Peptide library-based evaluation of T-cell receptor breadth detects defects in global and regulatory activation in human immunologic diseases.

Barber JS1, Yokomizo LK, Sheikh V, Freeman AF, Garabedian E, van Dijk E, Sokolic R, Candotti F, Weng NP, Sereti I, Milner JD.
Source: Proc Natl Acad Sci USA
Publication Date: (2013)
Issue: 110(20): 8164-9
Research Area:
Immunotherapy / Hematology
Basic Research
Culture Media:

Coculture Experiment:

CD14+ monocytes were purified from PBMC by positive selection with antibody-coated microbeads (Miltenyi Biotec) and cultured in R-10 with IL-4 (50 ng/mL, R&D Systems) and GM-CSF (100 ng/mL, PeproTech). After 5 d, TNF-a (10 ng/mL, R&D Systems) was added to activate DCs.

On day 7, naive (aqua-CD3-CD4-CD25-CD27-CD45RO-) or central memory (aqua- CD3-CD4-CD25-CD27-CD45RO-) cells were purified by FACS sorting and cocultured at a ratio of 50:1 with autologous DCs in X-Vivo 15 serum-free media (Lonza). Cells were then cultured for 7 d before restimulation with phorbol-12- myristate-13-acetate (20 ng/mL, Calbiochem) and ionomycin (1 µM, Calbiochem) in the presence of Brefeldin A (10 µg/mL, Sigma) for 6–12 h. Intracellular cytokine staining was then performed.


The ability of T-cells to respond to foreign antigens and to appropriately regulate this response is crucial for maintaining immune homeostasis. Using combinatorial peptide libraries, we functionally measured broad T-cell reactivity and observed impaired reactivity in established models of T-cell receptor repertoire restriction and in previously unrecognized disease contexts. By concurrently analyzing T-regulatory and T-effector cells, we show strong functional correlation between these subsets in healthy individuals and, strikingly, that alterations of this balance are associated with T helper type 2 (Th2)-mediated disease in a lymphopenic setting. Finally, we demonstrate that peptide-based priming of polyclonal naive cells with relatively low concentrations skews toward Th2 differentiation. These findings provide unique insight into the pathophysiology and functional consequences of abnormal T-cell repertoires and into differentiation of human naive T-cells.