/ml) or mouse IgG (Life Technologies, ) in 200 μl cold complete RPMI 1640 medium, and centrifuged at 300 rpm for 5min to facilitate contact followed by incubation at 37 C for 0, 30, 60 and 120 minutes. Cells were fixed by resuspending the pellet in 1 ml of ice-cold PBS containing 0.5% formalin. Unconjugated YT would therefore be red and unconjugated Cryptococcus would be green, whilst conjugates would coexpress both green and red. To assess NKp30 expression on NK subpopulations, primary human NK cells from healthy subjects were labeled with CD56 (BD, X) and anti-nkp30 (Abnova, PAB17794). Mouse IgG (BD, ) was used as control for CD56. The YT cells were washed, fixed and the fluorescent intensity was assessed using a Guava EasyCyte flow cytometer and CytoSoft v5.3 software (Guava Technologies). Data analyses were performed using FlowJo software (Tree Star, Inc.) and compared to the fluorescent intensity of YT cells labeled with anti-cd18 (ID Labs, IDAC1084). Visualization of capsules with India ink Cryptococcus neoformans were grown in YT media (RPMI 1640 medium supplemented with 10% FCS, 1% pen-strep, 1% sodium pyruvate) in the presence or absence of desferoxamine (80 μm final, Ciba-Geigy, Switzerland, kind gift from Dr. May Ho, University of Calgary) overnight in shaker at 32 C. Cells were harvested 24 hours later, and fixed with 1% formaldehyde, and concentrated and stained with Higgins Black India Waterproof Ink (Chartpak, Inc.), and imaged using an Olympus IX70 wide field microscope with 60X PlanApo NA 1.40 objective. Antifungal activity YT cells ( ) were co-cultured with C. neoformans or C. albicans at a starting effector to target ratio of 100:1 unless otherwise specified in 100 μl complete RPMI 1640 medium per well

9 S9 in 96-well plate (Costar) at 37 C for 24 hours. The mixture was resuspended, and an aliquot of 10 μl was added to 90 μl of distilled H 2 O to destroy YT cells, and from the latter, 10 μl was plated onto Sabouraud dextrose agar plate followed by incubation at 32 C. To test the ability of 1C01 to inhibit YT cell killing of C. neoformans or C. albicans, YT cells were pre-treated with mab 1C01 at 37 C for 30 min before Cryptococcus was added. The control Ab consisted of conditioned medium plus appropriate isotype IgG, i.e., mouse IgG (Life Technologies, ) or mouse IgG2a, κ (BD, ) for mab01 and rabbit polyclonal IgG (ab27472) for rabbit polyclonal anti-nkp30. Colony forming units (CFU) were counted after 24 hours of incubation. The anti-candida assay was validated using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2h-tetrazolium] measurement. MTS is used to measure cell metabolism by determining the bioreduced formazan in culture medium with the absorbance at 490nm. Briefly, part of the killing assay reaction was used for CFU, and the remaining part for MTS determination. YT cells were lysed with 0.03% SDS solution for 30 min at room temperature, and the Candida was washed before applying MTS reagent (CellTiter 96 AQ ueous One Solution Cell Proliferation Assay, Promega, Cat No G3580) after incubation with MTS at 37 C for 4 hrs. SDS solution (0.03%) had no effect on Candida growth (data not shown). NKp30 Knockdown in YT cells by sirna technology Three different sirnas for human NKp30 (Thermo Scientific, Cat , 4 and 5) were used to knock down NKp30 in YT cells. Non-targeting sirna (Thermo Scientific, Cat.D ) was used as control. Transfection was performed by suspending YT cells ( ) in Nucleofector solution V in an Amaxa cuvette (Lonza, Cat. VCA-1003). YT cells in solution V were mixed with the sirna and transfected with Program O-017. The cells were immediately transferred to a 6-well plate with warm complete RPMI 1640 medium followed by incubation at

10 S10 37 C for 24 to 48 hours before assessing the antifungal activity. The efficacy of knockdown was confirmed using immunoblot analysis. Perforin content in YT cells and in culture media YT cells were pre-treated with mab 1C01 or Mouse IgG2a, κ (BD, ) at 37 C for 30 min and cultured with or without C. neoformans (B3501) for various times. Cells and culture media were collected at the same time. Perforin in YT cells were analyzed by using Western blot and flow cytometry. Perforin released in the culture media was measured using an ELISA Kit (Abcam, ab46115) according to the manufacturer s instructions. Optic density readings were performed using SpectraMax M2 & M2e Multi-Mode Microplate Reader (Molecular Devices, LLC). The intensity of this colored product is directly proportional to the concentration of perforin present in the samples. Statistics analyses Statistical studies were performed using GraphPad Prism v5.0. Unless otherwise specified one-way ANOVA followed by Bonferroni comparison tests or unpaired T-test (two-tailed) with Welch correction was used to evaluate differences among conditions. In all cases p<0.05 was considered significant.