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Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Human ␥␦ T cells expressing the V␥9/V␦2 T-cell receptor have been previously found to proliferate in response to certain microorganisms and to expand throughout life, presumably because of extrathymic activation by foreign antigens. In vitro expansion of V␥9/V␦2 cells by mycobacteria has been previously shown to be dependent on accessory cells. In order to gain an insight into the mechanisms involved in the expansion of these cells, we have undertaken to identify the peripheral blood subset of cells on which proliferation of V␥9/V␦2 cells in response to mycobacteria is dependent. Contrary to their role in antigen presentation to ␣␤ T cells, professional antigen-presenting cells, such as monocytes, B cells, and dendritic cells, were unable to provide the cellular support for the expansion of V␥9/V␦2 cells. Selective depletion of T-cell subsets, as well as the use of highly purified T-cell populations, indicated that the only subset of peripheral blood cells that could expand V␥9/V␦2 cells were CD4 ؉ CD45RO ؉ CD7 ؊ ␣␤ T cells. These cells underwent distinct intracellular signaling events after stimulation with the mycobacterial antigen. Expansion of V␥9/V␦2 cells by ␣␤ T cells was dependent on cell-cell contact. This is the first evidence that a small subset of the memory helper T-cell population is exclusively responsible for the peripheral expansion of V␥9/V␦2 cells. These data illustrate a unique aspect of antigen recognition by ␥␦ T cells and provide new means to study their immune defense role.