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Autoimmunity, Intestinal Lymphoid Hyperplasia, and Defects in Mucosal B-Cell Homeostasis in Patients With PTEN Hamartoma Tumor Syndrome

2012; Elsevier BV; Volume: 142; Issue: 5 Linguagem: Inglês

10.1053/j.gastro.2012.01.011

1528-0012

Mario Heindl, Norman Händel, Joanne Ngeow, Janina Kionke, Christian Wittekind, Manja Kamprad, Anne Rensing‐Ehl, Stephan Ehl, J. Reifenberger, Christoph Loddenkemper, Jochen Maul, Albrecht Hoffmeister, Stefan Aretz, Wieland Kieß, Charis Eng, Holm H. Uhlig,

Immunodeficiency and Autoimmune Disorders

The Phosphatase And Tensin Homolog Deleted On Chromosome 10 (PTEN) regulates the phosphoinositol-3-kinase (PI3K)-AKT signaling pathway. In a series of 34 patients with PTEN mutations, we described gastrointestinal lymphoid hyperplasia, extensive hyperplastic tonsils, thymus hyperplasia, autoimmune lymphocytic thyroiditis, autoimmune hemolytic anemia, and colitis. Functional analysis of the gastrointestinal mucosa-associated lymphoid tissue revealed increased signaling via the PI3K-AKT pathway, including phosphorylation of S6 and increased cell proliferation, but also reduced apoptosis of CD20+CD10+ B cells. Reduced activity of PTEN therefore affects homeostasis of human germinal center B cells by increasing PI3K-AKT signaling via mammalian target of rapamycin as well as antiapoptotic signals. The Phosphatase And Tensin Homolog Deleted On Chromosome 10 (PTEN) regulates the phosphoinositol-3-kinase (PI3K)-AKT signaling pathway. In a series of 34 patients with PTEN mutations, we described gastrointestinal lymphoid hyperplasia, extensive hyperplastic tonsils, thymus hyperplasia, autoimmune lymphocytic thyroiditis, autoimmune hemolytic anemia, and colitis. Functional analysis of the gastrointestinal mucosa-associated lymphoid tissue revealed increased signaling via the PI3K-AKT pathway, including phosphorylation of S6 and increased cell proliferation, but also reduced apoptosis of CD20+CD10+ B cells. Reduced activity of PTEN therefore affects homeostasis of human germinal center B cells by increasing PI3K-AKT signaling via mammalian target of rapamycin as well as antiapoptotic signals. Patients with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) hamartoma tumor syndrome (PHTS; including Cowden and Bannayan-Riley-Ruvalcaba syndrome) harbor germline mutations in PTEN leading to profound defects in cellular phosphoinositol-3-kinase (PI3K) signaling.1Hollander M.C. et al.Nat Rev Cancer. 2011; 11: 289-301Crossref PubMed Scopus (351) Google Scholar Immune dysregulation including defects in T and B cell homeostasis, autoimmunity, intestinal lymphoid hyperplasia, and thymus hyperplasia and also thymoma and T-cell lymphoma were described in mice with heterozygous Pten deletions.2Podsypanina K. et al.Proc Natl Acad Sci U S A. 1999; 96: 1563-1568Crossref PubMed Scopus (844) Google Scholar, 3Di Cristofano A. et al.Science. 1999; 285: 2122-2125Crossref PubMed Scopus (492) Google Scholar Mouse models confirmed the functional role for Pten/Pi3k signaling in a broad variety of cell types including B cell subsets.4Baracho G.V. et al.Curr Opin Immunol. 2011; 23: 178-183Crossref PubMed Scopus (88) Google Scholar Immune dysfunction in humans carrying germline PTEN mutations has not been systematically analyzed. We studied 34 patients with pathogenic germline PTEN mutations (Figure 1A and B and Supplementary Materials). Among those, we detected histologically and/or serologically proven autoimmune lymphocytic thyroiditis (Hashimoto's thyroiditis), Coombs test-positive autoimmune hemolytic anemia, extensive adenoid lymphoid hyperplasia requiring steroid treatment and adenotomy, life-threatening thymic hyperplasia, and indeterminate colitis (Figure 1A). Gastrointestinal lymphoid hyperplasia was found in 16 patients (Figure 1A, C, and D). There was no indication to suspect increased susceptibility for infections (data not shown). In a subgroup of 14 patients, we studied gastrointestinal lymphoid hyperplasia (stomach [2/14], duodenum [2/14], ileum [3/14], as well as colon/rectosigmoid [12/14]). The lymphoid follicle size in PHTS patients was enlarged (Supplementary Figure 1A), but there was no indication of mucosa-associated lymphoid tissue (MALT) lymphoma. Helicobacter pylori was not detected in the two patients with aggregated gastric lymphoid follicles (Figure 1D). We reasoned that reduced PTEN activity would lead to AKT phosphorylation and subsequent mammalian target of rapamycin (mTOR) signaling. Indeed, within the MALT germinal centers (GC) of PHTS patients, we found reduced PTEN staining (Supplementary Figure 1B) as well as phosphorylated AKT (both p(Thr308)AKT and p(Ser473)AKT) as a downstream readout of PI3K and mTORC2 signaling (Figure 2A and B). We next analyzed activation of the mTOR pathway downstream of PI3K and AKT. Indeed, we found increased frequencies of phosphorylated S6 ribosomal protein and proliferating Ki-67+CD10+ GC B cells within the MALT of PHTS patients (Figure 2C and D). Because AKT signaling has an inhibitory role for several apoptotic pathways, we performed in situ apoptosis assays. There were lower frequencies of terminal deoxynucleotidyl transferase dUTP nick-end labeling (Tunel)-positive-CD10+ and activated caspase 3+CD10+ B cells in MALT from PHTS patients (Figure 2E and F). In contrast to B cells, we could not detect differences in proliferation and apoptosis in the T-cell areas between MALT of controls and PHTS patients. PHTS T-cell blasts exhibited apoptosis equivalent to controls (data not shown). In PHTS patients, blood leucocyte counts were largely normal, but there is a tendency toward lower lymphocyte numbers (Supplementary Figure 2A and B). Serum immunoglobulin (Ig) M, IgG, and IgA levels were within lower normal range (Supplementary Figure 2C–E). Interestingly, there were high frequencies of IgMhigh transitional and CD5+ and CD10+ B cells in PHTS patients (Supplementary Figure 2F). We investigated the effects of (preferential) mTORC1 inhibition in one patient. Within 4 months of rapamycin treatment (Supplementary Figure 3), we observed thymus involution. Whereas the absolute number of B cells was normal during the rapamycin treatment, switched memory and activated B-cell levels did not change significantly, but the number of IgMhigh transitional and CD5+ and CD10+ B cells rapidly dropped. In a series of 34 patients with heterozygous defects in PTEN, we observed immune dysregulation that can be classified into 2 groups: (1) autoimmunity or tissue inflammation and (2) lymphoid hyperplasia. Increased numbers of proliferating GC B cells were associated with increased mTOR signaling and reduced apoptosis. B-cell subsets changed in the peripheral blood, and autoantibodies were found in a fraction of patients. The high frequency of autoimmunity, the unusual early onset and severity of tonsil and thymus hypertrophy, and the extent of gastrointestinal lymphoid hyperplasia (anatomic distribution, follicle size, presence of aggregated lymphoid follicles, and absence of an otherwise explanatory condition) suggest that PTEN deficiency is driving this immune deviation. Previous case reports noted individual patients with PTEN mutation who developed tonsil lymphoid hyperplasia,5Sharma M.R. et al.Arch Otolaryngol Head Neck Surg. 2007; 133: 1157-1160Crossref PubMed Scopus (10) Google Scholar intestinal lymphoid hyperplasia,6Boccone L. et al.Am J Med Genet A. 2006; 140: 1965-1969Crossref PubMed Scopus (33) Google Scholar and autoimmune parotidis.7Raizis A.M. et al.Mol Pathol. 1998; 51: 339-341Crossref PubMed Scopus (16) Google Scholar Recent case series describe gastrointestinal lymphoid aggregates or inflammatory polyps8Heald B. et al.Gastroenterology. 2010; 139: 1927-1933Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar and lymphocytic infiltration in thyroidectomy samples.9Laury A.R. et al.Thyroid. 2011; 21: 135-144Crossref PubMed Scopus (68) Google Scholar Increased resistance of EBV-transformed B cells against Fas-mediated apoptosis has been shown as a case report.10Kanaseki T. et al.Pathobiology. 2002; 70: 34-39Crossref PubMed Scopus (5) Google Scholar Hereby, we describe several changes in B-cell homeostasis of patients with PHTS that are reminiscent of findings in mice. This underlines the essential role of the PI3K signaling pathway for several stages of B-cell development including pro-B cells and pre-B cells as well as mature B cells.4Baracho G.V. et al.Curr Opin Immunol. 2011; 23: 178-183Crossref PubMed Scopus (88) Google Scholar Indeed, Pten deficiency affects B-cell selection, apoptosis, differentiation, immunoglobulin class switching, and autoantibody formation.2Podsypanina K. et al.Proc Natl Acad Sci U S A. 1999; 96: 1563-1568Crossref PubMed Scopus (844) Google Scholar, 3Di Cristofano A. et al.Science. 1999; 285: 2122-2125Crossref PubMed Scopus (492) Google Scholar, 11Suzuki A. et al.J Exp Med. 2003; 197: 657-667Crossref PubMed Scopus (202) Google Scholar, 12Anzelon A.N. et al.Nature Immunol. 2003; 4: 287-294Crossref PubMed Scopus (170) Google Scholar, 13Browne C.D. et al.Immunity. 2009; 31: 749-760Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 14Cheng S. et al.Cell Res. 2009; 19: 196-207Crossref PubMed Scopus (35) Google Scholar A recent study in mice suggests that the GC B-cell response is not solely dependent on PI3K signaling on B cells but on follicular helper T cells.15Rolf J. et al.J Immunol. 2010; 185: 4042-4052Crossref PubMed Scopus (194) Google Scholar Increased frequencies of CD5+ B1 cells were described in Pten+/− and B cell-specific Pten−/− mice.3Di Cristofano A. et al.Science. 1999; 285: 2122-2125Crossref PubMed Scopus (492) Google Scholar, 11Suzuki A. et al.J Exp Med. 2003; 197: 657-667Crossref PubMed Scopus (202) Google Scholar, 12Anzelon A.N. et al.Nature Immunol. 2003; 4: 287-294Crossref PubMed Scopus (170) Google Scholar It is not clear whether the observed accumulation of CD5+ lymphocytes in PHTS patients represents a human B1 cell population. Our data provide evidence that PTEN deficiency in humans has a strong functional impact on B-cell homeostasis by modulating both mTOR and antiapoptotic signals ultimatively resulting in autoimmunity and lymphoid hyperplasia. The authors thank the patients who participated in the study and M. Ziegler, K. Bauer, and H. Knaack for technical help; W. Hirsch for kindly providing MRI images; and K. Maloy, F. Powrie, H. Chapel, M. Barnes, T. Schwerd, W. Heinritz, A. Merkenschlager, and K. Hauptmann for discussions on the manuscript or on patients. M.H. and N.H. contributed equally to this work. Patients were eligible for this study if a pathogenic phosphatase and tensin homolog deleted on chromosome 10 (PTEN) mutation was documented in their patient files, and informed consent was given to analyze clinical data. Series A represents an unselected patient cohort that are in the care of a single genetic referral center. The unselected series A was analyzed for clinical immunopathology only (clinical history, laboratory findings, leucocyte and lymphocyte counts, immunoglobulin level, endoscopies/operations, histopathology findings). Series B, C, and D were analyzed for clinical immunopathology but selected for the histologic analysis of gastrointestinal lymphoid hyperplasia. For the analysis of archived formalin-fixed paraffin-embedded gastrointestinal tissue, we selected patients from 3 different series: (series B) Pathology departments in Germany were asked to provide information facilitating contact with referring physicians of confirmed or suspected cases of PTEN hamartoma tumor syndrome (PHTS). Via their physicians, patients and parents were asked for their consent to provide clinical information and tissues. Series C and D represent subsets of prospectively collected cohorts that have been previously described.1Tan M.H. Mester J. Peterson C. et al.A clinical scoring system for selection of patients for PTEN mutation testing is proposed on the basis of a prospective study of 3042 probands.Am J Human Genet. 2011; 88: 42-56Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 2Heald B. Mester J. Rybicki L. et al.Frequent gastrointestinal polyps and colorectal adenocarcinomas in a prospective series of PTEN mutation carriers.Gastroenterology. 2010; 139: 1927-1933Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar Series C patients were prospectively enrolled according to relaxed International Cowden Consortium operational criteria, ie, presence of pathognomonic mucocutaneous lesion(s), at least 1 major criterion with or without minor criteria, or at least 2 minor criteria. Series D patients had at least 5 gastrointestinal polyps, at least 1 of which must have been hyperplastic or hamartomatous, and who also harbored germline PTEN mutation. The PHTS patients were originated from centers of North America and Europe. Gastrointestinal endoscopy was performed in all patients within the routine clinical setting, ie, because of symptomatic gastrointestinal complaints and/or to rule out polyps. Patients series B to D were analyzed for lymphoid hyperplasia (n = 14; 11 male; mean age, 33 years; range 3–56 years). Patient characteristics are provided in Supplementary Table 1.Supplementary Table 1Clinical Features of Patients Included in the Histologic Analysis: Patient Characteristics of Series B, C, and DPatientAge, ySexPTEN mutationClinical features117Mc.389G>A p.R130QMacrocephaly, intestinal lymphoid hyperplasia, lipomas, a lipomatous hemangioma, cutaneous teleangiectases, thyroid cysts, pigmented macule on the glans penis23Mc.80-?_1212+?delThymic hyperplasia, hyperplasia of the pharyngeal tonsils, intestinal lymphoid hyperplasia, abdominal lipomatosis, multiple lipomas356Mc.534T>G p.Y178XFollicular thyroid carcinoma, multiple dermatofibromas, multiple intestinal polypoid lesions, lymphoid hyperplasia of the gastric mucosa454Fc.334C>G p.L112_K164delMultiple benign hamartomatous lesions of the skin, papillomatosis of the lips and oral mucosa, polyposis coli, bilateral fibrocystic disease of the breast, bilateral breast carcinomas, and malignant skin melanomas514Mdel(10)(q23.2–23.31)Macrocephaly, numerous intestinal hamartomatous polyps, ganglioneuromatous polyps and adenomatous polyps, lipomas, oral mucosa papillomas, penile freckling637Mc.388C>TSNP c.1026+32T>G p.R130XMacrocephaly, trichilemmomas, oral mucosal papillomas, thyroid adenoma, numerous intestinal ganglioneuromatous polyps, colorectal cancer74Mc.26delT p.S10AfsX14Macrocephaly, multiple intestinal hamartomatous polyps, multiple hyperplastic and inflammatory polyps in the upper GI tract, glycogenic acanthosis853Mc.395G>A p.G132DMacrocephaly, thyroid adenoma, skin and oral mucosa papilloma, trichilemmomas, penile freckling, glycogenic acanthosis, renal cell carcinoma98Mc.491delA p.K164RfsX2Macrocephaly, multiple intestinal ganglioneuromatous polyps1053Mc.80-?_164+?delMacrocephaly, multiple inflammatory, hyperplastic and adenomatous intestinal polyps, thyroid adenoma1144Fc.49C>T p.Q17XMacrocephaly, thyroid adenoma and thyroiditis, fibrocystic breast disease1238Mc.634+2T>CMultiple intestinal hamartomatous polyps, trichilemmoma, oral and nasal mucosa papilloma, lipoma, skin fibroma1348Fc.389G>A p.R130QMacrocephaly, ductal breast cancer, lobular breast cancer (second primary), ductal and lobular breast carcinoma in situ, fibrocystic breast disease, glycogenic acanthosis, uterine fibroids, multiple hamartomatous, inflammatory and ganglioneuromatous intestinal polyps1437Mc.376G>C p.A126PMacrocephaly, thyroid adenoma, penile freckling, lipomas, numerous hamartomatous intestinal polypsF, female; M, male. Open table in a new tab F, female; M, male. Control tissues for histologic analysis (n = 27; 17 male; mean age, 21 years; range, 0.4–70 years) were derived from patients after appendectomy as well as upper and lower gastrointestinal endoscopy. We investigated mucosa-associated lymphoid tissue (MALT) tissue from patients with Helicobacter pylori gastritis (n = 6), ileum (n = 7), colon (n = 10), and appendix (n = 12). Ethical review was granted by the institutional ethical review boards (Leipzig University, Institutional Review Boards for Human Research Subjects' Protection of the Cleveland Clinic, the University of Bonn, and of the Albert-Ludwigs-University of Freiburg). Written consent was obtained for all patients and controls. The Clinical Ethics Committee of the University of Leipzig approved rapamycin treatment in 1 child as an individual trial. Flexible endoscopy as well as capsular endoscopy (Given Imaging, Yoqneam, Israel) was performed as a part of routine investigations. Chromoendoscopy with Indigocarmin has been performed to increase contrast. Genomic DNA was extracted from peripheral blood leukocytes. PTEN was analyzed with a combination of different methods.3Mutter G.L. Lin M.C. Fitzgerald J.T. et al.Changes in endometrial PTEN expression throughout the human menstrual cycle.J Clin Endocrinol Metab. 2000; 85: 2334-2338Crossref PubMed Scopus (135) Google Scholar, 4Zhou X.P. Waite K.A. Pilarski R. et al.Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway.Am J Hum Genet. 2003; 73: 404-411Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 5Reifenberger J. Rauch L. Beckmann M.W. et al.Cowden‘s disease: clinical and molecular genetic findings in a patient with a novel PTEN germline mutation.Br J Dermatol. 2003; 148: 1040-1046Crossref PubMed Scopus (42) Google Scholar, 6Teresi R.E. Zbuk K.M. Pezzolesi M.G. et al.Cowden syndrome-affected patients with PTEN promoter mutations demonstrate abnormal protein translation.Am J Hum Genet. 2007; 81: 756-767Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar This included polymerase chain reaction-based denaturing gradient gel electrophoresis (DGGE) as well as direct Sanger sequencing of all 9 exons, flanking intronic regions, and resequencing of the promotor. Multiplex ligation-dependent probe amplification (MLPA) assay was performed with the P158 MLPA kit (MRC Holland Amsterdam, the Netherlands) according to manufacturer's instructions.7Schouten J.P. McElgunn C.J. Waaijer R. et al.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2132) Google Scholar Tissue sections were stained with H&E using routine procedures. H&E overview images were constructed by electronic stitching of high-power images using BZ Viewer and Analyzer 2.5.1 software (Keyence, Osaka, Japan). Formalin-fixed, paraffin-embedded biopsy samples were cut (4 μm; Microm HM340E), mounted, and dried overnight at 40°C. Sections were deparaffinized and incubated at 85°C in antigen-retrieval solution pH 9 (Dako, Glostrup, Denmark) for 20 minutes. Endogenous peroxidase was blocked with 3% H2O2 and slides were incubated with normal donkey serum (Sigma-Aldrich, Saint Louis, MO). For immunohistochemistry, sections were stained for PTEN, p(Ser473)AKT, or p(Thr308)AKT followed by anti-mouse or anti-rabbit peroxidase (POD)-labeled antibodies. Staining was performed using 3,3′-diaminobenzidine tetrahydrochloride chromogen (diaminobenzidine; Dako) and Mayer's Hemalaun solution (Carl Roth, Karlsruhe, Germany). In addition, we performed PTEN staining using horseradish peroxidase polymer amplification system (Dako EnVision, Dako Glostrup, Denmark) according instructions of the manufacturer. For multicolor fluorescence microscopy, sections were sequentially stained using the thyramide fluorescence assay. As an example: incubation with anti-CD10 (clone 56C6; 1:50; Leica MS, Wetzlar, Germany) was followed by donkey anti-mouse POD and tyramide Cy5 amplification (PerkinElmer, Waltham, MA). POD was inhibited using H2O2 and sodium azide. Sections were subsequently incubated with mouse serum (Sigma), fluorescein isothiocyanate (FITC)-labeled mouse anti-Ki-67 (Mib-1; Dako), and mouse anti-FITC POD (Dianova, Germany) followed by tyramide FITC amplification (PerkinElmer). 4', 6'-Diamidino-2-phenylindole (DAPI) counterstaining was performed to visualize the tissue structure and to confirm the nuclear localization of the Ki67 signals. The following antibodies were applied for immunohistochemistry and immunofluorescence stainings: anti-CD3 (polyclonal rabbit; 1:50; Dako), anti-CD10 (clone 56C6; 1:50; Leica MS, Wetzlar), anti-CD20 (clone L26; 1:50; Dako), anti-Caspase-3 (Asp175) (clone 5A1E; 1:150; Cell Signaling Technology, Boston, MA), anti-PTEN (clone 138G6; Cell Signaling), anti-p(Ser473)-AKT (clone D9E; 1:50; Cell Signaling), anti-p(Thr308)-AKT (rabbit polyclonal AB66134; 1:50; Abcam, Cambridge, UK), anti-p(Ser235/236)-S6 ribosomal protein (clone D57.22E; 1:100; Cell Signaling), and anti-Ki-67 (clone Mib-1; unlabeled or anti-Ki-67-FITC; 1:100 Dako, respectively). Secondary antibodies directed against mouse immunoglobulin (Ig) G (polyclonal donkey POD labeled (1:100; Jackson Immunoresearch, Suffolk, UK) and anti-rabbit IgG (donkey polyclonal; POD labeled; 1:100; Jackson Immunoresearch) were used to detect primary antibodies. FITC was detected using mouse monoclonal anti-FITC (clone 1F8-1E4, POD labeled; 1:200; Jackson Immunoresearch). Phosphate-buffered saline, pH 7.4, with 0.05% (vol/vol) Tween 20 (Sigma) was used for washing and dilution of antibodies. Tris-buffered saline (TBS; pH 7.56) with 0.05% (vol/vol) Tween 20 (Sigma) was used for phospho-AKT and phospho-S6 stainings. To investigate apoptosis terminal deoxynucleotidyl transferase dUTP nick-end labeling (Tunel) assays were performed using the ApopTag peroxidase in situ apoptosis detection Kit (Chemicon, Billerica, MA). Paraffin-embedded sections (4 μm) were deparaffinized, rehydrated, and then treated with proteinase K (Dako) for 15 minutes. Slides were washed and treated with 3% H2O2 for 20 minutes to block endogenous peroxidase activity. Slides were incubated with equilibration buffer followed by TdT-Enzyme at 38°C for 1 hour. The reaction was terminated with stop/wash buffer dilution and followed by incubation with anti-digoxigenin-POD for 30 minutes at room temperature. Sections were stained using DAB+ substrate chromogen system (Dako) and counterstained with Mayer's hemalaun solution. Sections treated with DNAse I (Roche, Basel, Switzerland) served as positive and sections treated without TdT-enzyme as negative controls. To combine the TUNEL staining with immunohistochemistry, we initially performed the tyramide staining for CD3, CD10, or CD20, respectively. We next treated the sections with proteinase K, blocked the remaining peroxidase activity, and performed the TUNEL assay using tyramide fluorescence probes. Counterstaining was performed using DAPI. Fluorescence images were recorded using a Keyence BZ-8000 fluorescence microscope with filter combinations specific DAPI, FITC, Cyanine3, and Cy5. Images were recorded using a PlanApo objective (20×/0.75; Nikon) at 200×, and PlanApo VC (60×/1.40 oil; Nikon Osaka, Japan). BZ viewer and Analyzer 2.5.1 software were applied. Cell numbers were determined automatically using CellProfiler (version 1.0.5122, www.cellprofiler.org) as described8Handel N. Brockel A. Heindl M. et al.Cell-cell-neighborhood relations in tissue sections: a quantitative model for tissue cytometry.Cytometry A. 2009; 75: 356-361Crossref PubMed Scopus (6) Google Scholar or manually using SigmaScan 5.0 software (Systat Software, San Jose, CA). In vitro apoptosis assay was performed as previously described.9Rensing-Ehl A. Warnatz K. Fuchs S. et al.Clinical and immunological overlap between autoimmune lymphoproliferative syndrome and common variable immunodeficiency.Clin Immunol. 2010; 137: 357-365Crossref PubMed Scopus (51) Google Scholar After erythrocyte lysis (FACS Lysing Solution; BD Biosciences San Jose, CA), peripheral blood mononuclear cell samples were washed with phosphate-buffered saline, stained, and fixed with 1% formaldehyde solution. All analytical flow cytometry was performed on FACSCalibur (BD Biosciences) using CellquestPro Software (BD Biosciences). The following antibodies were used: anti-CD3 (clone SK7 or clone UCHT1; BD Biosciences), anti-CD4 (clone 13B8.2; Immunotech), anti-CD8 (clone SK1; BD), anti-CD9 (clone MEM-61; Exbio), anti-CD10 (clone HI10a; BD Biosciences), anti-CD16 (clone NKP15; BD Biosciences), anti-CD19 (clone SJ25C1; BD Biosciences), anti-CD20 (clone L27; BD Biosciences), anti-CD21 (clone B-ly4; BD Biosciences), anti-CD24 (clone SN3; Exbio, Vestec, Czech Republic), anti-CD25 (clone 2A3; BD Biosciences), anti-CD27 (clone M-T271; BD Biosciences), anti-CD38 (clone HB7; BD Biosciences), anti-CD45 (clone 2D1; BD Biosciences), anti-CD45RO (UCHL1; BD Biosciences), anti-CD56 (clone NCAM16.2; BD Biosciences), anti-IgA (polyclonal; Dianova, Hamburg, Germany), anti-IgD (clone IA6-2; BD Biosciences), anti-IgG (clone G18-145; BD Biosciences), anti-IgM (clone G20-127; BD Biosciences), and anti-Kappa light chain (clone A8B5; Exbio). Isotype-matched monoclonal antibodies served as controls. Internal validation data to define staining pattern, subset analysis, and internal normal reference range for lymphocyte subsets were established at the Institute of Immunology, University of Leipzig, and included healthy volunteers (n = 14; 4 male; mean age, 35 years; range, 25–49 years). Mann–Whitney U tests were applied for testing of differences between groups (GraphPad Prism 5 for MacOS X; GraphPad, San Diego, CA). Values of P < .05 were regarded as significant.Supplementary Figure 2Peripheral blood leucocyte and lymphocyte counts, serum immunoglobulin level, and B-cell subsets of PHTS patients. (A) Peripheral blood leucocyte and (B) lymphocyte counts. Lines represent the upper and lower reference according to Herklotz et al10. (C–E) Serum immunoglobulins M (C), G (D), and A (E). The serum immunoglobulin levels that we provide for 6 to 8 PHTS patients were derived from different certified clinical laboratories in Germany and the United States. These laboratories use standardized, certified, and validated tests but may base on different methods. Black lines indicate 2.5th and 97.5th percentile and red line the 50th percentile. Continuous lines denote male, and dotted lines denote female reference values. Reference values are presented according to Ritchie et al.11 Dots represent individual patients derived from series A to D. (F) B-cell subsets in patients with PHTS. Peripheral blood was analyzed using fluorescence-activated cell sorter. Three patients with prototypic mutations c.80-? 1212+?del, c.389G>A, and c.334C>G (age of analysis, 3.8, 18.3, and 66.5 years, respectively) were compared against an internal control group of 14 healthy volunteers (Institute of Immunology, University of Leipzig). There were no clear differences in other subsets including CD27− nonmemory B cells, IgD+CD27− naive B cells, CD27+ memory B cells, IgD+CD27+ nonswitched memory/marginal zone like B cells, IgD−CD27+ switched memory B cells, CD21lowCD38low activated B cells or IgM−CD38high plasmablasts. In comparison with pediatric normal values, the pediatric PHTS patient has equally high IgMhighCD38high transitional B cells (patient, 12.36%; normal range, 3.1%–12.3%; 5th to 95th percentile range according to Piatosa et al.12View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 3Thymus involution and normalization of B-cell subsets after rapamycin application in a patient with PHTS (c.80-?_1212+?del). (A) Reduction of thymus hyperplasia as indicated by magnetic resonance imaging (MRI). Thymus volume reduced from 180 mL before to 101 mL after rapamycin treatment. T marks thymus. (B–F) Cell numbers from peripheral blood were determined by fluorescence-activated cell sorter. Time point 0 denotes the start of rapamycin treatment. (B) T-cell populations. Absolute numbers (in 109/L). (C) CD19+ cell numbers. Absolute (in 109/L) and relative (percentage of lymphocytes). (D) Proportion of IgM+ transitional and CD10+ CD19+ B cells. (E) Proportion of IgD+IgM+, switched IgM−IgD− and CD38−CD21− B cells. (F) Percentage of CD5+ cells among CD19+ B cells. Bands indicate the normal range of the respective marker.View Large Image Figure ViewerDownload Hi-res image Download (PPT)