Longitudinal analysis of LTR cross-reactivity.

<p>NLV-specific cross-reactivity responses against B27 and A30 were measured against time after lung transplant in LTR5 ([A] top) and LTR8 ([B] bottom), respectively. Following a 6 hour ICS assay of day 13 T cell cultures, B*27:09 cross-reactivity significantly increased over time in LTR5, whereas A30 cross-reactivity remained stable post-transplant in LTR8. Percentages were based on IFN-γ production of NLV-specific +CD8+ T cells. The secondary axis represents the percentage of NLV-specific CD8+ T cells gated on total CD8+ T cells (circles).</p

HLA class I typing of cell lines.

<p>Molecular resolution of HLA class I antigens was available as indicated (4-digit).</p>a<p>C1R Parental cell line has no detectable surface expression of HLA-A, low levels of HLA-B35 and normal levels of HLA-Cw4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056042#pone.0056042-Zemmour1" target="_blank">[51]</a>.</p

Presence of CMV reactivation increases magnitude of cross-reactivity against the allograft.

<p>Comparison of CMV viral load in the BAL (grey circles, left y-axis) and NLV-specific cross-reactivity responses towards B27 and A30 (6 hour ICS assay using day 13 T cells) were measured against time after lung transplant in LTR5 (top) and LTR8 (bottom), respectively. A CMV reactivation episode was classified for viral titres above 10,000. LTR5 experienced CMV reactivation at day 270 post-transplant but had ceased after day 375. A steady increase in B*27:09-cross-reactivity response based on %IFN-γ production of tetramer+CD8+ gated cells (black bars, right y-axis) and %tetramer+CD8+ cells of total CD8+ T cells (white bars, right y-axis) were observed prior to CMV reactivation event at day 193 but had dropped to pre-transplant levels after CMV reactivation had ceased. For LTR8, BAL CMV viral titre was not detected post-transplant. In alignment, A30 cross-reactivity and %tetramer+CD8+ cells of total CD8+ T cells remained consistent post-transplant.</p

Cross-reactive NLV-specific CD8+ T cells via IFN-γ production.

<p>NLV-specific CD8+ T cells from HC5 were expanded for 13 days before performing a 6 hour ICS assay against a panel of transfected cell lines and EBV-LCLs encompassing 6 HLA-A and 13 HLA-B antigens. Cross-reactivity was measured by the production of IFN-γ in response to HLA antigenic stimulation after gating on tetramer+CD8+ T cells. Both the positive controls (C1R.A*02:01/NLV, NLV peptide) and negative controls (C1R Parental, C1R.A*02:01, T cells alone) responded as expected. No cross-reactivity was observed with the test panel, albeit C1R.B*27:05 which had a positive IFN-γ response well above background levels. IFN-γ responses towards 9009, 9026, T102, C1R.B*18:01, C1R.B*35:01, C1R.B*35:02, C1R.B*35:03, C1R.B*44:03, C1R.B*57:01, 721.221 Parental, 721.221.A*29:02, 721.221.A68 and 721.221.B53 were also negative (data not shown).</p

Functional analyses of new cross-reactivity towards B*27:05.

<p>Flow cytometric analyses of IFN-γ and CD107a expression were performed after 13 days of NLV-specific T cell expansion from HC5. T cell cultures were stimulated with C1R.A*02:01 (negative control), NLV-pulsed C1R.A*02:01 (positive control) or C1R.B*27:05 (cross-reactive target) for 6 hours in a combined CD107a staining and ICS assay revealing that cross-reactivity was mainly via cytokine production (IFN-γ+) and to a lesser extent dual cytokine/cytotoxic ability (IFN-γ+/CD107a+) (A). Both proliferation and cytokine production were measured in a parallel experiment after CFSE-labelled PBMCs from HC5 were cultured with autologous irradiated NLV-pulsed PBMCs for 13 days before performing a 6 hour ICS assay (B). The lymphocyte gate was based on side scatter versus forward scatter. CD8+ T cells were then gated from lymphocytes using side scatter versus anti-CD8 PE-Cy5.</p

Influence of cross-reactivity by B27 allelic subtypes.

<p>Site-directed mutagenesis of the pMIG.B*27:05 vector was performed to generate B*27:03- and B*27:09-specific retroviruses for transducing C1R Parental cells. Comparison of cross-reactive NLV-specific IFN-γ responses between B*27:05, B*27:03 and B*27:09 was then carried out following a 6 hour stimulation and ICS assay of day 13 NLV-specific CD8+ T cells (HC5) with the B27-specific cell lines as well as positive (C1R.A*02:01/NLV) and negative controls (C1R.A*02:01, Auto-T cells).</p

Healthy controls (HC) demographics and NLV expansion profiles.

<p>HLA class I typing, CMV serology status and NLV-specific T cell expansion profiles of healthy individuals. Molecular resolution of HLA class I antigens was available as indicated (4-digit). <i>In vitro</i> T cell cultures were derived by autologous stimulation of PBMC with NLV peptide (1 µM) for 13 days in the presence of IL-2 (20 U/ml). Percentages of A2/NLV-tetramer+ T cells were based on the total CD8+ T cell population. HC1 and 2 were excluded from the average, SD and range calculations.</p><p>Abbreviation: not tested (NT).</p

Expansion profiles of NLV-specific CD8+ T cells in LTR.

<p>NLV-specific CD8+ T cell frequencies were measured on day 0 (A) and after 13 days of NLV peptide stimulation (B) based on the total CD8+ T cell population, where available. CMV serostatus of the recipients and donors are indicated in the graphs.</p