Fig 1.
Construction and characterization of recombinant mCMV-NLV expressing the HLA-A2.1 restricted peptide NLV.
(A) Map of the major immediate-early (MIE) locus of mCMV, illustrating the integration of the sequence of the immunodominant HCMV peptide derived from protein pUL83/pp65 (bold face capital letters) or of the loss-of-function Ala variant (capital letters in italics) within mCMV ORFm128. Both peptide sequences were flanked by their authentic amino acid residues present in pUL83/pp65. Grey-shaded boxes represent exons, black bars and boxes symbolize the bidirectional promoters and enhancers, respectively. (B) Viral growth curves in vivo represented by the time course of numbers of infected IE1+ liver cells in representative 10-mm2 areas of liver tissue sections (left panel) or of virus titers in homogenates of spleen (center panel) and lungs (right panel) after intraplantar infection of NSG/HHD mice with 1x105 PFU of mCMV-Δm157 (filled circles) or mCMV-NLV (open circles). Symbols represent median values from 4 to 5 mice per group and time of assay. Log-linear regression lines (based on data from all individual mice) and their corresponding 95% confidence areas (bordered by dotted curves) are indicated. Viral doubling times (vDT) and their 95% confidence intervals (in parentheses) are given.
Fig 2.
Epitope recognition by NLV peptide specific CD8 T cells.
(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 gp100280-288-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 (KD) 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.
Fig 3.
Dose-dependent antiviral effect of NLV-peptide-specific CD8 T cells in mCMV-NLV infected NSG/HHD mice.
(A) Experimental strategy and schedule of adoptive transfer of NLV-specific CD8 T cells into infected NSG/HHD recipients that were preconditioned by total-body γ-irradiation with a dose of 2 Gy. Intraplantar infection was performed throughout with 1x105 PFU of chimeric mCMV. Recipients (n = 4–5 per group) were infected with mCMV-NLV (filled symbols) or mCMV-NLVAla (open symbols), followed by i.v. transfer of graded numbers of (B) murine or (C) human NLV-specific CD8 T cells (mCD8-NLV or hCD8-NLV CTLL, respectively). Infectivity was quantified at d11 post-transfer in spleen and lungs by standard plaque (plaque-forming unit, PFU, assay). In the livers of the same mice, infected cells (filled and open circles for mCMV-NLV and mCMV-NLVAla, respectively) and tissue-infiltrating CD8 T cells (filled and open squares, correspondingly) were stained by 2C-IHC and counted in representative 10-mm2 areas of tissue sections. Symbols represent individual mice and horizontal bars mark median values. Statistical analysis for group differences of most interest (bracketed) was performed after log-transformation using Student’s t-test (unpaired, two-sided; p<0.05 considered significant) with Welch’s correction.
Fig 4.
Protective NIF formation after adoptive transfer of NLV-specific murine and human CD8 T cells.
Corresponding to Fig 3, 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-NLVAla 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.
Fig 5.
Transgenic TCRNLV expression, structural avidity, and cytolytic activity of TCR-transduced human T-cell subsets.
(A) Immunomagnetically selected human CD8 T cells (left panels) and CD4 T cells (right panels) were retrovirally transduced with TCRNLV (CD8-TCRNLV and CD4-TCRNLV, respectively) or empty vector (CD8 mock and CD4 mock, respectively) and were drug-selected. After in vitro expansion with anti-CD3/CD28 beads for a period of 10d, cells were analyzed cytofluorometrically for expression of CD4 and CD8, as well as of TCRNLV using HLA-A2.1/NLV tetramers. Shown are 2D dot plots with the percentages of labeled cells indicated. (B) and (C) Characterization of CD8-TCRNLV and CD4-TCRNLV cells respectively. (Left panel) Structural avidity (KD) of antigen binding was quantified by dose-dependent HLA-A2.1/NLV tetramer binding determined by flow cytometry (see the legend of Fig 2B). (Right panels) Cytolytic activities of TCRNLV-transduced and mock-transduced T cells (filled and open circles, respectively) determined by a standard [51Cr]-release assay at the indicated effector (i.e. T cell) to target (i.e. MEF of NSG/HHD mice) cell ratios (E:T). NSG/HHD MEF were pre-treated with IFN-γ for 48h and were either exogenously loaded with a high dose of synthetic NLV peptide as a positive control, or infected for 12h with mCMV-NLV and, as a non-antigenic control, with mCMV-NLVAla. Data represent means of duplicate assay cultures. Error bars indicate the range.
Fig 6.
Antiviral effect of TCRNLV-transduced human CD4 and CD8 T cells upon adoptive transfer into mCMV-NLV infected NSG/HHD mice.
Virus titers were determined in the indicated organs of mCMV-NLV-infected NSG/HHD mice adoptively transferred with 1x107 mock-transfected (open circles) or TCRNLV-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-TCRNLV: filled circles). For controlling epitope specificity of the antiviral effect, virus titers were also determined in organs of mCMV-NLVAla-infected mice adoptively transferred with the 1:4 mixture of TCRNLV-transduced CD4 and CD8 T cells (Mixture-TCRNLV; 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 Fig 2.
Fig 7.
Survival of mCMV-NLV-infected NSG/HHD mice upon adoptive transfer of NLV-specific human T cells.
Groups of 10 γ-irradiated (2 Gy) NSG/HHD mice were infected (A) with 1x105 PFU or (B) with 1x103 PFU of mCMV-NLV, and in (A; B,b) they received the 1:4 mixtures (see the legend to Fig 6) of TCRNLV-transduced (solid graphs) or mock-transduced (dashed graphs) human CD4 and CD8 T cells on the day of infection as a pre-emptive therapy. (B,a) As a reference for comparison, recipients received 1x107 cells of CTLL hCD8-NLV (solid graph) or were left with no T cell transfer (dashed graph, w/o T cells). Survival rates over time are displayed as Kaplan Meier survival plots. Statistical significance of differences in survival was calculated using the log-rank test and the Gehan-Wilcoxon test. In the most efficient therapy with the mixture of TCRNLV-transduced CD4 and CD8 T cells (B,b), the median survival time was 26d (range: 21-27d) compared to 21d (range 16-22d) in the mock-transfected control group.
Fig 8.
Reduced viral spread and histopathology in liver tissue, differentiated by infected cell type, as correlate of protection by human TCRNLV-transduced T cells.
Corresponding to the survival data (Fig 7B and 7b), liver tissue sections were taken on day 20 after infection with 103 PFU of mCMV-NLV and adoptive transfer of mock-transduced (group: Control) or TCRNLV-transduced (group: Therapy) human T cells, each composed of 2x106 CD4 and 8x106 CD8 cells. Low-magnification IHC images show overviews of the extent of tissue infection (a1,b1; plaque-like foci of infected, red-stained IE1+ liver cells), and higher-magnification 2D-IHC images reveal infection of hepatocytes (iHc), which are distinctive by cytomorphology (red staining of intranuclear IE1 protein), combined with infected endothelial cells (a2, b2; iEC, co-expressing red-stained IE1 protein and black-stained CD31 antigen) or infected macrophages (a3,b3: iMΦ, co-expressing red-stained IE1 protein and turquoise green-stained F4/80 antigen). Bar markers represent 50 μm throughout.
Fig 9.
Immunohistological quantitation of infected and of apoptotic liver cells.
(A) Corresponding to the representative IHC images shown in Fig 8, infected cells were counted in absolute numbers on day 20, differentiated by cell type as indicated, in representative 10-mm2 areas of liver tissue sections. Symbols represent cell counts from individual mice, with the median values marked. Open circles, mice from the mock-transduction control group; closed circles, mice from the TCRNLV-transduction therapy group. (B) Corresponding to the representative IHC images shown in Fig 10 and S6 Fig, apoptotic (caspase 3+) liver cells were counted on day 20 in absolute numbers, differentiated by infected (IE1+) and uninfected (IE1-) liver cells. Further details as in subfigure (A). The calculation of P values for the significance of the observed trend to a reduction in apoptosis events in the therapy group makes no sense in view of the scientific question of the experiment, namely if xenogeneic T cells exert an immunopathology indicated by enhanced apoptosis, which was obviously not the case. Subfigure (C) points to the fact that on day 20 after infection and cell transfer, T cells (black staining of CD3ε antigen as in Fig 4) were no longer present in either experimental group. Symbols as in subfigure (A).
Fig 10.
Human NLV-specific T cells do not exert an immunopathology in terms of enhanced apoptosis or necrotic/necroptotic lesions outside of foci of infection.
Corresponding to the quantitative data provided in Fig 9B, representative 2C-IHC images of liver tissue sections show uninfected, apoptotic hepatocytes (IE1-caspase 3+, brown cytoplasmic staining), exclusively within IE1+ (red stained) foci of infection. (a1, b1) Low-magnification images for an overview of viral histopathology. Arrows in these overview images point to regions resolved to greater detail (a2, b2). Note that infected, apoptotic cells are difficult to find coincident with uninfected, apoptotic cells, because of the generally low incidence of apoptosis in this cell transfer model. An example for the existence of infected, apoptotic cells is shown in S6 Fig. Bar markers represent 50 μm throughout.