Detection of Low-Avidity CD4+ T Cells Using Recombinant Artificial APC: Following the Antiovalbumin Immune Response

Abstract

Subtle differences oppose CD4+ to CD8+ T cell physiologies that lead to different arrays of effector functions. Interestingly, this dichotomy has also unexpected practical consequences such as the inefficacy of many MHC class II tetramers in detecting specific CD4+ T cells. As a mean to study the CD4+ anti-OVA response in H-2d and H-2b genetic backgrounds, we developed I-Ad- and I-Ab-OVA recombinant MHC monomers and tetramers. We were able to show that in this particular system, despite normal biological activity, MHC class II tetramers failed to stain specific T cells. This failure was shown to be associated with a lack of cooperation between binding sites within the tetramer as measured by surface plasmon resonance. This limited cooperativeness translated into a low “functional avidity” and very transient binding of the tetramers to T cells. To overcome this biophysical barrier, recombinant artificial APC that display MHC molecules in a lipid bilayer were developed. The plasticity and size of the MHC-bearing fluorescent liposomes allowed binding to Ag-specific T cells and the detection of low numbers of anti-OVA T cells following immunization. The same liposomes were able, at 37°C, to induce the full reorganization of the T cell signaling molecules and the formation of an immunological synapse. Artificial APC will allow T cell detection and the dissection of the molecular events of T cell activation and will help us understand the fundamental differences between CD4+ and CD8+ T cells.

Clustering of T Cell Ligands on Artificial APC Membranes Influences T Cell Activation and Protein Kinase C θ Translocation to the T Cell Plasma Membrane

“…Such aAPC were very efficient in identifying class II-restricted Ag-specific T cells but their capacity to effectively stimulate T cells was limited to some highaffinity T cell hybridomas which do not require costimulation. As also suggested by elegant work from others (18), using similar technology, suboptimal stimulatory capacity was presumably due to the combination of several factors, including inappropriate orientation of MHC molecules, lack of organization, and insufficient local concentration of T cell ligands in the area of contact between the aAPC and T cell. Taken together, these data show that the efficiency of these artificial systems in inducing T cell activation may be influenced by the relative density of MHC-peptide complexes available at the point of interaction.…”

Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function

“…However, both flTCR-and scTCR-transduced primary T cells were stained poorly with the dimer (data not shown). In the future, we plan to try an improved tetramer technology 36 to identify the differential affinity between flTCR and scTCR. Holler et al 37 demonstrated that the low affinity of scTCR molecules could be dramatically increased by an in vitro selection system through site-directed mutagenesis.…”

Expansion of Functional Endogenous Antigen-Specific CD4+CD25+ Regulatory T Cells from Nonobese Diabetic Mice

“…2B, in general, 90% of the p31-I-A g7 -expanded T reg population failed to stain for the p31-I-A g7 multimer when analyzed by flow cytometry. It was possible that the unstained population was comprised of p31-I-A g7 -reactive cells with TCRs with too low of an affinity to be detected by flow cytometry (14). Alternatively, this population could be comprised of cells that were expanding in an Ag-nonspecific manner.…”

“…The 1040-31 peptide, specific for BDC2.5 TCR Tg ϩ T cells, consisted of amino acids YVRPLWVRME (9). The HEL [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] protein consisted of amino acids AMKRHGLDNYRGYSL. GAD 286 -300 was a gift from E. Sercarz (Torrey Pines Institute for Molecular Studies, San Diego, CA).…”