1. Volume 119, Issue 6, Pages 1745-1755 (December 2000)
Identical T-cell expansions in the colon mucosa and the synovium of a patient with enterogenic spondyloarthropathy 
Ekkehard May, *,‡, Elisabeth Märker–Hermann, ‡, Bianca M. Wittig, *, Martin Zeitz, *, Karl–Hermann Meyer Zum Büschenfelde, ‡, Rainer Duchmann, *,‡ 
Gastroenterology 
Volume 119, Issue 6, Pages (December 2000)
DOI: /gast
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
TCRB-CDR3 size analysis and spectratyping. mRNA was obtained from PBMCs (lane P), resected synovial membrane (lane S1), SFMCs (lane S2), and biopsy material from 5 sites of the colon (cecum, lane C1; ascending colon, lane C2; transverse colon, lane C3; descending colon, lane C4; and rectum, lane C5). Inflammation was present in the synovium and at all examined sites of the colon mucosa. Products from BV-specific PCR reactions (BV families are indicated by numbers heading the spectra; BV subfamily specificity of primer BV5.2 was TCRBV5S2, S3, and S7) were separated on a high-resolution gel and stained with silver nitrate. The representative BV families shown represent 4 different types of spectra: overall polyclonality (e.g., BV13.1), restriction of dominating bands (indicating expanded T-cell clones) to the peripheral blood (P), the synovium (S), the colon (C), and dominating bands that overlapped all compartments (PSC, BV18).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
T cells expressing TCRBJ1S5 and BJ2S1 dominate the synovial TCRBV18 spectrum. Bands that simultaneously dominated the synovium-, intestine-, and blood-derived BV18 spectra were reamplified with a BV18-specific primer and 13 BJ-specific primers (BJ1S1 to BJ2S7). TCRBJ-specific PCR products were separated by agarose gel electrophoresis. Highly restricted T-cell clonality in the synovial fluid (SF) and synovial membrane (SM) is illustrated by the presence of only 2 BJ-specific PCR products (BJ1S5 and BJ2S1). The same BJ segments were also overrepresented throughout the colon and present in the peripheral blood. Results of BV18-specific CDR3 spectra and BJ-specific PCR were confirmed by independent experiments.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
CDR3 from clonotypes 1815 and 1821 show primary sequence heterogeneity but physicochemical similarities. (A) BV18BJ1S5 and BV18BJ2S1 DNA amplified from the synovium, intestine, and peripheral blood of the patient were directly sequenced. Nucleotide sequence identity existed among all BJ1S5-specific PCR products (clonotype 1815) and among all BJ2S1-specific PCR products (clonotype 1821). Germline segment borders are indicated above the nucleotide sequences. N-region diversity nucleotides and hypervariable amino acid positions in the CDR3 are indicated in bold. Nucleotide and amino acid primary sequences showed only minor identity. These sequence data are available from EMBL/GenBank/DDBJ, accession numbers AF and AF (B) Charge distribution within CDR3 amino acid sequences was analyzed by generation and superposition of hydrophilicity plots according to the scaling of Hopp and Woods. The figure demonstrates physicochemical similarities in the core region of both CDR3s because of use of conserved amino acid residues (right side) at identical positions.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Expanded clonotypes 1815 and 1821 express CD8 and persist in the colon mucosa. (A) TCRBV18-specific CDR3 spectratyping was repeated with PBMCs and with purified peripheral blood CD4+ (purity 97.5%) and CD8+ (purity 92.4%) T-cell populations from the patient. The dominating band clearly segregated into the CD8+ T-cell subset. DNA sequencing from different spectratype analyses showed that the dominating bands and the forerunning bands contain identical clonotypic TCRB-DNA. The lower bands thus represent artifacts caused by renaturation of abundant clonotypic DNA. (B) TCRBV18-specific CDR3 spectratyping was repeated with biopsy specimens that had been obtained from the cecum of the patient 7 months after the initial examination. The initially dominating band was still present, indicating persistence of the expanded CD8+ T-cell clones.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Cytokine production of T-cell clones A6 (clonotype 1815) and 36 (clonotype 1821). Production of cytokines IFN-γ, IL-4, TNF-α, and IL-10 was determined by intracellular FACS. T-cell clones C1 and C2 are control CD8+ T-cell clones that were isolated in parallel and did not stain for VB18. Gates were adjusted to the large lymphoblastic population. Cells incubated with isotype-matched control or FITC-labeled secondary reagent [F(ab')2 fragment] were used as negative controls. Numbers indicate percentages of positive cells.
Gastroenterology  , DOI: ( /gast )
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