MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. self-antigens, which is usually generally lower than that of peptides from pathogens. NY-ESO-1 (ESO), a tumor-specific antigen of the malignancy/testis group frequently expressed in tumors of different histological types (6), is usually an important candidate for the development of generic malignancy vaccines (7). In a recent vaccination trial using a recombinant ESO protein (rESO) given with Montanide ISA 51 and CpG ODN 7909, we have observed induction of CD4+ T cell responses in all MC1568 vaccinated patients (8). By assessing vaccine-induced CD4+ T cells, we have recognized an immunodominant epitope (ESO119C143, core region ESO123C137) restricted by HLA-DR52b (and and and and and and gene encoding the prevalent -chain of the DR isotype. At variance with the -chain of the mouse I-E molecule (homolog to HLA-DR) that is usually encoded by a single gene, in humans, several additional genes encode other -chains, namely (DR52), (DR53), and (DR51). Whereas is usually present in all individuals, are only present in some of them, and are in strong linkage disequilibrium with defined alleles. These alternate DR MC1568 molecules are generally expressed at lower levels when compared with those encoded by alleles, is usually expressed by half of Caucasians. In recent years, an increasing number of studies have concentrated on alternate DR molecules, describing their structure and binding characteristics and peptide binding motifs have been defined for several of them (13, 26). Because of the failure of our initial attempts to generate efficient DR52b/ESO tetramers by peptide loading of DR52b molecules incorporating leucine zipper motifs, as previously explained MC1568 by Kwok et al. (10, 21), we designed a strategy that uses His-tagged peptides, enabling the isolation of folded away class II/peptide monomers by affinity purification before tetramer formation. Together, the data reported in this study clearly show that molecularly defined DR52b/ESO tetramers are reliable reagents for the detection, characterization, and isolation of ESO-specific CD4+ T cells. We obtained efficient staining of clonal and polyclonal ESO-specific DR52b-restricted CD4+ T-cell populations using concentrations of molecularly defined class II tetramers comparable to those generally used for class I/peptide tetramers (1C10 g/mL) (11, 12). Consistent with other reports (27, 28), and at variance with most class I/peptide tetramers that efficiently stain specific CD8+ T cells at 4 C or at 23 C, efficient staining with class II tetramers was optimally achieved upon incubation at 37 C. The molecular basis for this difference, which might be in relation with a lower functional avidity of CD4+ Rabbit polyclonal to ANKDD1A T cells or with a higher need for TCR clustering, remains to be fully elucidated. Importantly, we obtained efficient staining not only using tetramers incorporating the previously defined minimal peptide required for optimal T-cell acknowledgement (15 amino acids long, ESO123C137), but also using a His-tagged 25 amino acid-long peptide, ESO119C143, extended at both the N- and C-terminal ends of the core region. This obtaining indicates that there are no major limitations in the length of peptides that can be incorporated into DR52b molecules and implies that the use of molecularly defined DR52b tetramers incorporating long peptides from defined protein regions, possibly preselected on the basis of the presence of binding motifs or through functional binding assays, may be an efficient strategy, allowing the quick recognition of immunodominant DR52b epitopes from a large number of antigens. These findings are also compatible with the fact that, in contrast to the rigid length requirement of class I-bound peptides (8C10 mers) that need to perfectly fit a groove that is usually closed at both ends, often by adopting a kinked conformation, the class II binding groove, open at both ends, can very easily accommodate long peptides (15C25 mers) that hole MC1568 in an extended form (29, 30). It is usually noteworthy that, although we failed to detect a major effect of the His-tag MC1568 on the binding of ESO.