The preferred formation of a CDR3 motif that constrains TCR selection by requiring conservation of the V-D recombination site would seem to limit the possibilities for convergent recombination, suggesting that selection pressure may be imposed by conformational requirements of the TCR-peptide-MHC interface
The preferred formation of a CDR3 motif that constrains TCR selection by requiring conservation of the V-D recombination site would seem to limit the possibilities for convergent recombination, suggesting that selection pressure may be imposed by conformational requirements of the TCR-peptide-MHC interface. are lost in hybrids. A significant frequency of TCR CDR3 sequences in the DbM187C195 response have a distinct (D/E)WG motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but DbM187C195 showed a distinctive structure formed by Lys7. The data suggest that common recombination events in prevalent V genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines. peptide stimulation with mutant peptides. Lymphocytes were isolated as described previously (21) and stimulated with each of the mutant peptide at 2 g/ml along with the co-stimulatory antibodies (1 g/ml) to CD28 and CD49d for 5 h at 37 C. Cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate and 1 m ionomycin as a positive control. After the incubation, cells were fixed and permeabilized according to the manufacturer’s instructions (BD Biosciences-Pharmingen) and stained with fluorochrome-conjugated antibodies against CD3, CD8, IFN-, and TNF- (BD Biosciences-Pharmingen) for 20 min at 4 C. For tetramer analysis, cells were stained with KdM282C90 and DbM187C195 tetramers together with antibodies against CD3 and CD8. Tetramers that recognized the influenza NP366C374 were used as a negative control. Cells were washed after staining and analyzed Rabbit Polyclonal to Histone H2A by flow cytometry on a LSR-II system (BD Biosciences). Flow data were analyzed by FlowJo (version 6.3; Tree Star, San Carlos, CA). Using fluorochrome-conjugated antibodies against CD3, CD8, and IFN-, the effect of each amino acid mutation was expressed as a percentage reduction of the wild-type IFN- response. Bulk Clonotyping Sorted cells were lysed, mRNA was extracted (Oligotex kit, Qiagen), and non-nested, template switch-anchored RT-PCR was performed using a 3-TCR constant region primer (5-TGG CTC AAA CAA GGA GAC CT-3). Amplified products were ligated into pGEM-T Easy vector (Promega) and cloned by transformation of competent DH5 was generated with PyMOL 1.3 (PyMOL Molecular Graphics System, version 1.3, Schr?dinger, LLC.). Open in a separate window FIGURE 8. Modeling of M and M2 epitopes. (Asn5 and Ile9 in DbM187C195 and Tyr2 and Ile9 in KdM282C90). by a panel of mutant peptides along with wild- type peptides of KdM282C90 and DbM187C195. IFN- response was determined by intracellular cytokine staining. RESULTS Epitope-specific V Expression of CD8+ T Cells after Primary RSV Infection in Parent and Hybrid Strains of Mice The pre-existing TCR V repertoire of naive BALB/c, C57BL/6, and CB6F1/J mice was screened for TCR usage by staining naive murine splenocytes with a panel of V antibodies conjugated to FITC. We found that in all strains, TCR V13.2/13.3 (these two subtypes PF-06855800 are serologically indistinguishable) was present in the greatest frequency (Fig. 1). Next, we determined the pattern of TCR V utilization by RSV-specific CD8+ T cells during primary infection using the antibody panel to label tetramer-positive cells. Open in a separate window FIGURE 1. V profile of naive mice. Naive murine splenocytes isolated from BALB/c, C57BL/6, and CB6F1/J mice were stained with a panel of 15 PF-06855800 FITC-labeled anti-V mouse monoclonal antibodies. The percentage of each V subtype within the population of CD8+ CD3+ cells is shown. V 13.2/13.3 was the dominant response to KdM282C90 recognized by BALB/c parents and CB6F1/J hybrid mice. There were rare V29 responses in BALB/c and V13.1 responses in CB6F1/J hybrids. In C57BL/6 parents and F1 hybrids, the DbM187C195-specific CD8 T cells PF-06855800 were primarily V17 followed by V13.2/13.3, with rare responses detected to V 13.1, 14, and 19 in hybrid mice. Other responses were difficult to distinguish from background (Fig. 2 and supplemental Fig. 1). Open in a separate window FIGURE 2. V expression profile of KdM282C90- and DbM187C195-specific CD8+ cells following RSV infection. BALB/c, C57BL/6, and CB6F1/J mice were infected with RSV and sacrificed at day 7 post-infection. The KdM282C90 and DbM187C195 tetramer-stained lung lymphocytes were screened with a panel of FITC-labeled anti-V antibodies. The frequency of each V subtype within the population of KdM282C90- and DbM187C195-specific CD8 T cells is shown in BALB/c and C57BL/6 parent mice and CB6F1/J hybrid mice. V Repertoire of CD8+ T Cells after Primary RSV Infection in Parent and Hybrid Strains of Mice Next, we took two approaches to determining the molecular clonotype of the TCR repertoire responding to these two epitopes. First, a bulk clonotyping approach was used in which the tetramer-labeled cells were sorted and anchored RT-PCR was done to generate V amplicons. V fragments were cloned into pGEM-T Easy vector and transformed in and and 0.05) between the two responses based on a two-tailed test. To determine whether the CDR3 sequences were characterized by a particular.