Characterization of mucosal immune system by Immunofluorescence Linked Immunospot (ILISPOT): Immunofluorescence Digital Image Processing (IDIP) system

The mucosal immune system possesses several unique characteristics which are distinctively different from the systemic immune system. In order to invoke proper mucosal immunity to foreign antigens, immunization has to be initiated at the mucosa-associated lymphoreticular tissues (MALT). Therefore, different routes of mucosal vaccination have been extensively studied to provide the most effective mucosal immunity for the host. In order to investigate the outcome of antigen-specific immune response upon mucosal immunization, it is essential to analyze the immune response at the single cell level in both qualitative as well as quantitative ways.

As described in this dissertation, a new system, which utilizes a video microscope and digital image analysis was, developed for measurement of the amount of secreted immunoglobulin by single B cells. This system was designated as an Immunofluorescence Linked Immunospot (ILISPOT)-Immunofluorescence Digital Image Processing (IDIP) system, and was applied to enumerate both total and antigen-specific IgA producing cells in different parts of the intestine (e.g., small and large intestine).

By using the ILISPOT-IDIP system, several new findings were made. First, higher numbers of IgA producing cells were shown in the upper and middle parts of small intestine in comparison to the lower small intestine and large intestine. Second, high IgA producing cells were largely observed in the upper and middle region of small intestine. Third, increased numbers of antigen-specific IgA producing cells were mainly induced in the upper and middle parts of small intestine followed by the lower small intestine and large intestine in mice orally immunized with cholera toxin (CT). Fourth, the ratio of antigen-specific IgA producing cells in total IgA producing cells was equally distributed over the entire intestine when antigen was given by oral immunization. Fifth, in contrast to oral immunization, elevated numbers of CT-specific IgA producing cells with high frequency of high IgA secretors were predominantly observed in the large intestine but not in the small intestine for intracolonic immunization. Sixth, intracolonic immunization induced a high ratio of CT-specific IgA secreting cells in total IgA producing cells only in the large intestine.