Date Published: December 22, 2003
Publisher: Public Library of Science
Author(s): Yoav Soen, Daniel S Chen, Daniel L Kraft, Mark M Davis, Patrick O Brown
Abstract: The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease. We have developed and tested a method that uses arrays of peptide–MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells. CD4+ or CD8+ lymphocytes can be captured in accordance with their ligand specificity using an array of peptide–MHC complexes printed on a film-coated glass surface. We have characterized the specificity and sensitivity of a peptide–MHC array using labeled lymphocytes from T cell receptor transgenic mice. In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse. This approach should be useful for epitope discovery, as well as for characterization and analysis of multiple epitope-specific T cell populations during immune responses associated with viral and bacterial infection, cancer, autoimmunity, and vaccination.
Partial Text: Antigen-specific cellular immune responses are mediated by a diverse population of T cells, each capable of recognizing a specific peptide bound to a particular major histocompatibility complex (MHC) molecule on the surface of host cells. Recognition of peptides bound to class I or class II MHC molecules leads to the clonal expansion, activation, and maturation of T lymphocytes, resulting in effector populations of either cytotoxic (CD8+ CTL) or helper (CD4+) T cells, respectively. The presence of antigen-specific effector cells is diagnostic of an immune response specific to that antigen; detection of these antigen-specific cells is therefore critical for the characterization of the response and for understanding its natural course. In addition, a systematic survey of the global repertoire of T cell specificities, its dynamics over time and between individuals, could be of great value in elucidating the inner workings of the immune system and for designing efficient strategies for immunization, immunotherapy, and treatment of autoimmune disease.
Cellular immune responses to pathogens, allergens, deregulated or mutated proteins, and self-antigens play critical roles in health and disease. The ability of T cells to respond to the immense diversity of possible targets relies on the corresponding diversity of the repertoire of TCRs that can be generated by the immune system. The T cell population in each individual is diverse and dynamic. Even after exposure to a potent T cell antigen, an individual TCR clone seldom accounts for more than 5% of the total population of T cells in a normal human or mouse. Moreover, the specificities and phenotypes of the individual’s T cell repertoire may provide a rich picture of the immunological history, the physiological status, and perhaps the disease susceptibilities of that individual. A broad picture of the dynamic responses of the T cell repertoire to an immunological challenge should illuminate our understanding of the immune response and may point to individual-specific response patterns that can help guide design of immunological therapies.
To evaluate the effect of tetramer dilution on cell capture, we printed an OVA/kb tetramer dilution series and probed it with OT-1 cells (Figure S1A). The cells were suspended at 2.5 ×106 cell/ml (105 cells in 40 μl), which was below the concentration required for confluent coverage of the spot area. The cells were then incubated with the array (30 min at room temperature) and imaged following washout of unbound cells. In this specific example, the number of cells captured increased linearly with the amount of tetramer deposited (Figure S1B), with a binding threshold of approximately 0.05 ng/spot. The lack of plateau at high tetramer concentrations, together with the denser cell coverage on the anti-CD3 spots, suggests that a still further increase in tetramer concentration (without changing the suspension density) would result in higher numbers of bound cells. Indeed, a 4-fold increase in the amount of tetramer deposited leads to a significant increase in binding efficiency, which becomes comparable to binding via a mAb (data not shown). The lack of cell binding between spots or to noncognate peptide–MHC spots indicates that binding of as low as a few cells is attributable to peptide–MHC–TCR recognition. Note, however, that the linear shape of the binding curve (Figure S1B) should be considered as a special case and not as a general rule. Likewise, the minimal concentration for binding depends on the availability and functionality of the immobilized peptide–MHC, the peptide–MHC–TCR affinity, the type, density, and heterogeneity of the cells, the incubation temperature and duration, and the stringency of washing.