Streptococcus pneumoniae [ATCC 6303, serotype 3; 5 × 104 CFU per animal in 20 μl of Dulbecco’s PBS) was instilled intratracheally as before (22, 25). Blood was obtained for quantitative culture 24 hours after inoculation. Mice were monitored at least twice daily using a “humane end points” scoring system that included daily weight and temperature monitoring and observation of animals’ grooming, activity, behavior, and respirations. Animals that lost >20% of starting weight or that scored >5 on the clinical observation score were euthanized.

Tissues were harvested for either flow cytometry or for histology. Histological specimens were preserved in formalin (10%) and then embedded in paraffin. Sections were prepared and stained with hematoxylin and eosin by the Division of Comparative Medicine core facility at WUSM. Histological sections were reviewed by an independent veterinary pathologist.

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