Inflammatory bowel disease (IBD) can affect the lower part of the small intestine (ileum) and the colon. It can, however, occur in any part of the large intestine, small intestine, or stomach. Recent evidence suggests that several factors may tip the balance between homeostasis and intestinal inflammation, presenting future challenges for the development of new therapies for IBD.

In this study, we use normal small intestine as a model to identify multiple immune targets using mouse and rabbit monoclonal antibodies. Peyer Patches are lymphoid tissues in the wall of the small intestine that are involved in the development of immunity to antigens presented in that milieu. Small intestine and cases of IBD were evaluated as a potential model using immunohistochemistry (IHC) to identify categories of immune cells including cytotoxic T-cells, costimulatory cells (agonists), T-regulatory cells (TREGs), T-cell effector cells (regulators) and checkpoint inhibitors. cases of IBD were also examined for the utilization of single and double/triple stains.


Formalin-fixed paraffin-embedded cases of small intestine and IBD were cut to 4 micron thickness and stained with H&E. Mouse and rabbit monoclonal antibodies including CD56 (natural killer cells), CD163 (macrophages), CD8 and CD4 (cytotoxic T-cells and helper T- cells), CD103, OX40 and CD137 (costimulatory), FOXP3 and LAG3 (TREGs), T-bet and RORγT (T-cell effectors), and PD-1 and PD-L1 (immune checkpoints) were tittered for optimization and evaluated for IHC. Several IHC multiplex stains (double and triple stains) were also developed and compared with the relevant single stains.


Single, double and triple stains utilizing CD56, CD163, CD4, CD8, CD103, OX40, CD137, FOXP3, LAG3, T-Bet, RORγT, PD-1 and PD-L1 antibodies were successfully established using small intestine as staining model for IBD cases. All single stains versus double and triple stains gave equivalent results. Double stains and/or co-expression of CD8 + CD103, CD8 + PD-1, and triple stain CD8 + PD-1 + FOXP3 were easily visualized with blue, brown, red or black chromogen staining.


Small intestine is a good model to develop single or multiplex stains for a host of immune cell targets. Enhanced visualization of these targets is possible with the use of double and triple stains; thus, this technique may help facilitate acquisition of important prognostic information and help support new strategies for inflammatory bowel disease in targeted therapeutic treatment.

Note: This abstract was not presented at the meeting.

Citation Format: David Altree-Tacha, Wei Wei Yuan, George Yang. Deciphering the immune system in small intestine: Multiplex strategies for inflammatory bowel disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3817. doi:10.1158/1538-7445.AM2017-3817