Unexpected Modulation of Recall B and T Cell Responses after Immunization with Rotavirus-like Particles in the Presence of LT-R192G

Abstract: LT-R192G, a mutant of the thermolabile enterotoxin of E. coli, is a potent adjuvant of immunization. Immune responses are generally analyzed at the end of protocols including at least 2 administrations, but rarely after a prime. To investigate this point, we compared B and T cell responses in mice after one and two intrarectal immunizations with 2/6 rotavirus-like particles (2/6-VLP) and LT-R192G. After a boost, we found, an unexpected lower B cell expansion measured by flow cytometry, despite a secondary antibody response. We then analyzed CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and CD4⁺CD25⁺Foxp3⁻ helper T cells after in vitro (re)stimulation of mesenteric lymph node cells with the antigen (2/6-VLP), the adjuvant (LT-R192G) or both. 2/6-VLP did not activate CD4⁺CD25⁺Foxp3⁻ nor Foxp3⁺ T cells from non-immunized and 2/6-VLP immunized mice, whereas they did activate both subsets from mice immunized with 2/6-VLP in the presence of adjuvant. LT-R192G dramatically decreased CD4⁺CD25⁺Foxp3⁺ T cells from non-immunized and 2/6-VLP immunized mice but not from mice immunized with 2/6-VLP and adjuvant. Moreover, in this case, LT-R192G increased Foxp3 expression on CD4⁺CD25⁺Foxp3⁺ cells, suggesting specific Treg activation during the recall. Finally, when both 2/6-VLP and LT-R192G were used for restimulation, LT-R192G clearly suppressed both 2/6-VLP-specific CD4⁺CD25⁺Foxp3⁻ and Foxp3⁺ T cells. All together, these results suggest that LT-R192G exerts different effects on CD4⁺CD25⁺Foxp3⁺ T cells, depending on a first or a second contact. The unexpected immunomodulation observed during the recall should be considered in designing vaccination protocols.