Combinatorial HLA-peptide bead libraries for high throughput identification of CD8⁺ T cell specificity.

Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to CD8-null HLA-A*0201 on beads, we probed HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. We observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells.

Linking genotype to phenotype on beads: high throughput selection of peptides with biological function.

Although peptides are well recognised biological molecules in vivo, their selection from libraries is challenging because of relative low affinity whilst in linear conformation. We hypothesized that multiplexed peptides and DNA on the surface of beads would provide a platform for enhanced avidity and the selection of relevant peptides from a library (ORBIT bead display). Using human immunodeficiency virus (HIV-1) gp120 as a target, we identify peptides that inhibit HIV-1 replication in vitro through blocking of protein:protein interaction with the co-receptor CCR5. The bead display approach has many potential applications for probing biological systems and for drug lead development.

Cellular and cytokine correlates of severe dengue infection.

BACKGROUND: The occurrence of dengue haemorrhagic fever (DHF) is thought to result from a complex interplay between the virus, host genetics and host immune factors. Existing published data are not consistent, in part related to relatively small sample sizes. We set out to determine possible associations between dengue virus (DEN-V) NS3 specific T cells and cytokine and chemokine levels and the pathogenesis of severe disease in a large cohort of individuals with DHF. METHODOLOGY/PRINCIPAL FINDINGS: By using ex vivo IFNγ ELISpot assays we determined DENV-NS3 specific responses in patients with varying severity of DHF. Other cytokines produced by DENV-NS3 specific T cells were determined by using multiple bead array analysis (MBAA). We also determined the serum cytokine levels using MBAA, lymphocyte subsets and Annexin V expression of lymphocytes in patients with varying severity of DHF. Of the 112 DHF patients studied, 29 developed shock. Serum IL-10 and IP-10 levels positively and significantly correlated with T cell apoptosis while IL-10 levels inversely correlated with T cell numbers. In contrast, TGFß showed a very significant (P<0.0001) and positive correlation (Spearman's R = 0.65) with the platelet counts, consistent with platelet release. We found that whilst patients with severe dengue had lower total T cell numbers, the DV-NS3 specific T cells persisted and produced high levels of IFNγ but not TNFα, IL-3, IL-13, IL-2, IL-10 or IL-17. CONCLUSIONS/SIGNIFICANCE: Our data suggest that serum IL-10, TNFα and TGFß differentially associate with dengue disease severity.