Case Study:
ProImmune REVEAL® and ProVE® used to identify first HTLV-2 MHC class I epitope

Oliveira, AL. et al. (2009). High frequencies of functionally competent circulating Tax-specific CD8+ T cells in human T lymphotropic virus type 2 infection. J Immunol. 183(5): 2957-65. [PubMed ID: 19657093]

HTLV-2 is a retrovirus closely related to HTLV-1 but little is known about the immunological characteristics of the infection. Using ProImmune’s REVEAL® and ProVE® Rapid Epitope Discovery System, Oliveira et al mapped responses against the HTLV-2 Tax2 protein.

Overlapping 9-mer peptides spanning the entire Tax2 protein were synthesized as a Prospector PEPscreen® custom peptide library. Using these peptides, MHC-peptide binding assays were carried out against A*02:01, A*03:01 and B*35:01 alleles, which were commonly expressed in the population group studied. ProVE® Pentamers were synthesized for the peptides deemed positive in the MHC-peptide binding assays and staining of PBMCs from HTLV-2 infected donors clearly identified a single A*02:01 restricted epitope for Tax2 (11-19) (LLYGYPVYV) that was found at high frequencies in almost all asymptomatic carriers. This is the first report of any MHC class I HTLV-2 epitope.

Data showing results from the REVEAL™ MHC-peptide binding assay

Figure 1: Candidate peptides were assembled with A*02:01 and analyzed using the MHC-peptide binding assay to determine their level of incorporation into MHC molecules. Binding signal is shown as a percentage relative to the pass/fail control (yellow bar); peptide 11, LLYGYPVYV (red bar) was further validated by Pentamer staining.

Upon stimulation with Tax2 (11-19) (LLYGYPVYV) peptide further functional analyses of the antigen specific cells were carried out using a panel of CD8+ T cell functional markers. As a control, cells were also stimulated with a ProMix™ CEF-extended pool of peptides containing known CMV, EBV and Influenza epitopes. The majority of the antigen specific cells showed only single responses to the markers used in the panel, with those expressing IFN-gamma or MIP-1 beta being most prevalent. In addition, the cytolytic activity of the cells remained intact, as shown by combining custom Pro5®Pentamer staining with intracellular cytokine staining for granzyme B and perforin.

The services and reagents offered by ProImmune were pivotal to the identification of this first HTLV-2 Tax2 MHC class I epitope. The MHC-peptide binding binding assays allowed potential epitopes to be rapidly identified for a number of alleles of interest, whilst Pentamer staining confirmed and validated the epitope. ProImmune peptides were used for further functional analysis of the antigen-specific cells using intracellular cytokine staining.