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These data suggest that differences in immunodominance patterns might explain the incomplete …


Biology Articles » Immunobiology » Of mice and humans: how good are HLA transgenic mice as a model of human immune responses? » Methods

Methods
- Of mice and humans: how good are HLA transgenic mice as a model of human immune responses?

Peptides

Peptides were synthesized by A and A Labs and purified to >95% homogeneity by reverse-phase high-performance liquid chromatography (HPLC). Purity was determined using analytical reverse-phase HPLC and amino acid analysis, sequencing, and/or mass spectrometry. Peptides were radio-labeled with the chloramine T method as described [31].

Characteristics of study population

Healthy males and females between 18 and 59 years of age were used in this study. Exclusion criteria were body weight of < 45.4 kg and established pregnancy. Recruited donors were vaccinated by arm scarification with a VACV (Dryvax) vaccination as a prophylactic measure either because of their potential exposure to VACV in a laboratory or hospital setting or because of their enrollment into military and health worker vaccination program. The blood draw was performed within 1 year of the Dryvax vaccination. All experiments carried out with human subjects were in compliance with the Helsinki Declaration. Institutional Review Board approval (FWA# 00000032) and appropriate informed consent were obtained for this study.

PBMC isolation and HLA typing

PBMC were isolated from heparinized blood by gradient centrifugation with a Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) [32], suspended in fetal bovine serum containing 10% dimethyl sulfoxide, and cryo-preserved in liquid nitrogen. Donor's PBMC were typed for HLA-A by high-resolution polymerase chain reaction (Forensic Analytical Molecular Genetics, San Francisco, CA).

Mice

A2.1 Tg mice used in this study were the F1 generation derived from crossing homozygous Tg mice (H-2b haplotype) expressing a chimeric gene (A2.1/Kb) consisting of the α1 and α2 domains of HLA and the α3 domain of H-2Kb with BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) [4,33,34]. The HHD A2.1 strain [19] was kindly provided by Dr. Jack Bennink (NIAID, NIH, Bethesda, MD). All mice were bred and maintained following NIH guidelines and Institutional Animal Care and Use Committee-approved animal protocols (AAALAC# 000840 and OLAW# A3779-01).

Viruses and infection

The Western Reserve strain of VACV was obtained from Dr. Bernard Moss (NIAID). Mice were infected i.p. with 2 × 106 PFU of VACV. On day 7 post-infection, the mice were sacrificed and splenic CD8+ T cells were used in mouse IFN-γ ELISPOT assays.

Ex vivo IFN-γ ELISPOT

IFN-γ ELISPOT assays were performed as described [5,9]. In brief, for murine assays, 2 × 105 splenic CD8+ T cells were cultured with 105 human Jurkat cells (expressing the same A2.1/Kb construct as in the Tg mice) pulsed with 10 μg/ml of peptide. For human assays, 2 × 105 PBMC were incubated with 5 μg/ml of peptide. After a 20 h incubation at 37°C, plates were developed, and responses calculated as described [5,9,19]. Criteria for positivity were net spot-forming cells (SFC)/106 cells ≥ 20, stimulation index ≥ 2.0, and p-value ≤ 0.05 using a Student's t test in at least 2 out of 3 experiments.


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