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

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

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

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

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

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

Proliferation of NK cells in the presence of immunosuppressive drugs.

<p>MACS enriched NK cells from three healthy controls were labelled with CFSE and stimulated in culture for three days with a combination of IL-2, IL-12 and 721.221 cell lines in the presence or absence of immunosuppressants Cyclosporine A, MPA and Prednisolone. An example of the change in CFSE intensity as the cells proliferate is shown (A). NK cell proliferation is displayed in response to treatment with varying concentrations of the immunosuppressive drugs (B, <i>p</i>&lt;0.05 for all). Graphed data are presented as the mean ± SEM from three independent experiments. Symbols represent immunosuppressive drugs: •, Cyclosporine A; □, MPA; ▴, Prednisolone. Shaded area signifies therapeutic range.</p

NK cell and T cell IFN-γ production in the presence of immunosuppressive drugs.

<p>PBMC from 20 healthy controls were stimulated in culture with the cell line K562 or PMA-I in the presence of varying concentrations of immunosuppressive drugs. NK cell IFN-γ production measured in response to stimulation with K562 cell line (A) and PMA-I (B). T cell IFN-γ production measured in response to PMA-I stimulation (C). Statistical significance was defined as <i>p</i>&lt;0.001. Graphed data are presented as mean ± SEM. Symbols represent immunosuppressive drugs: •, Cyclosporine A; □, MPA; ▴, Prednisolone. Shaded areas signify therapeutic range.</p