HLA-mediated inhibition of NK cell recognition of HCMV infection.

<p>Experiments were performed with polyclonal NK cells (A, C, E, G) or NK cell clone #29 (B, D, F, G) from donor AJU. (A, B) Analysis of KIR expression by flow cytometry. (C, D) Killing by NK cells of the MHC class I-deficient cell lines K562, Daudi and L721.221, HLA-C*0602 or C*0702-transfected L721.221 cells, uninfected MRC-5 fibroblasts, or MRC-5 infected with CMV-wt at moi = 5, at an effector∶target ratio of 2. Data are shown as mean+SD of four replicates from one representative experiment out of four. (E, F) NK cell-mediated killing of uninfected (n.i.) and HCMV-infected fibroblasts over time after infection. Fibroblasts were or were not pretreated with IFN-γ before infection as indicated. (G) Blockade of NK cell mediated-killing by monoclonal antibodies specific for HLA-ABC or KIR2DL2/3 (both IgG2a) or a matched isotype control. Targets were pretreated with IFN-γ. Blockade of the non-KIR ligand HLA-A2 served as additional negative control. The HLA class I type of MRC-5 fibroblasts is HLA-A*0201, A*2902, B*0702, B*4402, C*0501, C*0702. HLA-C*0702 is the only ligand of KIR2DL3 expressed by MRC-5 cells. Killing was assessed at an effector∶target ratio of 2. Data are shown as mean+SD of triplicate samples from one out of two independent experiments.</p

Immunodominance of CRV/C*0702-specific T cells.

<p>(A) Frequencies of CRV-specific T cells in 15 HLA-C*0702-positive blood donors, tested with the CRV nonameric peptide in ELISPOT assays. Black parts of bars indicate CRV-specific signal, grey parts indicate background (no peptide). (B) Specific T cells in PBMCs of HLA-C*0702 carriers were quantified by fluorescent staining with HLA-C*0702/CRV streptamer or HLA-B*0702/TPR pentamer and anti-CD8 antibody. Donors LT12 and SA03 are HCMV-seropositive, donor ASM is HCMV-seronegative. (C) Distribution of T-cell targets within the IE-1 sequence for 15 HLA-C*0702-positive donors, tested with overlapping peptides covering the entire IE-1 sequence of strain AD169. The 120 peptides were divided into 10 subpools, each comprising 12 successive 15-mer peptides with an overlap of 11 amino acids. The C-terminal amino acid position of each subpool is indicated. (D) Frequencies of CRV/C*0702-specific and VLE/A*0201-specific T cells in HLA-C*0702/A*0201-positive donors (n = 6). (E) Comparison of IE-1-specific T cell frequencies in C*0702-negative (n = 13) vs. C*0702-positive (n = 15) donors. (A, C–E) IFN-γ ELISPOT assays were performed with 200 000 peptide-loaded PBMCs in each well and with 2–4 replicates per condition.</p

Effect of HCMV immunoevasins on epitope-specific T cell cytotoxicity.

<p>MRC-5 fibroblasts (HLA-A*0201, C*0702) were preincubated in medium with (A) or without (B) IFN-γ for 72 hours before infection at moi = 5 with AD169 (CMV-wt) or its derivatives CMV-Δall (ΔUS2/3/6/11), CMV-US11 (ΔUS2/3/6) or CMV-US2 (ΔUS3/6/11). Cytotoxicity was determined at 48 hours post infection in a 3.5-hour calcein release assay using an effector∶target ratio of 4. Fibroblasts that were not infected (n.i.) or peptide-loaded (n.i. +peptide) were negative and positive controls, respectively. Data are shown as mean+SD of three to four replicates.</p

Impact of individual HCMV immunoevasins on the recognition of IE-1 T cell epitopes.

<p>MRC-5 fibroblasts were infected with HCMV strain AD169 (wt), with AD169 derivative viruses that expressed only one of the four immunoevasins US2, US3, US6, or US11 as indicated, not infected (n.i.) or peptide-loaded (+pep), and their recognition by T cell clones specific for the IE-1 epitopes CRV/C*0702 and VLE/A*0201 was analyzed. Before infection, fibroblasts were precultured with IFN-γ for 72 hours, then infected at moi = 5 and cocultivated with T cells at 48 hours post infection (10 000 fibroblasts and 10 000 T cells per well). IFN-γ secretion was measured by ELISA. Data are shown as mean+SD of triplicate samples. Representative data are shown for one of 10 CRV-specific clones and one of 4 VLE-specific clones, assayed in two independent experiments.</p

Time course of pp65- and IE-1-specific CD8+ T cell recognition of HCMV-infected fibroblasts.

<p>pp65 and IE-1 T cell epitopes were analyzed for their HLA-A*0201 or -C*0702-restricted presentation to T cell clones at different time points post infection. MRC-5 fibroblasts (HLA-A*0201, C*0702) were not treated (A) or were treated (B) with IFN-γ for 72 hours prior to infection with HCMV AD169 at an moi of 5 or 10. At the indicated time points post infection, 10 000 fibroblasts were incubated with 10 000 T cells for 16–18 hours before measuring antigen-specific IFN-γ secretion by ELISA assay. Cells that were not infected (n.i.) or were peptide-loaded 48 hours post infection (+pep) served as controls. Data are shown as mean+SD of triplicate samples. One of three independent experiments with clones ALT#21 NLV, F61#38 VLE and AJJ#7 CRV is shown, representing experiments with a total of 3 NLV-, 6 VLE- and 12 CRV-specific T cell clones, from 3 different donors for each specificity.</p

Functional separation of epitope presentation and HCMV immunoevasion.

<p>WI-38 fibroblasts (negative for HLA-A*0201 and C*0702) were transfected with plasmids encoding HLA-A*0201, HLA-C*0702, or chimeric HLA class I heavy chains (HLA-A2/C7 or HLA-C7/A2) and subsequently infected with MVA-IE-1 or different HCMV derivatives. IFN-γ secretion was measured in ELISA after overnight incubation of 10 000 clonal T cells with 10 000 target cells. (A) Schematic representation of the native and chimeric HLA class I molecules that were tested. (B) HLA-transfected WI-38 cells were infected with MVA-IE-1, and presentation of IE-1 epitopes was detected by VLE- and CRV-specific T cell clones. Mean and SD of three replicates are shown for 3 T cell clones generated from 3 different donors for each specificity. (C) HLA-transfected WI-38 were infected with CMV-wt, CMV-Δall, CMV-US2 or CMV-US11, not infected (n.i.) or peptide-loaded (+pep). Three T cell clones generated from 3 different donors were used as effectors for each specificity. Data are shown as mean+SD of triplicate samples from one of two independent experiments.</p

Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

<div><p>Reactivation of human cytomegalovirus (HCMV) can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided 'proof of concept' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg^-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease.</p></div

Antiviral effect of TCR_NLV-transduced human CD4 and CD8 T cells upon adoptive transfer into mCMV-NLV infected NSG/HHD mice.

<p>Virus titers were determined in the indicated organs of mCMV-NLV-infected NSG/HHD mice adoptively transferred with 1x10⁷ mock-transfected (open circles) or TCR_NLV-transfected (filled circles) CD8 or CD4 T cells or with 1:4 mixtures of CD4 (2x10⁶) and CD8 (8x10⁶) T cells (mixture mock: open circles; mixture-TCR_NLV: filled circles). For controlling epitope specificity of the antiviral effect, virus titers were also determined in organs of mCMV-NLV_Ala-infected mice adoptively transferred with the 1:4 mixture of TCR_NLV-transduced CD4 and CD8 T cells (Mixture-TCR_NLV; open squares). Organs were sampled for the virus plaque assay on d11 post-infection and adoptive T-cell transfer. For spleen and lungs, data refer to the whole organ; for the liver data are normalized to 0.5g of tissue. Symbols represent individual mice (n = 5 per group) and horizontal bars mark the median values. For statistical analysis, see the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005049#ppat.1005049.g002" target="_blank">Fig 2</a>.</p

Protective NIF formation after adoptive transfer of NLV-specific murine and human CD8 T cells.

<p>Corresponding to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005049#ppat.1005049.g003" target="_blank">Fig 3</a>, 2C-IHC of liver tissue sections taken on d11 after transfer of (A) mCD8-NLV CTLL or (B) hCD8-NLV CTLL show mCMV-NLV or mCMV-NLV_Ala infected hepatocytes (iHc, red staining of intranuclear IE1 protein) with typical intranuclear inclusion bodies, and infiltrating CD8 T cells (CD8-T, black cytoplasmic and membrane staining of CD3ε), forming nodular inflammatory foci (NIF) or foci of infection in absence of CD8 T cells (IF), respectively. Upper row images give overviews (A,B; a1, b1), higher-magnification lower row images (A, B; a2, b2) reveal details. Bar markers represent 50 μm throughout.</p

Epitope recognition by NLV peptide specific CD8 T cells.

<p>(A) IFN-γ spots, each representing a CD8 T cell responding with IFN-γ production, formed upon sensitization of NLV peptide-specific murine (mCD8-NLV; left panel) and human (hCD8-NLV; right panel) cytolytic CD8 T-cell lines (CTLL) by NLV-peptide-pulsed NSG/HHD MEF at an effector-to-stimulator cell ratio of 0.1:1. MEF were pretreated with murine IFN-γ for 48h (filled bars) or left untreated (open bars) and were exogenously loaded with synthetic NLV- or non-cognate gp100_280-288-peptide at the indicated concentrations. Data are shown as mean of duplicates from one experiment representative of two performed. Error bars represent the range. (B) Structural avidity of antigen binding to mCD8-NLV and hCD8-NLV CTLL was quantified by dose-dependent HLA-A2.1/NLV tetramer binding in flow cytometry. The respective dissociation constant (K_D) was calculated from half-maximal tetramer binding obtained by Scatchard plot analysis. Dotted curves border the 95% confidence regions of the log-linear regression lines. MFI, mean fluorescence intensity. (C) HLA-A2.1 restricted presentation of NLV epitope by NSG/HHD MEF pretreated with murine IFN-γ for 48h (filled bars) or left untreated (open bars) and infected at an MOI of 4 with the indicated viruses for a total time of 22h until the end of the assay. Peptide presentation on the infected MEF during that period was detected in an IFN-γ ELISpot assay with mCD8-NLV (left panel) and hCD8-NLV (right panel) CTLL at an effector-to-stimulator cell ratio of 0.1:1. Data are shown as mean of duplicates from one experiment representative of two performed. Error bars represent the range.</p