Supplementary material
T cell memory to evolutionarily conserved and shared Hemagglutinin epitopes of H1N1
viruses: A pilot scale study.
Venkata R. Duvvuri
1,3 # *
, Bhargavi Duvvuri2 *, Veronica Jamnik2, Jonathan B. Gubbay 4,5,6,7,
Jianhong Wu3, Gillian E. Wu1, 2
* Joint first authorship
1
Biology Department, York University, Toronto, Ontario, Canada
Kinesiology & Health Science, York University, Ontario, Canada
3
Centre for Disease Modeling, York Institute of Health Research, Canada
4
Ontario Agency for Health Protection and Promotion, Toronto, Canada
5
Mount Sinai Hospital, Toronto, Canada
6
University of Toronto, Toronto, Canada
7
The Hospital for Sick Children, Toronto, Canada (Secondary Affiliation)
2
1. Peripheral blood mononuclear cells (PBMC) and plasma isolation:
Blood was diluted with an equal amount of 1X Mg++ and Ca++ free phosphate buffered saline
(PBS) room temperature (RT) and was mixed gently. 15 ml of RT Ficoll was added to a sterile
50 ml conical tube. Ficoll was gently overlaid with 30 ml of diluted blood using a sterile
serological pipette. Mixing of two phases was kept to the minimum followed by centrifugation at
591 relative centrifugal force (RCF) for 30 minutes at RT with brake off to ensure that
deceleration does not disrupt the density gradient. Using a Pasteur pipette PBMC (cloudy layer)
from the diluted plasma/Ficoll interface was collected and cells were placed into a sterile 50 ml
conical tube. Interface from a maximum of two 50 ml tubes were combined into one wash tube.
While collecting cells, care was taken to aspirate as little Ficoll as possible since lower cell
number will pellet if the proportion of Ficoll is too high in the wash tube. 1X PBS was added to
the PBMC layer to bring up the volume to 45 ml followed by mixing gently up and down.
Mixture was centrifuged at 330 RCF for 10 minutes at RT with brake ON.
Following
centrifugation supernatant was removed and discarded without touching the pellet. Pellet was
loosened by tapping the tube with finger and 5ml of sterile 1X PBS+1% fetal bovine serum
(FBS) (Freezing media A) was added and carefully mixed by gently pipetting up and down. All
individual suspensions were pooled into a single sterile 50 ml tube. Cells were mixed and 20 µl
of the cell suspension was taken out for cell counting. Total number of cells was counted. Cell
suspension was centrifuged at 330 RCF for 10 minutes at RT with brake ON. Supernatant was
discarded and to the pellet appropriate amount of Freezing media A was added to adjust the cell
concentration to 20 x 106/ml. Cells were re-suspended by tapping the tube with finger until no
pellet was visible. Slowly, drop by drop an equal volume of Freezing media B (20% Dimethyl
sulfoxide (DMSO) in FBS) was added to the side of tube containing Freezing media A
containing the PBMCs. To mix the cells, cell suspension was gently mixed by pipetting up and
down and avoiding bubbles. The final concentration was maintained to be 10 x 106cells/ml. Once
mixing was complete PBMC suspension was aliquoted into the pre-labled crovials, 0.5-1ml in
1.8 ml vial (~5-10 million cells per vial and no more than 10 million cells per vial). Cells were
gently pipetted to minimize the sheer forces. Cryovials were kept in -70oC freezer for at least 12
hours and followed by final transfer to liquid nitrogen for indefinite storage.
2. IFN-γ ELISPOTPRO assay:
Enzyme-linked immunospot assay kits (human IFN-γ ELISPOTPRO kits, Mabtech, (Nacka
Strand, Sweden)) were used to determine the frequency of epitope-specific IFN-γ secreting T
cells. PBMCs (2 x 105 per well) were pulsed with peptides (10 µM) O/N in polyvinlylidene
difluoride plates pre-coated with anti-human IFN-γ mAb at 370C in 5% CO2. After O/N
incubation, wells were washed with filtered PBS to remove the non- adherent cells. Following
washing, each well was treated with 100 µl of one step-detection reagent (alkaline phosphatase
(ALP)-conjugated detection IFNγ antibody) 1:200 in filtered PBS containing 0.5% FBS, for 2
hrs at 370C. Plates were washed five more times with filtered PBS before chromogenic
development with 100 µl/well “ready to use” 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) /
nitro-blue tetrazolium chloride (NBT). BCIP/NBT-added plates were incubated for 15min at RT
during which time the blue spots developed. Each spot-forming unit (SFU) corresponds to one
IFNγ-secreting T cell. The numbers of antigen specific T cells are presented as SFU per total
number of PBMCs added. The negative control was PBMCs in medium without peptide
stimulation and was used to assess the spontaneous secretion of IFNγ. The positive control was
polyclonal activator anti-CD3 mab (>500 SFU/2 x 105 PBMCs are expected) and was used in
determining T cell numbers, viability and functionality of the immunoassay. Each peptide was
incubated O/N in ELISPOT plates without PBMCs to detect non-specific reactions. A well was
considered positive when it contained
at least 10 SFU and twice as many SFU as the negative control
wells. Each data point is the mean of duplicate wells.
3. Determination of flu exposures with Human IgG Enzyme-linked immunosorbent assay
(IgG ELISA):
Indirect ELISA (from MABTECH) was used to quantify specific IgG antibodies to the seasonal
H1N1 and the 2009 pandemic swine H1N1 in the donor plasma samples. Hemagglutinin antigens
of seasonal H1N1 (A/Brisbane/59/2007, Catalogue Number: 11052-V08H) and 2009 pandemic
H1N1 (A/California/4/2009, Catalogue Number: 11055-V08H) were procured from the Sino
Biological Inc, China. ELISA plates were coated overnight at 40C with 100ul per well of 5μg/ml
of the appropriate influenza virus antigens dissolved in phosphate buffered saline, pH 7.4 (PBS).
After overnight incubation, plates were washed twice with PBS. Plates were blocked by adding
200μl per well of *PBS with 0.1% of fetal bovine serum (FBS) (*incubation buffer) and
incubated for 1 hour at room temperature (RT). The plates were washed and incubated with
100μl per well of plasma (1:1 diluted in incubation buffer) for 2hrs at RT. After washing, plates
were incubated for 1 hr at RT with 100μl per well of IgG isotype-specific mouse anti-human
conjugated to alkaline phosphatase. After five washes, the plates were incubated with 100μl per
well of substrate solution: p-nitrophenyl-phosphate (pNPP) for 30 minutes in the dark.
Absorbance at 405nm was measured immediately with an ELISA microplate reader. Absorbance
values correspond to the binding strength of IgG to influenza HA antigens. This reaction can be
stopped by adding equal volume of 0.75 M NaOH if necessary. The positive control was a
human IgG standard in the well to validate the assay performance. The negative control was PBS
without antigen in the well. The control wells were treated the same as the experimental wells.
IgG content (concentration) of samples was determined from the standard curve based on the
human IgG standard. Absorbance values two times higher than the absorbance of the negative
control were considered to be positive.