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We discovered by flow cytometry a cell population expressing both T cell (CD3) and monocyte (CD14) surface markers in the live singlet cell population of PBMC from human subjects. Imaging experiments revealed that CD3+CD14+ cells are T cell:monocyte complexes in such strong interaction that flow cytometry acquisition did not break them apart. The T cell:monocyte complexes can be detected in frozen and fresh PBMC samples at similar frequencies. High resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting they form in vivo and represent true biological interactions between immune cells. Moreover, T cell:monocyte complex frequencies were increased after immune perturbations. They were found at high levels at diagnosis in subjects with active tuberculosis and decreased upon treatment, varied as a function of disease severity in acute dengue infection, and increased in a time dependent fashion post tetanus, diphtheria and pertussis (Tdap) vaccination. Intriguingly, the CD4 vs CD8 phenotype of T cells that paired with the monocyte within the complexes varied with the nature of the immune perturbation, and CD4-CD8- T cells showed consistently the highest constant of association with monocytes. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

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