PMC

Sensitivity of PBL to CD95-mediated apoptosis. Lymphocytes were isolated from the peripheral blood from four healthy donors (filled circles) and from five CSS-patients (open circles). After treatment with PHA (2.4 μg/ml) for 24 hours, the lymphocytes were incubated with an agonistic anti-CD95 antibody at various concentrations. After another 24 hours, the lymphocytes were subjected to TUNEL analysis as described in Materials and Methods and percentages of apoptotic lymphocytes were counted. Data are given as means ± SE.

Preferential usage of distinct TCR-Vβ and Jβ genes in T cells from five CSS patients. Fluorescent profiles of the Vβ21-Jβ1.2 rearrangements are shown in samples from the peripheral blood of one healthy donor and five CSS patients (WI, VN, GM, LM, and SJ). The peripheral blood samples of these five CSS patients contained clonally expanded T cells carrying a gene from the Vβ21 gene family rearranged to the Jβ1.2 gene with a dominant CDR3-size peak corresponding to a CDR3 of 11 aa in length (arrows).

Identification of T cells as CD95L-expressing cells in ulcerative gastric mucosa in patient WI. Cryosections from ulcerative gastric mucosa from patient WI were double-stained using CD3-specific and CD95L-specific antibodies as described in Materials and Methods. In A and B, one cryosection from infiltrated muscularis propria and in C and D one from infiltrated mucosa is shown. Double fluorescence staining for CD3 and CD95L was performed on the same cryosection. In A and C, signals are specific for CD3 (FITC-labeled), in B and D, for CD95L expression (CY3-labeled). CD3-expressing cells (ie, T lymphocytes) and CD95L expression were colocalized (arrows).

T-cell repertoire diversity and clonal expansions in PBL from Churg-Strauss syndrome patient LM. Using the Immunoscope RT-PCR technique, TCRVβ chain segments in PBL from patient LM was amplified using a panel of 24 Vβ subfamily-specific PCR primers. Clonal expansions was detected in several Vβ-Cβ profiles. Clonality of Vβ12- and Vβ21-specific transcripts is shown here. Samples were analyzed from the peripheral blood prior to (PBL before) and after immunosuppressive treatment (PBL after). Clonal expansions are indicated by arrows. Clonality of the Vβ-Cβ profiles was confirmed using Jβ-specific primers. For both Vβ-Cβ profiles, each of 13 Vβ-Jβ recombinations was selected to demonstrate the strongest deviation from normal polyclonal gaussian-like peak size distribution, ie, Vβ12-Jβ2.2 for the Vβ12 family and Vβ21-Jβ1.2 for the Vβ21 family.

T cell repertoire diversity and clonal expansions in gastric IEL and PBL from Churg-Strauss Syndrome patient WI. Using the Immunoscope RT-PCR technique, TCRVβ chain segments in IEL and PBL from patient WI were amplified using a panel of 24 Vβ family-specific PCR primers. Clonal expansions were detected in several Vβ-Cβ profiles. Clonality of Vβ11- and Vβ21-specific transcripts is shown here. Samples were analyzed from infiltrated ulcerative gastric mucosa (IEL before) and the peripheral blood before (PBL before) and after immunosuppressive treatment (PBL after) as well as after recurrence of CSS (PBL recurrence). The peak sizes of clonal expansions are indicated by arrows. Clonality of the Vβ-Cβ profiles was further analyzed using Jβ-specific primers. For both Vβ-Cβ profiles, each one of 13 Vβ-Jβ recombinations was selected to demonstrate the strongest deviation from normal polyclonal gaussian-like peak size distribution, ie, Vβ11-Jβ2.1 for the Vβ11 gene family and Vβ21-Jβ1.2 for the Vβ21 gene family.

Distribution of CD95 receptor isoform mRNA expression in peripheral blood T cells (PBL), gastric intraepithelial T cells (IEL) and eosinophils. A: cDNAs derived from PBL of one healthy donor, patient WI 1 day prior to (CSS, day −1) and 14 days after gastrectomy and immunosuppressive treatment (CSS, day 14) were amplified using CD95 isoform- and HPRT-specific primers as described in Materials and Methods in 30 PCR cycles. Amplification products for membrane-bound CD95 (CD95Tm, 392 bp), soluble CD95 (CD95Sol, 330 bp), and HPRT (237 bp) were separated on a 2% agarose gel. B: CD95 membrane-bound and soluble isoform mRNA expression was analyzed in eosinophils from healthy donors (representative for three donors), one patient with infectious eosinophilia (patient DA, 25% eosinophilic counts), and one CSS patient (patient PF, 32% eosinophilic counts). C: CD95 isoform mRNA expression in ulcerative and non-ulcerative gastric mucosa was studied in samples from gastric antrum and corpus of patient WI at the time of gastrectomy.

A: Immunoscope-based analysis of the clonality of TCRβ VDJ-transcripts. T cell receptor β (TCRβ) transcripts are reverse transcribed and amplified using a panel of 24 Vβ- and Cβ-specific primers. Thus, the first step of the Immunoscope technique involves 24 RT-PCR reactions run to saturation (top). In an initial low-resolution analysis, a dye labeled Cβ-specific primer is used to visualize the amplified products in run-off reactions (middle). If higher resolution is required, run-off experiments are carried out using 13 dye-labeled Jβ-specific primers in theoretically 24 × 13 = 312 run-off reactions (bottom). After electrophoresis on an automated sequencer and subsequent analysis, the different size peaks are separated and their CDR3 size in amino acids (aa) and area are calculated. B: Quantification of CD95 ligand and receptor isoform mRNA levels by quantitative competitive RT-PCR. The cDNA to be assayed (WT) was co-amplified with known amounts (10⁸, 10⁷, … 10 copies) of an internal DNA standard (Δ4), which was apart from a deletion of four nucleotides identical to the corresponding fragment of the assayed cDNA. PCR products were specifically labeled in run-off reactions, loaded on an acrylamide gel, and analyzed by an automated sequencer. The fluorescent profiles were recorded and the profile areas were analyzed. For co-amplifications with 10 and 10 copies of the CD95L standard, respectively, the peak area ratios for CD95L wild-type (CD95L WT) and standard (CD95L Δ4) were calculated. The number of CD95L WT copies in the cDNA sample was calculated as the mean of CD95L WT/CD95L Δ4 peak area ratios at two standard dilutions (eg, for the sample shown here: (0.262 × 10⁶ + 2.931 × 10⁵)/2, or 277,550 copies).