1. Impact of clonal competition for peptide-MHC complexes on the CD8+ T-cell repertoire selection in a persistent viral infection
by Katherine K. Wynn, Zara Fulton, Leanne Cooper, Sharon L. Silins, Stephanie Gras, Julia K. Archbold, Fleur E. Tynan, John J. Miles, James McCluskey, Scott R. Burrows, Jamie Rossjohn, and Rajiv Khanna
Blood
Volume 111(8):
April 15, 2008
©2008 by American Society of Hematology

2. The HCMV-encoded CD8+ T-cell epitopes CPS and FPT are exclusively recognized by HLA B*3508 individuals.
The HCMV-encoded CD8+ T-cell epitopes CPS and FPT are exclusively recognized by HLA B*3508 individuals. (A) Healthy HCMV seropositive donors (shown as D1-D8) who expressed different HLA B35 subtypes were assessed for CPS- and FPT-specific T-cell responses using ELISPOT assays as described in “Methods.” The results are expressed as spot forming cells (SFC) per 106 PBMC. (B) In vitro stimulation of T cells with CPS and FPT epitopes confirmed effector-memory responses exclusively in HLA B*3508 individuals. Polyclonal CTL cultures were generated from 2 HLA-B*3508+ and 3 HLA-B3501+ healthy HCMV-exposed individuals by stimulation with CPS or FPT peptide epitopes. CTLs were tested as effectors at a range of E:T ratios for recognition of autologous phytohemagglutinin (PHA) blast target cells pretreated with the peptide used to stimulate the CTLs or left untreated. The HLA-A/B types of the donors were as follows: donor 1: HLA A2, A2, B*3508, B57; donor 2: HLA A23, A32, B*3508, B49; donor 3: HLA A1, A3, B8, B*3501; donor 4: HLA A11, A32, B*3501, B44, and donor 5: HLA A11, A24, B*3501, B60.
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology

3. Ex vivo analysis of TRBV usage by CPS- and FPT-specific T cells.
Ex vivo analysis of TRBV usage by CPS- and FPT-specific T cells. PBMCs from healthy virus carriers were co-stained with pMHC pentamers (CPS or FPT) and a panel of TRBV monoclonal antibodies. Data based on fresh PBMCs is presented in panels A and B (for CPS-specific T cells) and panels C and D (for FPT-specific T cells). After in vitro culture these T cells were re-analyzed using pMHC-pentamers and a panel of TRBV monoclonal antibodies. Data for CPS-specific T-cell cultures is presented in panels E and F, while data for FPT-specific T cells is displayed in panels G and H. The results are expressed as percent pMHC-pentamer-specific cells. Data presented in panels A, C, E, and G are from donor 1; data in panel B and D are from donor 2; while data in panels F and H are from donor 6 (HLA A30, A32, B*3508, B42).
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology

4. Immunoscope analysis of the CDR3 region of TCR specific for CPS- and FPT-specific T cells. cDNA samples from PBMCs or in vitro–expanded T cells cultures were used for PCR reaction with Vβ specific primers as outlined in “Methods,” and results were analyzed ...
Immunoscope analysis of the CDR3 region of TCR specific for CPS- and FPT-specific T cells. cDNA samples from PBMCs or in vitro–expanded T cells cultures were used for PCR reaction with Vβ specific primers as outlined in “Methods,” and results were analyzed using ABI PRISM GeneScan software. Panel A shows the representative immunoscope profile of CPS-specific CD8+ T cells sorted from fresh PBMCs using pMHC-peptide pentamer; panels B and C show data based on in vitro–expanded T-cell lines. Data in panel A was obtained from donor 2, while data in panels B and C was acquired from donors 1 and 2, respectively. Panel D shows representative data for FPT-specific T cells sorted from fresh PBMCs using pMHC-peptide pentamer; panels E-I show data based on in vitro–cultured T-cell lines. Data presented in panels D and E are from donor 1; data in panels F-H were from donor 2; while data in panel I was from donor 6, respectively. Panel J shows a comprehensive summary of multiple CDR3 profiles for CPS- and FPT-specific T cells. Data presented in panel J is based on the analysis carried out in 3 different HLA B* individuals.
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology

5. Nucleotide and amino acid sequences of the CDR3 regions of the TRBV and TRAV chains expressed by CPS- (A) and FPT- (B) specific T cells.
Nucleotide and amino acid sequences of the CDR3 regions of the TRBV and TRAV chains expressed by CPS- (A) and FPT- (B) specific T cells. Data presented in this Figure is based on either clonal or pMHC-pentamer sorted CPS- and FPT-specific T cells from fresh PBMCs or polyclonal T-cell lines (as indicated in left-hand column). Sequences from pMHC-pentamer sorted cells were carried out using pooled cells from an individual donor. *Donor 2 PBMCs were used for TRBV sequencing only.
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology

6. Crystal structures of HLA B*3508CPS and HLA B*3508FPT.
Crystal structures of HLA B*3508CPS and HLA B*3508FPT. Panels A and C showing the electron density 2mfo-Dfc at 1σ of the FPT (orange stick) or CPS (purple stick) epitope bound to HLA B*3508. Panel B represents the structures of the featureless FPT peptide (orange) superposed with the one of the bludged CPS epitope (purple), which made one helix turn in its c-terminus. Panels D and E show surface view of the CPS and FPT epitopes bound to the HLA B*3508 molecule. The solvent-exposed surface of the peptide, which is accessible to the TCR, is represented by the green surface. The FPT epitope (panel E) has a smaller solvent-exposed surface than the CPS epitope (panel D).
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology

7. CD8+ T-cell recognition of target cells sensitized with CPS or FPT epitopes using either viral infection or synthetic peptides.
CD8+ T-cell recognition of target cells sensitized with CPS or FPT epitopes using either viral infection or synthetic peptides. (A) MRC-5 cells expressing HLA B*3508 were infected with HCMV at an MOI of 5:1 and then exposed to CPS- or FPT-specific T cells (responder to stimulator ratio of 2:1) at different time intervals (indicated on x-axis). At each time point, T cells were assessed for IFN-γ expression using intracellular cytokine assay. Data presented here is summary of 3 different experiments based on 3 different HLA B*3508+ donors. (B) CTL recognition of HLA B*3508 expressing target cells sensitized with synthetic CPS and FPT peptide epitopes. MRC-5 cells expressing HLA B*3508 were sensitized with serial dilutions of the peptides and then exposed to CPS- or FPT-specific CTL lines. These clonal T cells were derived from donor 1. (C) MRC-5 cells expressing HLA B*3508 were sensitized with synthetic CPS- or FPT-peptide epitopes (1 μg/mL) and then exposed to CPS- or FPT-specific T cells (responder to stimulator ratio of 2:1) for 16 to 18 hours. T cells were assessed for IFN-γ expression using intracellular cytokine assay. Data presented here is a summary of 3 different experiments. (D) HCMV-infected MRC-5 cells expressing either wild-type or mutated (to disallow CD8 co-receptor binding) HLA*B3508 were exposed to CPS- or FPT-specific T cells at 2 different responder-to-stimulator ratios, and IFN-γ expression measured using an intracellular cytokine assay. These data are representative of 3 different experiments and shows mean fluorescence intensity (MFI) of IFN-γ expression in CPS- or FPT-specific T cells. (E) MRC-5 cells expressing HLA B*3508 were pre-sensitized with synthetic peptide epitopes (CPS or FPT) and then exposed to CPS- or FPT-specific T cells (responder to stimulator ratio of 2:1). These T cells were assessed for IFN-γ expression as described above. MFI indicates mean fluorescence intensity.
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology

8. Ex vivo phenotypic analysis of CPS- and FPT-specific T cells.
Ex vivo phenotypic analysis of CPS- and FPT-specific T cells. Panel A: PBMC from HCMV-infected healthy individual were co-stained with pMHC-pentamers and antibodies specific for CD27, CD57 and CD62L. Data presented here is a summary of 3 different experiments. Panel B: To determine any functional differences between CPS and FPT-specific T cells, these effector cells were exposed to MRC-5 cells expressing HLA B*3508 and co-infected with HCMV at an MOI of 0.5:1. 2 different responder to stimulator ratios (80:1 and 20:1) were used in these assays. Following incubation, these cells were co-stained with pMHC pentamer, anti-IFN-γ and anti-CD107a as outlined in the Material and Methods section. This data are representative of 3 different experiments.
Katherine K. Wynn et al. Blood 2008;111:
©2008 by American Society of Hematology