An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

To stimulate lymphocytes via Haemophilus influenzae — a pathogenic bacterium, take a suspension of isolated peripheral blood mononuclear cells, or PBMCs, including lymphocytes and antigen-presenting cells or APCs.

Add a bacterial suspension and incubate at physiological temperature.

Pattern recognition receptors on the APCs bind to specific components of the bacterial cells, triggering phagocytosis. The phagosome — containing internalized bacteria — fuses with the lysosome, causing bacterial degradation and processing into peptide fragments.

Major histocompatibility complex or MHC molecules bind to the peptide fragments and present them on the cell surface as antigens. T lymphocytes bind to the presented antigen via the T cell receptor.

Add co-stimulatory antibodies that bind to specific surface receptors on T lymphocytes. The synergistic binding induces downstream signaling, activating T lymphocyte cytokine production.

Add a Golgi-blocking compound — blocking vesicular protein transport from the endoplasmic reticulum to the Golgi — to inhibit cytokine secretion from T lymphocytes, facilitating intracellular cytokine accumulation.

Add a fixative to preserve cellular morphology and detergent to permeabilize the cell membrane. Add a cocktail of fluorescently labeled antibodies, including antibodies binding to the T lymphocyte-specific surface markers — labeling the cell surface — and cytokine-specific antibodies that penetrate inside the cell to bind intracellular cytokines.

Perform flow cytometry to detect cells with dual fluorescence — indicating cytokine production by stimulated T lymphocytes.