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.

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

α1(V) peptide binding to HLA-DR1 and HLA-DR15.

<p>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.</p

Candidate α1(V) peptide screen in col(V)-responsive patients.

<p>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-A^b binding peptides (white bars) are shown. (Data represents the mean of 2-4 separate assays).</p

Candidate α1(V) peptide screen in col(V) immunized HLA-transgenic mice.

<p>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.</p

Unusual pattern of µ1(V) peptide response in a DR1⁺ lung recipient with a DR15⁺ transplant donor.

<p>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-A^b binding peptides (white bars) are shown. Data represents the mean of 2-5 independent experiments. </p

Th17 Responses to Collagen Type V, kα1-Tubulin, and Vimentin Are Present Early in Human Development and Persist Throughout Life

T helper 17 (Th17)-dependent autoimmune responses can develop after heart or lung transplantation and are associated with fibro-obliterative forms of chronic rejection; however, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we investigated whether removal of regulatory T cells or blockade of function reveals a similar autoantigen bias. We found that Th17 cells specific for collagen type V (Col V), kα1-tubulin, and vimentin were present in healthy adult peripheral blood mononuclear cells, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1[V]), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. Although the latter responded well to α1(V) fragments and peptides in an HLA-DR-restricted fashion, Th17 cells from healthy persons responded in an HLA-DR-restricted fashion to fragments but not to peptides. Col V, kα1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, preexisting Th17 response that is MHC class II restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis