Supplemental Digital Content 3
Supplementary Materials & Methods
Study Population
Patients were recruited from the BMT units at Queen Elizabeth Hospital Birmingham
and Birmingham Heartlands Hospital and had undergone transplantation between
March 1999 and July 2007. HLA alleles of donor and recipient were determined using
a standard PCR-based DNA typing method at the National Blood Service Laboratory,
Birmingham, UK. Patients were recruited at least 6 months after transplantation and
the diagnosis of cGVHD was made on the basis of standard clinical criteria (29). The
patients’ clinical characteristics are summarised in Table 1. 30 healthy volunteer
subjects with no past medical history of auto-immune disease acted as controls to
define the ‘normal range’ of ELISPOT response in the assays described below. In all
cases 20ml of blood was taken and the peripheral blood mononuclear cells were
purified by density centrifugation.
Informed consent was obtained for collection of samples and usage of data was
obtained in accordance with the Declaration of Helsinki. The study was part of a
research protocol approved by South Birmingham Research Ethics Committee.
Peptide synthesis
15mer peptides were synthesized (NeoMPS, Strasbourg, France) using Fmoc
chemistry. The selection of peptides for study was based on class 1 HLA derived
sequences, which had been screened for the binding to common HLA class 2 alleles
and previously found to induce immune responses in sensitised patients awaiting
renal transplantation (8). The sequences chosen in this study were those
demonstrating little or no polymorphism, (except for three peptides p19 to p21, which
encompass a well-described allogeneic epitope from HLA-A2). In the case of each
peptide derived from the 3 domain of class 1 HLA (p39 to p53) a naturally occurring
analogue peptides was also synthesised. One or other of these analogues is
represented in the vast majority of HLA-A and B molecules (see Table 2). These
sequences were invariably identical in donor and recipient because they were HLA
matched. If the peptide sequence to which an immune response was observed was
identical to donor-recipient HLA then this response was defined as autologous.
Enzyme-Linked Immunosorbent Spot Assay
Peripheral blood mononuclear cells (PBMC’s) were isolated from peripheral blood of
patients and normal controls by Ficoll density-gradient centrifugation prior to use.
Viable cells were enumerated by trypan blue exclusion. A -interferon ELISPOT assay
was used according to the manufacturer’s instructions (Mabtech, Nacka Strand,
Sweden). A total of 5x105 PBMCs were added to each well in a final volume of 100l
of ‘complete medium’: 95% RPMI 1640 medium (Sigma, Poole, UK)/5% human AB
serum
(PAA
laboratories,
Somerset,
UK),
with
L-glutamine
and
penicillin/streptomycin (Sigma) along with peptide at a final concentration of 20gml. Peptides were used either singly or for some as pairs if the sequences were offset by
1
2 amino acids. This concentration of peptide had been established as optimal in a
previous study (8) and is consistent with that used in other studies using similar
experimental designs (30, 31)). This includes the use of T cell clones to identify
optimal peptide concentrations using peptides as antigen (32).
In addition to peptide, wells containing PPD (SSI, Copenhagen, Denmark) at a final
concentration of 10μgml-1 and anti CD3 (CD3-2) supplied by the manufacturer of the
ELISPOT assay used at 100ng/ml were used as ‘positive’ controls. Peptide containing
wells were analysed in replicates of 4. Negative control wells contained responder
PBMC’s plus medium alone in replicates of 6.
PBMC were cultured at 370C, 5% CO2 for 48 hours, then discarded and the plate
washed 6 times with PBS. Plates were then incubated at room temperature for 2 hours
with the one-step detection reagent (alkaline phosphatase-conjugated detection
monoclonal antibody 7-B6-1, prepared by diluting to 1:200 in filtered PBS containing
0.5% fetal calf serum (Sigma)). This was then discarded and plates washed 5 times
with PBS. Filtered ready-to-use chromogenic alkaline phosphatase substrate
((nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate
(BCIP/NBT-plus))
provided by manufacturer was added at a volume of 100μl/well. The plates were
allowed to develop and reaction terminated once spots emerged. Plates were washed
extensively under tap water and then air-dried in darkness for at least 12 hours before
analysis. The number of spots in each well was then counted using an AID ELISPOT
plate reader (Strassberg, Germany). The mean number of γ-interferon producing cells
per well were calculated as the mean number of spots from quadruplicates (for each
peptide combination) and the corrected mean was calculated by subtracting the mean
of the medium control wells. ELISPOT numbers were compared between different
populations by Mann Whitney U statistic. The number of responders in each group
were compared by Fisher’s exact test.
Intracellular -interferon staining
The flow-cytometric identification of peptide-specific T cells on the basis of interferon production was used according to the method of Litjens and colleagues (33).
2.5×106 freshly isolated PBMC’s were cultured for 6 hours in RMPI 5% human AB
serum at 37°C with 5% CO2 in 12×75-mm polystyrene tubes (BD Falcon™) containing
peptide at a final concentration of 20gml-1 with 1µg/106 PBMC’s of monoclonal
antibodies αCD28 (L293) and αCD49d (L25) (BD-Pharmingen,). Over the final five
hours of culture Golgiplug (BD Pharmingen), 1 μl/106 PBMC’s was added.
Subsequently, cells were treated with 2 mM EDTA for 15 min at room temperature
followed by a wash with PBS 0.5% w/v FCS. The following monoclonal antibodies
were used to stain the cell surface by co-incubation for 30 min at room temperature:
peridinin chlorophyll protein (PerCP)-labelled CD8 (SK1, BD), allophycocyanin
(APC)-labelled CD3 (SK7, BD), fluorescein isothiocyanate (FITC)-labelled anti-CD4
(RPA-T4, BD). Cells were then fixed by adding 1 ml of Fixation and Permeabilization
solution™ (BD) and incubated for 10 min at room temperature. Cells were
subsequently washed with Permeabilization/Wash Buffer (BD) and incubated for 30
min at room temperature with phycoerythrin (PE)-labelled anti IFN-γ antibody (BD).
After washing, the cells were re-suspended in PBS 0.5% w/v FCS. Cells were then
analyzed on a FACS-Calibur flow cytometer using CELLQuest software (Becton
Dickinson).
MHC class 2 restriction of ELISPOT responses
The MHC restriction of responses in ELISPOT was investigated by undertaking interferon ELISPOT assays in the presence of antibody against HLA-DR (0.5 - 5
µgml−1) (L243, Becton Dickinson) in the absence or presence of additional antibody to
MHC class 2 (5 µgml−1) (Tu39 Becton Dickinson, Oxford, UK).