PHA-L

PBMCs from freshly isolated blood samples or cryopreserved samples (denoted with †) were twice washed with 1× PBS and stained using CellTrace® Violet (CTV, Life Technologies) at a final concentration of 2.5 μM for 10 min at room temperature. The reaction was quenched by adding cold FBS. CTV-labelled PBMC in RPMI containing 10% human AB serum (Sigma), 1 mM Pen/Strep and 2mM l-Glut were plated in 48 or 96-well round-bottom plates at 500,000 and 250,000 cells, respectively, and stimulated with peptide pools from SARS-CoV-2, FEC-T, HCV NS3 or HCV core protein (1 μg/ml per peptide). Media containing 0.1% DMSO (Sigma) representing DMSO content in peptide pools were used as negative control and 2 μg/ml phytohemagglutinin L (PHA-L, Sigma) as used as a positive control. Cells were subsequently incubated at 37 °C, 5% CO₂, 95% humidity for 5 days without media change or 7 days with media change on day 4 if cultures were kept beyond 5 days. At the end of incubation, cells were subjected to flow cytometry staining as described below. Responses above 1% were considered true positive. To determine the breadth of antigenic response targeted by T cells, the number of peptide pools that each volunteer responded to was counted. To determine the magnitude of the total response to structural and accessory proteins, the average number of cells proliferating in response to any of the peptides M, N, ORF3, 6, 7, 8 was obtained as a function of their respective CD4⁺ or CD8⁺ T cell population and then expressed as a percentage. The background was then subtracted from the total response for each volunteer.

The day before culling, Merk Multiscreen 96 well Filter Plates (Merck) were incubated with primary antibody (INFγ mAb clone AN18, Mabtech, 3321-3-1000) diluted 1:200 in sterile PBS (Gibco), at 4 °C. The next day, the antibody was removed, plates were washed four times with PBS at 250 µl/well, then blocked with 200 µl/well R10 (RPMI [Gibco] supplemented with 10% heat-inactivated FCS, Non-essential amino acids, L-Glutamine and penicillin/streptomycin (all from Sigma) for 2 h at 37 °C). Mice were culled, their spleens removed, and passed through a 40 µm cell strainer (Falcon) and the single cell suspension pelleted by centrifugation. The splenocytes were resuspended in 3 ml ACK lysis buffer (Lonza) for 3–5 min to lyse the red blood cells, then stopped with 20 ml PBS, followed by centrifugation at 400 g, 5 min at room temperature. The splenocyte pellet was resuspended in 5 ml R10, counted and the cell concentration adjusted to 4 × 10⁵/ml. Blocking buffer was removed and replaced with 50 µl of cells which were stimulated with the respective individual peptides (50 µl of peptide at 15 µg/ml) that the group had been vaccinated with. Each peptide was tested in duplicate. Negative control wells contained DMSO only while cells were stimulated with PHA-L (11249738001, Roche, dilution 1:200) as a technical control. The plates were incubated overnight (15–20 h) in a 37 °C (5% CO₂) incubator. The cells and peptides were removed and the wells washed 7 times with sterile PBS. Secondary antibody (biotin conjugated anti-INFγ, MabTech, 3321-6-100, clone R4-6A2-Biotin) diluted 1:2000 in assay diluent (AD) (25 mg/ml BSA in PBS), was added (50 µl/well) and incubated for 2 h at room temperature. The plates were then washed four times with PBS then 50 µl of streptavidin-alkaline phosphatase (Mabtech, 3310-10-1000) diluted 1:750 in AD was added and incubated for 2 h at room temperature. Plates were washed four times with PBS then 50 µl BCP/NBT substrate was added to each well and allowed to develop for 5–10 min until spots were visible in the positive control wells. Reaction was stopped by rinsing the plates in DI water three times. The rubber bottom was removed and the membrane was rinsed on both sides with DI water then allowed to dry. The spots were quantitated on an ELISpot counter (AID ELISpot software v7, Autoimmun Diagnostika).

1 × 10⁶ cells from each cell line were harvested, washed twice with PBS, re-suspended in staining buffer (PBS + 0.2% FBS) and blocked (FcR Blocking Reagent, Miltenyi Biotec). Cells were then incubated with PHA-L lectin (GlycoMatrix) at a concentration of 10 µg/mL for 30 min at 4 °C. Following incubation, cells were washed twice with PBS, and re-suspended in staining buffer. 30,000 events were recorded for each tube using a BD LSRII Cell Analyzer. All data was further processed using FlowJo software.

To validate the results of the lectin microarray, serum samples from different groups were randomly chosen from the lectin microarray analysis cohort and a new cohort of 50 RA-ILD patients was included. Briefly, to determine the location of IgG in immunoblotting, 1:100 diluted serum proteins mixed with loading buffer (CW biotech, Beijing, China) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electro-transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking non-specific binding sites with 10 × Carbo-Free Blocking Solution (1:10; Vector Laboratories Inc., US) at room temperature for 2 h, the membranes were incubated with 20 μg/mL Cy3 (1:1,000; GE Healthcare, Chicago, IL, USA)-labeled lectins including SBA, STL, PHA-E, SNA, Jacalin, SNA-I, MNA-M, AAL, ConA, PHA-L, DBA (EY Laboratories, Inc., US and Vector Laboratories Inc. US) at 4 °C overnight in the dark. Excess lectins were removed by washing three times with PBST. The washed and dried membranes were detected by a fluorescence signal system of Typhoon FLA 9500 (GE Healthcare, Chicago, IL, USA). Finally, ImageJ software was used for signal intensity analysis.