Combined Blockade of costimulation and adhesion pathways overcomes heterologous alloimmunity.

<p>A) Kaplan-Meier survival curves comparing skin graft survival between non-infected, CoB treated mice (solid black line, MST 22 d, n = 48, 8 independent experiments), non-infected mice treated with anti-LFA-1 and anti-VLA-4 antibodies (dotted black line, MST 24 d, n = 12, 2 independent experiments), MHV68-infected, CoB treated mice (solid red line, MST 13.5 d, n = 40, 7 independent experiments), and MHV68-infected mice treated with anti-LFA-1 and anti-VLA-4 antibodies (solid orange line, MST 27 d, n = 14, 2 experiments). When non-infected, CoB treated mice were compared to non-infected, anti-LFA-1+ anti-VLA-4 treated mice with the log-rank test, no significant difference was observed (p = n.s.). Comparison of MHV68-infected, anti-LFA-1+ anti-VLA-4 treated mice to non-infected mice treated with either regimen also yielded a non-significant p-value. MHV68-infected, anti-LFA-1+ anti-VLA-4 treated mice demonstrated significantly different skin graft survival compared to MHV68-infected, CoB-treated animals (p = .002). B) Kaplan-Meier survival curves comparing skin graft survival between the following groups: MHV68-infected mice treated with CoB (solid red line, MST 13.5, n = 40, 7 independent experiments); MHV68-infected mice treated with anti-LFA-1/anti-VLA4 (solid orange line, MST 27 d, n = 13, 2 independent experiments); MHV68-infected mice treated with CoB+anti-LFA1 (dotted grey line, MST 16, n = 12, 2 independent experiments); MHV68-infected mice treated with CoB+anti-VLA4 (dotted blue line, MST 20 d, n = 11, 2 independent experiments); MHV68-infected mice treated with CoB+anti-LFA1/anti-VLA4 (solid green line, MST>100 d, n = 25, 3 independent experiments). Statistical comparisons were made with the log-rank test. In infected animals, comparison of combined CoB and dual adhesion blockade to CoB alone yielded p<.0001. Comparison between MHV68-infected animals treated with CoB alone, CoB+anti-LFA-1, and CoB+anti-VLA-4 revealed no significant differences (p = 0.103). C) Kaplan-Meier survival curves comparing skin graft survival between the following groups: Non-infected mice treated with CoB (solid black line, MST 22 d, n = 48, 8 independent experiments); Non-infected mice treated with CoB+anti-LFA-1/anti-VLA-4 (dotted black line, MST>100 d, n = 26, 3 independent experiments); MHV68-infected mice treated with CoB (solid red line, MST 13.5, n = 40, 7 independent experiments); MHV68-infected mice treated with CoB+anti-LFA1/anti-VLA4 (solid green line, MST>100 d, n = 25, 3 independent experiments). Statistical comparisons of skin graft survival between groups were made with the log-rank test. In non-infected animals, comparison of combined CoB+dual adhesion blockade to CoB alone resulted in p<.0001. In MHV68-infected animals, comparison of CoB+anti-LFA-1/anti-VLA-4 treatment to CoB treatment yielded p<.0001. (D) Maintenance adhesion blockade (anti-LFA1/anti-VLA4) therapy was discontinued from 8 MHV68-infected mice (MHV68 WT or MHV68 M1.STOP) treated with CoB+anti-LFA1/anti-VLA4 and who had stable surviving allografts after 90-100 days of treatment with dual adhesion blockade. Following discontinuation of therapy, all grafts failed with a median time of 34 d. (E) Mice treated with combined costimulation and adhesion blockade demonstrated a decreased frequency of KLRG1+/CD127+ CD8+ T cells relative to mice treated with costimulation blockade alone. Gray columns: MHV68-infected mice treated with CoB. Red columns: MHV68-infected mice treated with CoB+antiLFA1/anti-VLA4. Y axis: % of KLRG1+/CD127+ CD8+ T cells expressed as a percentage of total CD8+ T cells. After accounting for multiple testing using the Holm method, this effect was significant at 28 days after skin grafting. In mice treated with CoB alone (n = 6), 33.3% of CD8+ T cells were KLRG1+/CD127+ versus 19.9% in mice with combined treatment (n = 4, p = .042*). Error bars represent the standard error of the mean.</p

MHV68 results in the expansion of a unique T cell population, CD8^dim.

<p>A) Flow cyotmetric plots showing CD8 versus CD3 staining after gating on lymphocytes by forward and side scatter followed by gating on CD3+/CD8+ cells. Flow cytometry was performed on peripheral blood samples drawn 6 weeks post-infection or in similarly aged non-infected animals. Each plot shows the combined results from equal numbers of cells from 5 animals. B) Longitudinal analysis in MHV68-infected animals of the absolute numbers of total CD8 (dotted black line), CD8^bright (dashed blue line), and CD8^dim (solid red line) T cells following infection. Data include 10 mice from 2 independent experiments except at 1 week where 5 mice from a single experiment are displayed. Error bars represent 1 standard deviation. C) Longitudinal analysis in non-infected B6 mice of the percentage of CD8 T cells that are CD8^dim after placement of allogeneic skin grafts. The solid black line represents mice not receiving immunosuppressive therapy (n = 4). The dashed gray line shows mice receiving CTLA-4-Ig+anti-CD154 therapy (n = 6). Error bars show 1 standard deviation. D) Flow cytometric analysis at 6 weeks after infection comparing the relative expression of CD44, KLRG1, Bcl2, and CD127 with histograms between CD8 T cells from non-infected animals (gray filled area), CD8^bright (solid black line) from MHV68-infected animals, and CD8^dim (solid red line) from MHV68-infected animals. The plot in the bottom right shows the expression of CD127 versus KLRG1 for CD8^dim in MHV68-infected animals. Each cell type for each plot shows the combination of 5 individual mice.</p

Latent infection with MHV68 results in abbreviated skin allograft survival.

<p>A) Kaplan-Meier survival curves showing skin graft survival for the following cohorts: Black solid line - Non-infected B6 recipients of BALB/C skin allografts, receiving CTLA-4-Ig+anti-CD154 CoB. N = 48, 8 independent experiments, MST 22 d. Red solid line - MHV68-infected B6 recipients of BALB/c skin allografts receiving CTLA-4-Ig+anti-CD154 CoB. N = 40, 7 independent experiments, MST 13.5 d. Grey box - indicates MST from two historical control cohorts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221-Coley1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221-Reisman1" target="_blank">[23]</a> of non-infected B6 recipients receiving BALB/c skin grafts without any immunosuppression (MST 13 d). Statistical Analysis: Log-rank comparison of MHV68-infected/CoB treated versus non-infected/CoB treated yielded p<.0001. Log-rank comparison of MHV68-infected/CoB treated to non-infected/no immunosuppression yielded a non-significant p-value. B) Representative skin allografts from mice 12 days after graft placement. The graft on the non-infected animal remains healthy and pristine while the graft on the animal infected with latent MHV68 demonstrates necrosis, scarring, and erythema.</p

CD8^dim exhibit decreased expression of costimulatory molecules and increased expression of adhesion molecules.

<p>A) Histograms from flow-cytometry based-phenotyping at 6 weeks after infection (or age-matched, non-infected controls) compare the relative expression of the costimulatory molecules CD28, ICOS, and 4-1BB between CD8 T cells from non-infected animals (gray filled area), CD8^bright (solid black line) from MHV68-infected animals, and CD8^dim (solid red line) from MHV68-infected animals. Each cell population represents the combined results from 5 individual mice. Similar results (not shown) were observed 3 weeks after infection. B) Flow-cytometric phenotyping from mice 6 weeks after infection (or age-matched, non-infected controls) compare the relative expression of the adhesion molecules LFA-1 and VLA-4 between CD8 T cells from non-infected animals (gray filled area), CD8^bright (solid black line) from MHV68-infected animals, and CD8^dim (solid red line) from MHV68-infected animals. Each cell population represents the combined results from 5 individual mice. Similar results (not shown) were observed 3 weeks after infection. C) Comparison of gene expression profiles between CD8+ T cells from non-infected animals, CD8^bright from infected animals, and CD8^dim from infected animals (additional data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221.s002" target="_blank">Table S2</a>). Data shown from 3 independent experiments, with 1–3 biologic replicates and 1–4 technical replicates each. D) Flow cytometric analysis of CD8+ T cells from MHV68-infected animals at 6 weeks after infection identifies MHV68-specific CD8 T cells by the use of both MHC tetramers and peptide-stimulated IFN-γ release. The plots show MHV68-specificity versus CD8 surface expression. Each plot represents combined results from 5 mice. E) Flow cytometric analysis of CD8+ T cells from MHV68-infected mice at 6 weeks after infection demonstrating TCR Vβ4 staining versus CD8 staining. Each plot represents combined results from 5 mice. F) Longitudinal analysis of total CD8^dim cells (dotted red line), CD8^dim Vβ4- (solid green line), and CD8^dim Vβ4+ (dashed gray line) from MHV68-infected mice 6 weeks after infection (n = 10, except for week 1; n = 5). Error bars represent 1 standard deviation.</p

The addition of adhesion blockade to costimulation blockade increases viral replication.

<p>MHV68 viral loads were measured>100 days after treatment with CoB +/− adhesion blockade by PCR for MHV68 ORF50 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221-Moorman1" target="_blank">[19]</a>. Recipients treated with CoB alone (red data points) showed a median value of 313 copies/mL (n = 5). Recipients treated with CoB+anti-LFA-1 alone (gray data points) showed a median value of 1027/mL (n = 4, p = n.s. compared to CoB alone). Recipients treated with CoB+anti-VLA-4 alone (blue data points) showed a median value of 9255/mL (n = 5, p = n.s. compared to CoB alone). Recipients treated with CoB+anti-LFA-1+ anti-VLA-4 (green data points) showed a median value of 20278/mL (n = 5, p<.05 compared to CoB alone). Plots for each group show the median viral load with range from a single experiment.</p

Comparative Effect of CTLA-4-Ig and anti-CD154+ adhesion blockade in MHV68-infected and non-infected mice.

<p>A) Kaplan-Meier survival curves comparing skin graft survival between the following groups of MHV68-infected mice: MHV68-infected mice treated with CoB (solid red line, MST 13.5 d, n = 40, 7 independent experiments); MHV68-infected mice treated with anti-LFA-1+ anti-VLA-4 (solid orange line, MST 27 d, n = 14, 2 independent experiments); MHV68-infected mice treated with anti-CD154+ anti-LFA-1/anti-VLA-4 (solid violet line, MST>100 d, n = 8, one experiment); MHV68-infected mice treated with CTLA-4-Ig+anti-LFA-1/anti-VLA-4 (solid magenta line, MST 41 d, n = 8, one experiment); MHV68-infected mice treated with CoB+anti-LFA-1/anti-VLA-4 (solid green line, MST>100 d, n = 25, 3 independent experiments). Skin graft survival between groups was compared using the log-rank test. In infected animals, the combination of anti-LFA-1 and anti-VLA-4 was compared to CoB alone resulting in p = .0023. CTLA-4-Ig+anti-LFA-1/anti-VLA-4 compared to CoB alone yielded p = .0002. The combination of anti-CD154+ anti-LFA-1/anti-VLA-4 was significantly different from CoB alone (p<.0001) and not significantly different from dual CoB (anti-CD154+ CTLA-4-Ig)+dual adhesion blockade (p = .73). B) Kaplan-Meier survival curves comparing skin graft survival between the following groups of non-infected mice: non-infected mice treated with CoB (dotted red line, MST 22 d, n = 48, 8 independent experiments); non-infected mice treated with anti-LFA-1+ anti-VLA-4 (dotted orange line, MST 24, n = 12, 2 independent experiments); non-infected mice treated with anti-CD154+ anti-LFA-1/anti-VLA-4 (dotted violet line, MST>100 d, n = 8, one experiment); non-infected mice treated with CTLA-4-Ig+anti-LFA-1/anti-VLA-4 (dotted magenta line, MST>100 d, n = 8, one experiment); non-infected mice treated with CoB+anti-LFA-1/anti-VLA-4 (dotted green line, MST>100 d, n = 26, 3 independent experiments). Graft survival between groups in non-infected animals was compared with the log-rank method. Survival in the CoB only and anti-LFA-1+ anti-VLA-4 groups was not significantly different (p = .87). Relative to CoB alone, survival was significantly prolonged in non-infected animals treated with anti-CD154+ anti-LFA-1/anti-VLA-4 (p<.0001), CTLA-4-Ig+anti-LFA-1/anti-VLA-4 (p<.0001), or dual CoB (anti-CD154+ CTLA-4-Ig)+anti-LFA-1/anti-VLA-4 (p<.0001).</p