Volume 128, Issue 7, Pages 1879-1889 (June 2005) Human Intestinal IgA Response Is Generated in the Organized Gut-Associated Lymphoid Tissue but Not in the Lamina Propria  Laurent Boursier, John N. Gordon, Sivashankari Thiagamoorthy, Jonathan D. Edgeworth, Jo Spencer  Gastroenterology  Volume 128, Issue 7, Pages 1879-1889 (June 2005) DOI: 10.1053/j.gastro.2005.03.047 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 RT-PCR analysis of AID expression. (A) Analysis of AID message in whole duodenal biopsies. Lanes 1 and 2: duplicate examples from a biopsy that gave a positive result. Lane 3: example of a biopsy giving a negative result. (B and C) No AID message in either 35 cryostat sections of duodenal LP from 1 biopsy (lane 7) or in isolated colonic CD138+ cells (lanes 8–10) but identification of AID message in a section through an ILF (lane 11). Tonsil positive control was strongly positive in each experiment, as illustrated in lane 4 (T) in A. RT-PCR negative control (cDNA template omitted) was consistently negative, as illustrated in lane 5, A. GAPDH was strongly positive in each case. Expected PCR product sizes are shown in brackets. MWM, molecular weight marker. (D and E) Frozen sections stained using immunoperoxidase with CD20 monoclonal antibody. A section used to screen the duodenal mucosa before analysis of AID expression (lane 7, B) is shown in D, and a section through an ILF that gave a strong positive result for RT-PCR analysis of AID expression is illustrated in E. Gastroenterology 2005 128, 1879-1889DOI: (10.1053/j.gastro.2005.03.047) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Sections of ileal mucosa stained using Ki-67 proliferation marker (brown nuclear stain) and CD20 pan B cell marker (blue surface stain). (A) Low-power view of organized GALT and adjacent i-LP illustrating abundant proliferation of B cells in the germinal center (arrowheads) and scattered proliferating cells in the surrounding marginal zone (MGZ) of B cells (arrows). Dividing B cells are not present in the i-LP. Proliferating epithelial cells at the bases of the crypts are apparent. Note the infiltration of marginal zone B cells between the crypts adjacent to the follicle. (B) High-power of proliferating marginal zone B cells (arrows) as they extend from the follicle, infiltrating between the crypts. Gastroenterology 2005 128, 1879-1889DOI: (10.1053/j.gastro.2005.03.047) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Distribution of clonally related cells identified by shared H-CDR3 DNA sequences of IgVH5DJH-CH transcripts in numbered tissue sections of ileal and colonic LP of case 1. These tissue sections were confirmed to be free of organized lymphoid tissue and cellular proliferation by analysis of adjacent sections using immunohistochemistry. Six clones were identified in 2 or more sections from the same block, but no clones were detected in both blocks. Blue rectangles: IgVH5DJH-Cμ, red rectangles: IgVH5DJH-Cα functional transcripts. Gastroenterology 2005 128, 1879-1889DOI: (10.1053/j.gastro.2005.03.047) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Distribution of clonally related cells identified by shared H-CDR3 DNA sequences of IgVH5DJH-CH transcripts in numbered tissue sections of colonic LP of case number 2. These tissue sections were confirmed to be free of organized lymphoid tissue and cellular proliferation by analysis of adjacent sections using immunohistochemistry. Fifteen clones were identified in 2 or more sections from the same block. Among these, 10 clones were identified in both blocks of colonic mucosa, located 5 cm apart in the surgical specimen. Blue rectangles: IgVH5DJH-Cμ, red rectangles: IgVH5DJH-Cα functional transcripts. Gastroenterology 2005 128, 1879-1889DOI: (10.1053/j.gastro.2005.03.047) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Details and analysis of the related group of sequences labeled μα4 in Figure 4. Block (A and B) and sequence numbers (2, 3, 9, and 11) relate to those in Figure 4. (A) Illustrates nucleotide sequence alignment of IGHV5-51DJH4B-Cμ and -Cα transcripts. Replacement mutations (uppercase) and silent mutations (lowercase) identity with the germ-line sequences above (dashes) are indicated. Location of H-CDR3 “clonal signature” is indicated. RNA splice sites (5′ end of CH exons) are shown with asterisks. Annealing sites of PCR primers are shown in italics. One deletion identified is marked “d.” (B) B-cell lineage tree built on the 4 DNA sequences shown in A. Ig sequences from different blocks of tissue were more closely related to each other than to their neighbors in the same block. x, hypothetical intermediate; mut, point mutation(s). Gastroenterology 2005 128, 1879-1889DOI: (10.1053/j.gastro.2005.03.047) Copyright © 2005 American Gastroenterological Association Terms and Conditions