Dominik Meier, Guillermo Docena, Diego Ramisch, Gabriel Gondolesi, Martín Rumbo

Instituto de Trasplante Multiorganico - Programa de Inmunobiología e Investigación Traslacional en Trasplante, Hospital Universitario - Fundación Favaloro, Ciudad Autonoma de Buenos Aires, Buenos Aires, Argentina

Introduction: Isolated lymphoid follicles (ILF) are organized structures that function as inductive sites contributing to intestinal IgA production. Increased numbers of lymphoid aggregates were detected in inflammatory bowel disease. However, no studies have been conducted about the behavior of ILF after intestinal transplantation (ITx) in humans. We aimed to isolate ILF from human intestinal allografts and to elucidate the impact of immunosuppressive therapy on ILF structure and function.

Material and Methods: 10 full thickness biopsies of distal ileum were obtained, 5 at the time of the ileostomy closure, (ITx group, all under steroids and tacrolimus); and 5 at ileal segmental resection in non-Tx patients (Control group). To isolate ILF, mucosa and submucosa were dissected from the muscular layer, then the epithelial monolayer were removed by EDTA treatment  to finally obtained the ILFs. ILF surface density was measured. ILF were collected and homogenized for immune cell analysis by flow cytometry (markers used: CD3, CD4, CD8, CD19, CD56, CD69) and activation induced cytidine deaminase (AID), a gene that is critical for class switch recombination, was measured by qPCR. Formaldehyde-fixed and paraffin-embedded tissue was used for immunohistological analysis.

Results: A non-significant increase of ILF numbers/cm² between  ITx group (mean3±1.8) and Control group (mean 1.84±0.9) was observed. No significant changes among the different percentages of subpopulations (CD4⁺ and CD8⁺ T, CD19⁺ B, CD56⁺ NK, CD3⁺ CD56⁺ NKT cells) were seen in both groups. The relative expression of AID was increased in ILF compared to lamina propria in both groups (mean fold increase was 21±15 for ITx group vs. 106±56 for Control group). Finally, immunohistological analysis showed a similar distribution of IgA positive plasma cells in lamina propria from ITx and Control groups sections.

Conclusions: Our results reveal for the first time that ILF can be isolated and further analyzed in samples obtained after ITx. The standard immunosuppressive therapy does not seem to affect cellularity and functional capacity of immunoglobulin class switch of ILF. We could not detect any sign of increased activation in ILF from allograft specimen. Further studies of allograft ILF would contribute to better understand the immunobiology of intestinal transplantation.