Article Figures & Data
Figures
- FIG. 1.
Identification of an antigenic peptide in ORF m164 of mCMV strain Smith. (A) A search for the Dd binding motif xGPxxxxx[L, I, F] was performed for all ORFs of the full-length genomic sequence of mCMV strain Smith (GenBank accession no. MCU68299 ), and the corresponding synthetic nonapeptides were used at the indicated molar concentrations for the generation of short-term microculture CTLLs. Data represent the cytolytic activity of individual microcultures tested on P815 target cells that were pulsed with the corresponding peptides at the corresponding concentrations. The genomic positions of the nonapeptide-coding sequences are given by the positions of the first nucleotides (n) according to the listing by Rawlinson et al. (43). C, complementary strand. (B) List of nonameric Dd-binding motifs. ORFs of mCMV that are sequence homologs of hCMV ORFs are indicated by capital M. Scores for Dd binding strengths were provided by Stefan Stevanovic (Institute for Cell Biology, Department of Immunology, University of Tuebingen, Tuebingen, Germany). (C) HindIII and EcoRI genetic map locations of ORF m164 peptide C 222881 257AGPPRYSRI265 drawn to scale. Amino acid positions refer to the deduced protein sequence according to Rawlinson et al. (43).
- FIG. 2.
HPLC retention of naturally processed ORF m164 peptide. Naturally processed peptides derived from fetal fibroblasts in the L phase of mCMV replication were separated by HPLC (run 1). HPLC fractions were used for repeated restimulation of memory spleen cells to generate short-term microculture CTLLs. (A) Cytolytic assay of HPLC fraction-specific CTLLs with P815 target cells that were pulsed with the naturally processed peptides contained in the corresponding HPLC fractions. (B) Cytolytic assay of HPLC fraction-specific CTLLs with P815 target cells that were pulsed with 10−7 M synthetic m164 peptide aa 257 to 265. Excess of synthetic peptide was washed off before use of the target cells in the cytolytic assay. Bars represent mean values of triplicate CTLL cultures. The dashed line indicates the background lysis of target cells that were not exposed to peptide.
- FIG. 3.
Generation of long-term CTLLs. Memory spleen cells were restimulated with synthetic peptides IE1 (aa 168 to 176) and m164 (aa 257 to 265) at the indicated molar concentrations for the generation of IE1-CTLL and m164-CTLL, respectively. After three rounds of restimulation, a cytolytic assay was performed at an E/T cell ratio of 15 with P815 target cells that were pulsed with the indicated molar concentrations (abscissa) of the respective peptides. The target peptide concentration for half-maximal lysis (background subtracted) is indicated.
- FIG. 4.
Frequencies of IFN-γ-secreting cells in CTLLs determined by ELISPOT assays. Long-term CTLLs were generated by repeated restimulations of memory spleen cells with the respective peptide at a concentration of 10−9 M. (A) Photodocumentation of ELISPOT filters. One representative filter of triplicate assay cultures with 100 cells seeded is shown. Ø, P815-B7 stimulator cells with no peptide added; Cognate peptide, P815-B7 stimulator cells pulsed with the respective peptide at a concentration of 10−8 M; αCD3, stimulation with 145-2C11 hybridoma cells that produce anti-mouse CD3ε MAb. (B) Data of triplicate assay cultures for three cell numbers seeded. Solid circles, stimulation with cognate peptide; open circles, stimulation with anti-CD3ε hybridoma. (C) HPLC retention of naturally processed peptides detected by IE1-CTLL and m164-CTLL. ELISPOT assays were performed with 100 CTLL cells and with P815-B7 stimulator cells that were pulsed with naturally processed peptides contained in HPLC fractions (run 2). Control stimulations were done as described above. Bars represent mean values of triplicate assay cultures.
- FIG. 5.
In vivo antiviral function of CTLLs. Graded numbers of CTLs were transferred intravenously into BALB/c recipients under lethal conditions of infection (6.5 Gy of total-body gamma irradiation followed by intraplantar infection with 105 PFU of mCMV). Ø, no adoptive cell transfer. Virus titers in homogenates of spleen, lung, and liver were determined on day 12 after infection. The virus plaque assay was performed under conditions of centrifugal enhancement of infectivity. Accordingly, titers of infectious virus are expressed as PFU*. Dots represent virus titers in numbered individual mice. Asterisks at the numerals mark individual mice for which the liver histopathology is documented in Fig. 6. The median values are marked by horizontal bars. The dotted line indicates the detection limit of the plaque assay.
- FIG. 6.
Clearance of infection in the liver by cytoimmunotherapy with CTLLs. Immunohistochemical analysis specific for the intranuclear IE1 protein pp89 of mCMV (black staining) was performed on liver sections from mice for which virus titers are documented in Fig. 5 (see numerals marked by an asterisk). (A1 to A3) Infection of liver parenchyma in the absence of protective CTLs. Panel A1 gives an overview at low magnification. The arrow points to a site that is resolved to greater detail in panels A2 and A3, highlighting intranuclear inclusion bodies in infected hepatocytes. These so-called “owl’s eyes” are characteristic of the L phase of the productive cycle. (B and C) Protection mediated by adoptive transfer of 105 cells of IE1-CTLL and m164-CTLL, respectively. Light counterstaining was performed with hematoxylin. Bars, 50 μm.
- FIG. 7.
Frequencies of memory CD8 T cells determined by IFN-γ-based ELISPOT assays. (A) Responder cells were CD8 T cells isolated from the spleens of unprimed BALB/c mice at 5 months of age. (B) Responder cells were CD8 T cells isolated from age-matched BALB/c mice at 3 months after intraplantar infection with 105 PFU of mCMV. For peptide-specific stimulation of responder cells, P815-B7 cells were pulsed with a 10−8 M concentration of antigenic peptides IE1 (aa 168 to 176) and m164 (aa 257 to 265). αCD3, polyclonal stimulation with 145-2C11 hybridoma cells that produce MAb anti-mouse CD3ε; Ø, P815-B7 cells with no peptide added. Dots represent data from triplicate assay cultures. The median values are marked by vertical bars. For 104 CD8 T cells seeded, one representative filter of each of the triplicates is documented as a photograph.
- FIG. 8.
Frequencies of acutely sensitized CD8 T cells present in the draining PLNs during the primary immune response. Stimulation conditions for the IFN-γ-based ELISPOT assays were as described for Fig. 7. (A) Primary immune response in the ipsilateral PLN at day 8 after intraplantar infection with 106 PFU of mCMV. (B) Primary immune response in the ipsilateral PLN at day 8 after intraplantar inoculation with 106 PFUUV of inactivated mCMV virions.
- FIG. 9.
Frequencies of CD8 T cells present in acute and persisting pulmonary infiltrates. Stimulation conditions for the IFN-γ-based ELISPOT assays were as described for Fig. 7. (A) Pulmonary CD8 T cells isolated from infected lungs at 1 month after BMT and intraplantar infection with 105 PFU of mCMV. (B) Interstitial CD8 T cells isolated accordingly from the lungs of mice at 3 months after BMT and intraplantar infection, a time point at which productive infection of the lungs was resolved and latent infection was established.
- FIG. 10.
Empirical immunome analysis. The frequencies of CD8 T cells responding to known antigenic peptides or to antigenic peptides processed naturally in fetal fibroblasts during the L phase of mCMV infection were determined by IFN-γ-based ELISPOT assays. P815-B7 stimulator cells were pulsed either with the respective synthetic peptides or with HPLC fractions (run 2). (A) Responder cells for the first scan were CD8 T cells isolated from memory spleens at 5 months after intraplantar infection with 105 PFU of mCMV. (B) Responder cells for the second scan were CD8 T cells isolated from draining PLNs on day 8 after intraplantar infection with 106 PFU of mCMV. Controls (left panels) include stimulations at a saturating peptide concentration of 10−8 M with all currently known antigenic, H-2 d-restricted peptides of mCMV, namely IE1 (aa 168 to 176) presented by Ld, m164 (aa 257 to 265) presented by Dd, M83 (aa 761 to 769) presented by Ld, M84 (aa 297 to 305) presented by Kd, and m04 (aa 243 to 251) presented by Dd. Ø, P815-B7 cells with no peptide added; αCD3, polyclonal stimulation with 145-2C11 hybridoma cells that produce a MAb directed against mouse CD3ε. Throughout, bars represent mean values of triplicate assay cultures.
