Journal:

Article Title: Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2 d Haplotype

doi: 10.1128/JVI.76.1.151-164.2002

Figure Lengend Snippet: 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).

Article Snippet: An immune response to mCMV was primed by subcutaneous (intraplantar) infection of female, 8- to 10-week-old BALB/c (haplotype H-2 d ) mice at the left hind footpad with 10 5 or 10 6 PFU of cell culture-propagated and sucrose gradient-purified mCMV strain Smith (ATCC VR-194/1981) ( 32 ).

Techniques: Binding Assay, Sequencing, Activity Assay

Journal:

Article Title: Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2 d Haplotype

doi: 10.1128/JVI.76.1.151-164.2002

Figure Lengend Snippet: 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. ​Fig.6.6. The median values are marked by horizontal bars. The dotted line indicates the detection limit of the plaque assay.

Article Snippet: An immune response to mCMV was primed by subcutaneous (intraplantar) infection of female, 8- to 10-week-old BALB/c (haplotype H-2 d ) mice at the left hind footpad with 10 5 or 10 6 PFU of cell culture-propagated and sucrose gradient-purified mCMV strain Smith (ATCC VR-194/1981) ( 32 ).

Techniques: In Vivo, Infection, Irradiation, Plaque Assay, Histopathology

Journal:

Article Title: Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2 d Haplotype

doi: 10.1128/JVI.76.1.151-164.2002

Figure Lengend Snippet: 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 CD3ɛ; 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.

Article Snippet: An immune response to mCMV was primed by subcutaneous (intraplantar) infection of female, 8- to 10-week-old BALB/c (haplotype H-2 d ) mice at the left hind footpad with 10 5 or 10 6 PFU of cell culture-propagated and sucrose gradient-purified mCMV strain Smith (ATCC VR-194/1981) ( 32 ).

Techniques: Enzyme-linked Immunospot, Isolation, Infection, Concentration Assay

Journal:

Article Title: Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2 d Haplotype

doi: 10.1128/JVI.76.1.151-164.2002

Figure Lengend Snippet: 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. ​Fig.7.7. (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.

Article Snippet: An immune response to mCMV was primed by subcutaneous (intraplantar) infection of female, 8- to 10-week-old BALB/c (haplotype H-2 d ) mice at the left hind footpad with 10 5 or 10 6 PFU of cell culture-propagated and sucrose gradient-purified mCMV strain Smith (ATCC VR-194/1981) ( 32 ).

Techniques: Enzyme-linked Immunospot, Infection

Journal:

Article Title: Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2 d Haplotype

doi: 10.1128/JVI.76.1.151-164.2002

Figure Lengend Snippet: 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. ​Fig.7.7. (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.

Article Snippet: An immune response to mCMV was primed by subcutaneous (intraplantar) infection of female, 8- to 10-week-old BALB/c (haplotype H-2 d ) mice at the left hind footpad with 10 5 or 10 6 PFU of cell culture-propagated and sucrose gradient-purified mCMV strain Smith (ATCC VR-194/1981) ( 32 ).

Techniques: Enzyme-linked Immunospot, Isolation, Infection

Journal:

Article Title: Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2 d Haplotype

doi: 10.1128/JVI.76.1.151-164.2002

Figure Lengend Snippet: 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-2d-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 CD3ɛ. Throughout, bars represent mean values of triplicate assay cultures.

Article Snippet: An immune response to mCMV was primed by subcutaneous (intraplantar) infection of female, 8- to 10-week-old BALB/c (haplotype H-2 d ) mice at the left hind footpad with 10 5 or 10 6 PFU of cell culture-propagated and sucrose gradient-purified mCMV strain Smith (ATCC VR-194/1981) ( 32 ).

Techniques: Infection, Enzyme-linked Immunospot, Isolation, Concentration Assay