Objective: The balance between mucosal CD4+ T cells producing IFN-γ or IL-10 is essential in the maintenance of intestinal homeostasis. We aimed to investigate how in situ activated T cells were expanded in vitro according to a new cell culture protocol and if it selected for continuous clonal CD4+ T cells capable of producing mainly IFN-γ or IL-10. Methods: T cell cultures were established from colonic biopsy specimens of 27 patients with Crohn’s disease and from 10 healthy controls in a medium supplemented with IL-2 and IL-4 but without addition of exogenous antigen or mitogen. Cytokine production was measured after stimulation (IL-12, super antigen) and inhibition (ciclosporin and methylprednisolone). Results: Cytokine production was increased in cultures from patients with Crohn’s disease compared to controls (IFN-γ, p = 0.005; IL-10, p = 0.003; TNF-α, p = 0.03). Early cultures were highly responsive to IL-12 stimulation. We established CD4+ T-cell clones escaping cellular senescence with an inflammatory or regulatory cytokine profile. Discussion: The data indicate that cultures of in situ activated inflammatory and regulatory subpopulations of intestinal T lymphocytes with pathogenic importance in Crohn’s disease can be established preserving their functional properties during growth.

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