Analysis of cytokine patterns produced by individual CD4+ lymph node cells during experimental murine leishmaniasis in resistant and susceptible mice

The concept of subdividing CD4+ T cells into Th0, Th1 and Th2 cells is based on the cytokine pattern produced by long-term in vitro cultured T cell lines. However, there exists uncertainty whether this classification can also be applied to CD4+ T cells in vivo. Herein it was investigated whether and at which frequency Th0, Th1 and Th2 cells are induced in vivo during an infection of mice with Leishmania major. Cytokine co-production in single IFN-gamma+ or IL-4+ CD4+ T cells as well as the frequency of such cells were assessed in the lymph nodes (LN) of infected mice. For this purpose, T cells derived from the draining LN were activated by phorbol myristate acetate (PMA)/ionomycin, and the intracellular cytokines IL-2, IL-4, IL-5, IL-10 and IFN-gamma were analyzed by immunofluorescence. One week after infection, a strong, but comparable increase of IFN-gamma+ CD4+ and IL-4+ CD4+ cells (up to 7% of all CD4+ cells) in the LN was observed in resistant C57BL/6 mice and susceptible BALB/c mice. IFN-gamma and IL-4 were not co-produced by single cells ('Th0 cells'). At later stages of the infection, the number of IL-4+ CD4+ cells decreased in C57BL/6, but not in BALB/c mice. All IL-4+ CD4+ cells showed an unexpected phenotype, because at least half of these cells co-produced IL-2, and the majority of the IL-4+ CD4+ did not co-produce the Th2 cytokines IL-5 and IL-10. Similar cytokine profiles were obtained when the CD4+ T cells were stimulated by Leishmania major-antigen instead of PMA/ionomycin. This study demonstrates that 'classical' Th1 cells (IFN-gamma+IL-2+), but no 'classical' Th2 cells (IL-4+IL-5+IL-10+) and no Th0 cells (IFN-gamma+IL-4+) are generated during L. major infection of mice in vivo.