STIMULATION OF ANTIGEN-SPECIFIC T-CELL AND B-CELL MEMORY IN LOCAL AS WELL AS SYSTEMIC LYMPHOID-TISSUES FOLLOWING ORAL IMMUNIZATION WITH CHOLERA-TOXIN ADJUVANT

In the present study we investigated immunological memory at the cellu lar level following oral immunization using cholera toxin (CT) as the mucosal adjuvant. We found that memory cells, isolated from mice orall y primed with keyhole limpet haemocyanin (KLH) admixed with CT adjuvan t 8 months earlier, responded by increased proliferation to antigen-ch allenge in vitro. In contrast, unstimulated memory cells or KLH-stimul ated cells from naive mice did not respond. Memory cells were isolated from different lymphoid tissues; spleen (SP), mesenteric lymph nodes (MLN), Peyer's patches (PP) as well as the intestinal lamina propria ( LP). Thus, oral immunization using CT adjuvant promoted the generation of memory cells that were present in both systemic and local intestin al lymphoid tissues. The demonstration of lymphokine production in the KLH-responsive cultures indicated the presence of antigen-specific me mory T cells. Lymphokine production early in culture was dominated by interleukin-2 (IL-2), which peaked on day 2-3, followed by IL-5 and, i n particular, interferon-gamma (IFN-gamma) which increased over time. Lamina propria memory cells were found to proliferate poorly to recall antigen in vitro compared to lymphocytes from SP or MLN. In contrast, very significant production of IL-5 and, in particular, IFN-gamma was demonstrable in LP cell cultures. The use of CT adjuvant also stimula ted the generation of antigen-specific memory B cells following oral i mmunization. This was evidenced by KLH-specific antibody production in antigen-challenged memory lymphocyte cultures. The memory B cells pro duced IgM anti-KLH, while no detectable antigen-specific IgG or IgA wa s found. Unstimulated memory cells or naive cells failed to produce an ti-KLH antibodies. These in vitro findings provide evidence that oral immunization using CT adjuvant stimulates both antigen-specific memory T and B cells. Furthermore, our data suggest the existence of memory B cells following oral CT adjuvant immunization which have retained th e ability to produce IgM and which therefore probably have not undergo ne terminal isotype switch differentiation to other isotypes and thus have not deleted the mu constant heavy-chain gene. Finally, our data a lso suggest that memory T and B cells, either sessile in the various l ymphoid tissues or recirculating, can be activated by antigen in situ in, for example, lymph nodes and spleen and, more importantly, in the intestinal LP itself.