A High Throughput Whole Blood Assay for Analysis of Multiple Antigen-Specific T Cell Responses in Human Mycobacterium tuberculosis Infection

• Published in: The Journal of immunology (1950) 2018-04, Vol.200 (8), p.3008-3019
• Main Authors: Whatney, Wendy E, Gandhi, Neel R, Lindestam Arlehamn, Cecilia S, Nizam, Azhar, Wu, Hao, Quezada, Melanie J, Campbell, Angela, Allana, Salim, Kabongo, Mbuyi Madeleine, Khayumbi, Jeremiah, Muchiri, Benson, Ongalo, Joshua, Tonui, Joan, Sasser, Loren E, Fergus, Tawania J, Ouma, Gregory Sadat, Ouma, Samuel Gurrion, Beck, Allison A, Mulligan, Mark J, Oladele, Alawode, Kaushal, Deepak, Cain, Kevin P, Waller, Lance, Blumberg, Henry M, Altman, John D, Ernst, Joel D, Rengarajan, Jyothi, Day, Cheryl L
• Format: Article
• Language: English
• Subjects: Antigens; Assaying; Blood; CD4 antigen; CD8 antigen; Cross-Sectional Studies; Dilution; Evaluation; Hematologic Tests - methods; High-Throughput Screening Assays - methods; Human behavior; Humans; Immune response; Immunologic Techniques - methods; Immunology; Infections; Longitudinal Studies; Lymphocytes; Lymphocytes T; Mycobacterium tuberculosis; Optimization; Phenotypes; Reproducibility of Results; Standardization; T cell receptors; T-Lymphocytes - immunology; Tuberculosis; Tuberculosis - blood; Tuberculosis - immunology; Vaccines; γ-Interferon
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.1701737

Antigen-specific CD4 and CD8 T cells are important components of the immune response to , yet little information is currently known regarding how the breadth, specificity, phenotype, and function of -specific T cells correlate with infection outcome in humans. To facilitate evaluation of human -specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of -unexposed healthy adults, foreign-born adults with latent infection residing in the United States, and tuberculosis household contacts with latent infection in a tuberculosis-endemic setting in Kenya. The -specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating -specific T cell responses across different states of infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of -specific T cell responses associated with infection outcomes.