Use of MHC class II tetramers to investigate CD4⁺ T cell responses: Problems and solutions

Abstract

MHC-class I tetramers technology enabled the characterization of peptide-specific T cells at the single cell level in a variety of studies. Several laboratories have also developed MHC-class II multimers to characterize Ag-specific CD4 1 T cells. However, the generation and use of MHC-class II multimers seems more problematic than that of MHC-I multimers. We have generated HLA-DR*1101 tetramers in a versatile empty form, which can be loaded after purification with peptides of interest. We discuss the impact of critical biological and structural parameters for the optimal staining of Agspecific CD41 T cells using HLA-DR*1101 tetramers, such as: (i) activation state of CD41 T cells; (ii) membrane trafficking in the target CD41 T cells; (iii) binding characteristics of the loaded CD4 epitope. Our data indicate that reorganization of TCR on the plasma membrane upon CD41 T cell activation, as well as an homogenous binding frame of the CD4 epitopes to the soluble HLA-DR monomer, are critical for a stable TCR/MHC-class II tetramer interaction. These factors, together with the low frequencies and affinities of specific CD41 T cells, explain the need for in vitro expansion or ex vivo enrichment of specific T cells for the optimal visualization with MHC-class II tetramers. ' International Society for Advancement of Cytometry Key terms CD4+ T cells; tetramers; HLA-DR1101THE MHC tetramer technology was developed to investigate the dynamic of T cell response at the single cell level. Until the advent of these reagents (1), identification of antigen-specific T cells was only possible using either limiting dilution analysis or by tracking the TCR V repertoire at the molecular level. However, none of these techniques allow the direct phenotypic and functional characterization of the antigenspecific T cells. In this scenario, MHC class I tetramers appeared immediately powerful, enabling the direct visualization of CD8 1 T cells and clarifying relevant aspects of antigen-specific response in viral infections and cancer (2-7) (Chattopadhyay et al., Cytometry, in press [this issue]). By contrast, the use of MHC class II tetramers turned out to be more problematic, both for the possibility to obtain stable peptide-MHC class II complexes and the ability to detect specific CD4 1 T cells directly ex vivo.We will try to highlight the advantages and potentials, as well as the limitations and problems that are still under solution, of MHC class II tetramer technology.

The CD4+ T‐cell epitope‐binding register is a critical parameter when generating functional HLA‐DR tetramers with promiscuous peptides

“…3A, two different p57/58-specific T-cell clones, but not an irrelevant T-cell line, were stained by HLA-DR Ã 1101 tetramers loaded with p57/58C3AL peptide. Consistent with published data [8][9][10], the tetramers did not stain the two T-cell clones with the same intensity, in agreement with the different TCR avidity exhibited by the two T-cell clones for the p57/58 peptide (data not shown); 6.22, the T-cell clone with a higher avidity of recognition was stained more brightly by the HLA-DR Ã 1101 loaded with the p57/58C3AL peptide than clone 2E5.53.…”

“…3A, two different p57/58-specific T-cell clones, but not an irrelevant T-cell line, were stained by HLA-DR Ã 1101 tetramers loaded with p57/58C3AL peptide. Consistent with published data [8][9][10], the tetramers did not stain the two T-cell clones with the same intensity, in agreement with the different TCR avidity exhibited by the two T-cell clones for the p57/58 peptide (data not shown); 6.22, the T-cell clone with a higher avidity of recognition was stained more brightly by the HLA-DR Ã 1101 loaded with the p57/58C3AL peptide than clone 2E5.53.HLA-DR*1101 tetramers loaded with anchor-tailored p58 peptide stain MAGE-A3-specific CD4 1 T-cell clonesIn the p58 peptide, we maintained the single putative anchor residue Met 290 and replaced the two additional putative Val 286 and Leu 287 anchor residues with two Ala [39]. The resulting p58AA analogue was tested for its ability to form functional HLA-DR Ã 1101 tetramers.…”

“…Two problems at the bases of this difficulty can be identified in the low frequency and low affinity of CD4 1 T cells [7,8]. Furthermore, the activation state of CD4 1 T cells determines the staining ability of MHC class II tetramers [12,15] unlike CD8 1 T-cell staining by MHC class I tetramers [43], making more difficult the detection ex vivo of quiescent CD4 1 T cells.…”

“…It is estimated, however, that only up to 50% of empty recombinant MHC class II molecules can be loaded with an exogenous synthetic peptide [8]. This clearly impacts the overall avidity, and therefore staining ability, of the tetramers.…”

“…The identification of primary antigen-specific CD4 1 T cells by peptide-pulsed MHC class II tetramers seems, however, less straightforward than that of CD8 1 T cells by peptide-loaded MHC class I tetramers [7,8]. Several biological and structural characteristics of the CD4 1 T-cell response that differ from those displayed by the CD8 1 one seem to affect the capacity of MHC class II tetramers to optimally stain specific CD4 1 T cells ex vivo: (i) the lower frequency of peptide-specific CD4 1 T cells, (ii) their apparent lower affinity for the peptide-MHC class II complexes [9][10][11][12][13], and (iii) the requirement for an activation-induced TCR reorganization to bind with sufficient avidity cognate peptide-MHC class II tetramers [12,14,15].…”