EBV-specific CD4+ T cells differ in their tumor-protective potential.

<p>(A) Survival of mice after adoptive transfer of different EBV-specific CD4+ T-cell clones. 1×10⁷ LCL and 1×10⁷ T cells were consecutively i.p. injected and mouse survival analyzed. As exemplified by the EBNA1-specific T-cell clone 1C3 and the EBNA3B-specific clone B9, injection of latent antigen-specific T cells had no effect on mouse survival, except for EBNA3C-specific CD4+ T cells that showed a trend towards delaying tumor growth (group sizes: EBNA1-1C3: LCL n = 20, LCL + T cells n = 11; EBNA3B-B9: LCL n = 20, LCL + T cells n = 7; EBNA3C-3H10: LCL n = 11, LCL + T cells n = 4). Adoptive transfer of the BLLF1-specific CD4+ T-cell clone 1D6 prolonged mouse survival (group sizes: LCL n = 10; LCL + T cells n = 10; summarized results of 2 independently performed experiments). (B) CFSE-labeled BLLF1- and EBNA1-specific T cells were i.p. injected into mice that had received autologous LCL 25 days before. Single cell suspensions of tumors were analyzed 24, 48, or 72 hours post injection by FACS for the presence of CFSE-labeled T cells as well as human CD20-expressing tumor cells. BLLF1- but not EBNA1-specific T cells infiltrated tumors and led to a decrease in the percentage of CD20+ cells. (C) Immunostaining of tumor sections from mice described in (B). Cryo-embedded tumor sections were double-stained with FITC- and BLLF1-specific antibodies to detect tumor infiltrated CFSE-labeled T cells (brown) and BLLF1-expressing tumor cells (blue). BLLF1-specific T cells infiltrated tumors and were found in proximity to antigen expressing cells while no EBNA1-specific T cells were found to infiltrate the tumors. Two immunostainings of two separate tumor sections are shown in each case. (D) Injection of the EBNA1-specific T-cell clone 3E10 and the BNRF1-specific T-cell clone 1H7 led to faster tumor development and shortened mouse survival (group sizes: EBNA1-3E10: LCL n = 20, LCL + T cells n = 10; BNRF1-1H7: LCL n = 20, LCL + T cells n = 4).</p

Mouse survival is mediated by non-virus-specific T cells.

<p>T-cell lines were generated by four rounds of stimulation <i>in vitro</i> with LCL cultivated in media containing human serum (HS), fetal calf serum (FCS), or fetal calf serum plus acyclovir (FCS-ACV) to prevent the expression and presentation of virion antigens. (A) Specificity analysis of the generated T-cell lines. Autologous PBMC were pulsed with recombinant EBV latent proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004068#ppat.1004068-Adhikary3" target="_blank">[20]</a> for 24 h and then probed with the T cells. Whereas all T-cell lines recognized LCL, none specifically responded against PBMC pulsed with any of the latent proteins of EBV. (B) Following i.p. injection with autologous LCL as tumor inducing cells, all three T-cell lines prolonged mouse survival significantly (*** p<0.0001). Group sizes: LCL n = 20; LCL + T-cell line HS n = 9; LCL + T-cell line FCS n = 12; LCL + T-cell line FCS-ACV n = 10 (summarized results of two independent experiments). (C) Injection of T-cell line HS together with LCL Z(-) significantly prolonged mouse survival, demonstrating that virion antigen-specific T cells are not required for the tumor protective effect. Group sizes: LCL Z(-) n = 7; LCL Z(-) + T-cell line HS n = 8 (*** p<0.0001).</p

Induction of human PTLD-like tumors in immunodeficient mice.

<p>(A) Intraperitoneal injection of SCID mice with 1×10⁷ LCL of four different donors led to tumor development with an incidence of 75 – 100% and a latency of 20 and 46 days (group sizes: LCL FL and LCL MF n = 4; LCL GB n = 11; LCL JM n = 20; days p.i.: days post injection). (B) Injection of 1×10⁷ LCL, 1×10⁷ LCL Z(-), or 5×10⁷ PBMC from the same donor led to tumor development in all animals but with different latency (group sizes: LCL JM n = 20; PBMC JM n = 6; LCL Z(-) JM n = 6). All survival curves and donor dependent incidences and latencies were reproduced in several independent experiments. (C) Different numbers of LCL from the same donor were injected in mice and the survival determined. Results are depicted in a Kaplan-Meier curve (group sizes: 1×10⁷ n = 20; 5×10⁶ n = 4; 1×10⁶ n = 6; median survival 28, 30, and 38 days). (D) Developing tumors were confirmed as PTLD-like lymphomas. Formalin-fixed, paraffin-embedded tumor slides (3–8 µm) were stained with H&E (first row) and with antibodies against the human B cell marker CD20 (second row), as well as antibodies against the EBV latent proteins EBNA1 (third row) and EBNA2 (fourth row), whose co-expression is characteristic of PTLD.</p

Later passage T-cell preparations show increased virion antigen specificity but are less tumor-protective.

<p>(A) Reactivity of the T-cell lines against virion antigens. With increasing numbers of stimulation, the T-cell lines progressively responded against virion antigens transferred by viral particles. T-cell specificity was tested by cytokine secretion upon stimulation with autologous LCL Z(-). The target cells were either left untreated or loaded with virus particles for presentation of structural antigens (mainly late lytic antigens). (B) Tumor protection by early and late passage T-cell lines. 1×10⁷ LCL and 1×10⁷ T cells stimulated with autologous LCL four (p4) or ten (p10) times <i>in vitro</i> were simultaneously injected into SCID mice (group sizes: LCL n = 20; LCL + T cells p4 n = 13; LCL + T cells p10 n = 13; depicted results are combined from two independently performed experiments). Later passage T-cell lines prolonged mouse survival less efficiently.</p

LCL Z(-)- as well as miniLCL-stimulated T-cell lines recognize autoantigens and prolong mouse survival.

<p>(A) Recognition of autoantigens by LCL Z(-) or miniLCL-stimulated T cells. Specificity analysis of the T-cell line JM-W3 was performed using autologous LCL and LCL Z(-) as well as HLA-matched EBV-negative and EBV-positive BL30 cell lines. T-cell recognition of the EBV-negative BL30 cell line, but barely of BL30 cells that had been infected with the B95.8 or the P3HR1 EBV strains, demonstrated that these T cells recognized a non-viral antigen(s). Recognition of viral antigens by the GB-W3 T cells was excluded by probing the cells with the HLA-matched, EBV-negative Hodgkin lymphoma cell line L428 pulsed with recombinant latent proteins of EBV. (B) Analysis of the tumor-protective potential of these autoreactive T-cell lines <i>in vivo</i>. 1×10⁷ LCL Z(-) or miniLCL were i.p. injected in combination with 1×10⁷ autologous T cells and tumor development assessed (LCL Z(-) n = 6; LCL Z(-) + T cells JM-W3 n = 4; miniLCL n = 4; miniLCL + T cells GB-W3 n = 10; * p<0.05; *** p<0.001) (C) Phenotypic characterization of the autoreactive T cells GB-W3 by FACS. Autoreactive T cells displayed a CD3+CD4+ effector T-cell phenotype (CD62L-CCR7-) of differentiated T cells (CD27-CD28+CXCR3+CCR4-CCR6+/-), and produced granzyme A (black line) and B (grey line) (D).</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

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

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

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

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