In vivo analysis of CD4⁺ T cell responses to oral immunization

An adoptive transfer model was used to study antigen-induced cytokinc expression, surface phenotype modulation, and anatomic localization of a clonal population of naive CD4 T cells in vivo following oral delivery of antigen. Ovalbumin (OVA)-specific CD4+ T cells isolated from DO 10 aβ TCRtransgenic mice were adoptively transferred into syngeneic (BALB/c) mice and OVA was delivered by gastric gavage in a multiple emulsion vehicle two days later. The clonotype-positive population of CD4 T cells in Peyer's patch (PP), mesenteric lymph node (MLN), and spleen (SP) were analyzed over a time course. Cytokine expression by transgenic lymphocytes in tissue cryosections was determined using an enzymatic in situ hybridization technique. PP cytokine responses were limited to infrequent IL-2 and IL-10 positive cells, and seen only at the earliest time points. MLN CD4+ T cells showed higher frequency expression of IL-2 and IL-10 and infrequent IFN-g at early time points. Cytokine responses in the spleen were delayed, and showed increased IFN-g relative to PP and MLN, but included significant numbers of IL-10 positive cells. This phenotype is distinct from delivery of OVA in the same vehicle I.P., where IL-2 and IFN-g were dominant, and IL-10 was rarely detected. Triple-color flow cytometric analysis of the transgenic T cell population showed increased expression of CD25, CD69 and CD40L compared to the endogenous, nontransgenic CD4+ T cell population. These data suggest that in vivo cytokinc responses are shifted by route of antigen delivery, and that primary CD4+ T cell responses are not limited to IL-2 in vivo.