Development of a 96-well murine whole blood intracellular cytokine staining (ICS) assay for preclinical tuberculosis immune monitoring.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), remains the leading cause of death from a single infectious agent. The only approved vaccine for TB, Bacille Calmette-Guerin (BCG), provides variable and incomplete protection from disease, underscoring the need to improve vaccine development. Preclinical murine models are widely used to triage TB vaccine candidates, but immunological endpoints in mice frequently rely on splenocytes or infected tissues, whereas human clinical studies commonly evaluate peripheral blood mononuclear cells or whole blood. This difference in sample type creates a methodological gap that can limit cross-species comparison of antigen-specific T cell responses. Here, we describe the development of a 96-well, small-volume murine whole blood intracellular cytokine staining assay designed to better align preclinical immune monitoring with blood-based clinical endpoints. Iterative optimization identified blood handling and red blood cell processing as key determinants of assay performance. Heparinized collection combined with red blood cell lysis before culture enabled detection of mitogen-induced cytokine responses in murine blood, while stimulation with a broad M. tb epitope pool enabled detection of low-frequency infection-driven antigen-specific CD4+ and CD8+ T cell responses. In contrast, low-frequency ID93-specific recall responses following subunit vaccination were readily detected in splenocytes but remained difficult to detect reproducibly in blood. These results define the utility and sensitivity limits of murine whole blood ICS for TB vaccine studies and provide a practical workflow for infection-driven immune monitoring that more closely parallels clinical assay formats.