A high throughput whole blood assay for analysis of multiple antigen-specific T cell responses in human Mycobacterium tuberculosis infection1

Antigen (Ag)-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis (Mtb), yet little information is currently known regarding how the breadth, specificity, phenotype and function of Mtb-specific T cells correlate with Mtb infection outcome in humans. To facilitate evaluation of human Mtb-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay (RSA) 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 Mtb Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50µl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and inter-assay variability, and implemented testing of the assay in diverse cohorts of Mtb-unexposed healthy adults, foreign-born adults with latent Mtb infection (LTBI) residing in the U.S., and TB household contacts with LTBI in a TB-endemic setting in Kenya. The Mtb-specific T cell RSA further enhances the immunological toolkit available for evaluating Mtb-specific T cell responses across different states of Mtb 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 Mtb-specific T cell responses associated with Mtb infection outcomes.