Monitoring of NY-ESO-1 specific CD4 ⁺ T cells using molecularly defined MHC class II/His-tag-peptide tetramers

Abstract

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. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4 + T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/ NY-ESO-1 tetramers, we could demonstrate that in DR52b + cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccineinduced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4 + T cells, include central and transitional memory polyfunctional populations, and do not include CD4 + CD25 + CD127 − regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.S oluble MHC-peptide tetramers, allowing the direct visualization, characterization, and isolation of antigen-specific T cells, have become essential tools for T-cell analysis. MHC class I tetramers incorporating short CTL peptide epitopes, originally developed by Altman and Davis (1) have been generated for a large number of murine and human alleles, incorporating a variety of peptides of microbial, tumor, and self-antigen origin (2). The development of MHC class II tetramers, however, and particularly of those incorporating peptides from tumor and self-antigens, has been far less successful (3-5). One limiting factor is the high polymorphism of the human MHC class II molecules, especially those encoded by the DRB1 locus, the most frequently studied. Another limiting factor is the binding affinity of antigenic peptides derived from tumor and self-antigens, which is generally lower than that of peptides from pathogens.NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group frequently expressed in tumors of different histological types (6), is an important candidate for the development of generic cancer vaccines (7). In a recent vaccination trial using a recombinant ESO protein (rESO) administered with Montanide ISA 51 and CpG ODN 7909, we have observed induction of CD4 + T cell responses in all vaccinated patients (8). By assessing vaccineinduced CD4 + T cells, we have identified an immunodominant epitope , core region ESO 123-137 ) restricted by HLADR52b (DRB3*0202), an allele expressed by half of Caucasians (9). DRB3-, DRB4-, and DRB5-encoded molecules are less polymorphic than those encoded by DRB1, and are therefore attractive candidates for the development of generic MHC class II tetramers.Our initial attempts to const...

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer

“…In contrast, we obtained no significant in vitro priming from EM populations ( Figure 2B + DC, generated from isolated circulating CD14 + monocytes as described, 31 with a recombinant full-length ESO protein, and assessed antigen recognition by the lines by measuring IFN-g secretion. 28 Similar to vaccine-induced ESO-T H , in vitro primed ESO-tetramer + -T H lines efficiently recognized the recombinant ESO protein processed and presented by DC, whereas they failed to recognize DC incubated with a control protein ( Figure 3C). …”

“…28 Ex vivo sorted CD4 + T-cell subpopulations (3-5x10 6 ) were stimulated in vitro with peptide ESO119-143 (2 mM) in the presence of irradiated autologous CD4 -cells (3-5x10 6 ) and were cultured in the presence of rhIL-2 (100 U/mlL, Chiron). Day 12 cultures were incubated with tetramers at a final concentration of 3 g/mL for 1 h at 37°C and then stained with anti-CD4 mAb and analyzed by flow cytometry.…”

“…TCR variable b chain (BV) usage by ESO-specific cells was determined by flow cytometry analysis following staining of Day 12 cultures with ESO-tetramers and anti-BV2 mAb (Immunotech), as previously described. 28 ESO-specific T cells were isolated from peptide-stimulated cultures by tetramerguided flow cytometry cell sorting, and expanded by stimulation with PHA and irradiated allogeneic PBMC in the presence of IL-2, as previously described. 28 The specificity of the obtained polyclonal T H lines was assessed by tetramer staining and flow cytometry analysis.…”

“…[25][26][27] Through a strategy that combines the use of Histidine (His)-tagged peptides with isolation of MHC/peptide monomers by affinity purification, we have recently generated MHC class II tetramers incorporating an ESO immunodominant peptide (ESO-tetramers). 28,29 We have shown that MHC class II/ESO tetramers specifically and avidly bind to ESO-specific CD4 + T cells, allowing their highly sensitive detection and isolation from polyspecific populations. In this study, we have used ESO-tetramers to implement an in vitro priming platform, allowing the generation of ESO-monospecific polyclonal T H lines from CD4 + T cells of non-immune individuals.…”

Viral-specific adoptive immunotherapy after allo-SCT: the role of multimer-based selection strategies

“…44 However, there are still a number of aspects to improve regarding the applicability of these reagents, in particular the strength of the TCR/pMHC II interactions. 45,46 TRANSLATIONAL APPLICABILITY OF THE MULTIMER TECHNOLOGY There are many strategies to select antigen-specific CD8 þ T cells for adoptive transfer; however, multimer-based technology has the potential to select antigen-specific T cells from healthy seropositive donors and transfer them directly to the patient without any further manipulation.…”