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. 2013 Nov 12;8(11):e79601.
doi: 10.1371/journal.pone.0079601. eCollection 2013.

Epitope analysis of the collagen type V-specific T cell response in lung transplantation reveals an HLA-DRB1*15 bias in both recipient and donor

Melissa R Keller  1 , Lynn D Haynes, Ewa Jankowska-Gan, Jeremy A Sullivan, Vrushali V Agashe, Scott R Burlingham, William J Burlingham
Affiliations

Epitope analysis of the collagen type V-specific T cell response in lung transplantation reveals an HLA-DRB1*15 bias in both recipient and donor

Melissa R Keller et al. PLoS One. .

Abstract

Background: IL-17-dependent cellular immune responses to the α1 chain of collagen type V are associated with development of bronchiolitis obliterans syndrome after lung transplantation, and with idiopathic pulmonary fibrosis and coronary artery disease, primary indications for lung or heart transplantation, respectively.

Methodology/principal findings: We found that 30% of the patients awaiting lung transplantation exhibited a strong cell-mediated immune response to col(V). Of these, 53% expressed HLA-DR15, compared to a 28% HLA-DR15 frequency in col(V) low-responders (p=0.02). After transplantation, patients with HLA-DR1 and -DR17, not -DR15, developed anti-col(V) responses most frequently (p=0.04 and 0.01 vs. controls, respectively). However, recipients of a lung from an HLA-DR15(+)donor were at significantly elevated risk of developing anti-col(V) responses (p=0.02) and BOS (p=0.03). To determine the molecular basis of this unusual pattern of DR allele bias, a peptide library comprising the collagenous region of the α1(V) protein was screened for binding to HLA-DR0101, -DR1501, -DR0301 (DR17) or to HLA-DQ2 (DQA1*0501: DQB1*0201; in linkage disequilibrium with -DR17) and -DQ6 (DQA1*0102: DQB1*0602; linked to -DR15). Eight 15-mer peptides, six DR-binding and two DQ-binding, were identified. HLA-DR15 binding to two peptides yielded the highest binding scores: 650 (where 100 = positive control) for p799 (GIRGLKGTKGEKGED), and 193 for p1439 (LRGIPGPVGEQGLPG). These peptides, which also bound weakly to HLA-DR1, elicited responses in both HLA-DR1(+) and -DR15(+) col(V) reactive hosts, whereas binding and immunoreactivity of p1049 (KDGPPGLRGFPGDRG) was DR15-specific. Remarkably, a col(V)-reactive HLA-DR1(+)DR15(neg) lung transplant patient, whose donor was HLA-DR15(+), responded not only to p799 and p1439, but also to p1049.

Conclusions/significance: HLA-DR15 and IPF disease were independently associated with pre-transplant col(V) autoimmunity. The increased risk of de novo immunity to col(V) and BOS, associated with receiving a lung transplant from an HLA-DR15(+) donor, may result from presentation by donor-derived HLA- DR15, of novel self-peptides to recipient T cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Differences in TV-DTH reactivity pre-transplant to col(V) in patients with end-stage lung disease by disease type and by prevalence of DRB1*1501.
A significant number of patients with IPF had col(V) reactivity (net swelling ≥ 25 x 10-4 inches, dotted line; *ANOVA p<0.0007). Independently, patients with DRB1*1501 (red symbols) were enriched in col(V) responders (net swelling ≥ 25 x10-4 inches, Chi Square p= 0.01). AAD - α1 -antitrypsin disease; CF - cystic fibrosis; COPD - chronic obstructive pulmonary disease; IPF - idiopathic pulmonary fibrosis. Patients with lung diseases: n=53 (previous publication, [14]), n=45 new subjects, and n=1 subject who donated samples to both studies (average values of 2 time-points). Healthy subjects (n=14) with known HLA-DR types were tested and used as controls.
Figure 2
Figure 2. HLA-DR frequencies in col(V) reactive lung transplant recipients after transplantation.
HLA-DR frequencies in very high col(V) reactive (TV-DTH net foot pad swelling > 25 x 10-4 inches) lung transplant recipients (A, black bars) or their donors (B, black bars) were compared to the HLA-DR frequency of controls (1200 subjects, open bars) and non-col(V) reactive or col(V)low (≤ 25 x 10-4 inches) lung transplant recipients (A, grey bars) or their donors (B, grey bars). HLA-DR frequencies of very high col(V) reactive recipients (and their donors) who later developed BOS 2 or 3 (hatched bars) were also compared to HLA-DR frequency of controls. There were no significant differences in the frequencies of any HLA-DR alleles between normal control subjects from the University of Wisconsin (n=1200) and lung transplant recipients/donors (n= 281 recipients) or patients screened using the TV-DTH assay (n=54), data not shown. The frequency of HLA-DR1 (33%) and –DR17 (46%) was significantly higher in transplant recipients with very high col(V) responses (>25 x10-4 inches, A) than in normal controls (16% and 22%, respectively). HLA-DR13 was significantly less represented in col(V) responsive recipients compared to normal controls (4% vs 23% in controls). The frequency of HLA-DR15 was significantly higher in the donors of subjects who developed a high response to col(V) (50%) as well as in the donors of subjects with BOS 2/3 incidence (63%) compared to the frequency in the normal controls (26%). The frequency of HLA-DR17 was significantly higher in the donors of patients that did not have very high col(V) responses. *=p< 0.05, **= p<0.01.
Figure 3
Figure 3. α1(V) peptide binding to HLA-DR1 and HLA-DR15.
ProImmune REVEAL MHC-peptide binding assay screen of 5-amino acid overlapping peptide library (101 total peptides) of the α1(V) helical domain to HLA-DR15 (A), and HLA-DR1 (B). Each MHC:peptide binding score is based on known binding of a positive control peptide and a weaker binding positive control peptide. MHC:peptide binding scores above 12% are considered positive and have potential for immunological activity. Lack of bar for a peptide represents a score of 0%. Peptide number corresponds to the location in the α1(V) AA sequence. Blue shaded areas indicate epitope regions where only HLA-DR15 bound, red shaded areas where only HLA-DR1 bound, and yellow shaded areas indicate both HLA-DR1 and –DR15 bound within that helical domain.
Figure 4
Figure 4. α1(V) peptide binding to HLA-DQ2, HLA-DQ6 and mouse H-2 I-Ab.
ProImmune REVEAL MHC-peptide binding assay screen of 5-amino acid overlapping peptide library (101 total peptides) of the α1(V) helical domain to HLA-DQ2 (A), HLA-DQ6 (B) and H-2 I-Ab (C). Each MHC:peptide binding score is based on known binding of a positive control peptide and a weaker binding positive control peptide. MHC:peptide binding scores above 12% are considered positive and have potential for immunological activity. Lack of bar for a peptide represents a score of 0%. Peptide number corresponds to the location in the α1(V) AA sequence. Green shaded areas indicate where HLA-DQ2, -DQ6 and H-2 I-Ab similarly bound within the helical domain.
Figure 5
Figure 5. Candidate α1(V) peptide screen in col(V)-responsive patients.
PBMC from (A) CAD patient 20 (HLA-DR15, 17; -DQ2,6), (B) End-stage lung disease patient L138 (HLA-DR13,15; -DQ5,6) (C) Heart transplant recipient H76 (HLA-DR1,4; -DQ3,5, with donor HLA-DR9,11; -DQ3,-), (D) CAD patient 23 (HLA-DR11,17; -DQ2,7) and (E) End-stage lung disease patient PPG25 (HLA-DR7,11; -DQ2,7) were screened for reactivity to ProImmune binding peptides by TV-DTH. Results from HYP and non-HYP p629, p599 were combined. Patient response to intact col(V) (black bar), HLA-DR1-specific peptide (light gray bar), HLA-DR1/15-specific peptides (hatched bars), HLA-DR15-specific peptide (dark gray bar), and HLA-DQ/H-2 I-Ab binding peptides (white bars) are shown. (Data represents the mean of 2-4 separate assays).
Figure 6
Figure 6. Candidate α1(V) peptide screen in col(V) immunized HLA-transgenic mice.
Splenocytes and ILN cells from col(V) immunized HLA-A*0201/DRB1*0101 Tg (open bars) and HLA-DRB1*1501 Tg (hatched bars) mice were pooled and co-injected with col(V), col(I), or candidate α1(V) peptides into footpads of CB-17 SCID mice to determine peptide-specific swelling responses ; n=3-6 mice were used for each test. * p=.026; # p=.057 comparing peptide responses in DRB1*0101 vs. DRB1*1501 Tg mice using an unpaired Mann-Whitney U test. Responses to col(I) negative control and col(V) positive control were not significantly different between the two Tg mouse strains.
Figure 7
Figure 7. Unusual pattern of µ1(V) peptide response in a DR1+ lung recipient with a DR15+ transplant donor.
PBMC from lung transplant recipient (L86, HLA-DR1,11; -DQ3,5, with donor: HLA-DR1,15; -DQ5,-) was assayed by TV-DTH for reactivity to α1(V) peptides or intact col(V). Results from HYP and prolinated p629, p599 were combined. Patient response to intact col(V) (black bar), HLA-DR1-specific peptide (light gray bar), HLA-DR1/15-specific peptides (hatched bars), HLA-DR15-specific peptide (dark gray bar), and HLA-DQ/H-2 I-Ab binding peptides (white bars) are shown. Data represents the mean of 2-5 independent experiments.
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