Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices.

Local differentiation of eosinophil precursors occurs in the human thymus. Thymic eosinophils are often positioned in the corticomedullary junction between the CD4+ CD8+ double-positive (DP) thymocytes and the CD4+ or CD8+ single-positive (SP) thymocytes. The aims of this study were to (1) determine if there are distinct thymic eosinophil populations that differ from the blood eosinophil populations and (2) evaluate the capacity of thymic eosinophils to promote the development of SP thymocytes from DP thymocytes. Thymic and blood eosinophils from thymectomized infants (n = 7) were compared regarding the expression of 34 molecules using cytometry by time-of-flight (CyTOF). In addition, FACS-sorted thymic eosinophils were co-cultured with autologous CD3/CD28-stimulated DP, CD4 SP, and CD8 SP thymocytes and analysed by flow cytometry and CyTOF. X-shift clustering analysis and viSNE dimensionality reduction were performed. Seven eosinophil populations were identified within the blood and thymus, respectively, five of which were specific for either tissue. Whereas the blood eosinophil populations varied between individuals, the thymic eosinophil populations were more uniform. The eosinophil-thymocyte co-cultures resulted in (1) an increase in CD4 SP thymocytes when eosinophils were cultured with DP thymocytes, (2) decreased frequency of CD8 SP thymocytes when these were cultured with eosinophils, and (3) a more mature thymic phenotype when eosinophils were cultured with CD4 SP thymocytes. Thymic eosinophils are a specialized population of eosinophils with a distinct phenotype that separates them from their blood counterparts, and in vitro they appear to favour CD4 SP thymocyte development to the detriment of CD8 SP thymocytes.

Eosinophils differentiate and mature in the thymus, outside of the bone marrow, in healthy individuals. Locally developed thymic eosinophils may contribute to the maturation and selection of human thymocytes.

A unique memory B-cell population characterized by the lack of or low expression of CD21, termed CD21–/low, has been described in tonsils and peripheral blood (PB) of healthy individuals.1Thorarinsdottir K. Camponeschi A. Cavallini N. Grimsholm O. Jacobsson L. Gjertsson I. et al.CD21(−/low) B cells in human blood are memory cells.Clin Exp Immunol. 2016; 185: 252-262Crossref PubMed Scopus (50) Google Scholar, 2Ehrhardt G.R. Hsu J.T. Gartland L. Leu C.M. Zhang S. Davis R.S. et al.Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells.J Exp Med. 2005; 202: 783-791Crossref PubMed Scopus (248) Google Scholar CD21–/low B cells are expanded in patients with chronic infections and autoimmune conditions,3Thorarinsdottir K. Camponeschi A. Gjertsson I. Mårtensson I.L. CD21−/low B cells: a snapshot of a unique B cell subset in health and disease.Scand J Immunol. 2015; 82: 254-261Crossref PubMed Scopus (56) Google Scholar but their role in health and disease is unclear. In the human and mouse thymus, B cells are present at very low percentages.4Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 5Perera J. Zheng Z. Li S. Gudjonson H. Kalinina O. Benichou J.I.C. et al.Self-antigen-driven thymic B cell class switching promotes T cell central tolerance.Cell Rep. 2016; 17: 387-398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar These cells express tissue-restricted antigens orchestrated by the autoimmune regulator (AIRE) transcription factor and in mice, the thymic B cells are efficient antigen-presenting cells (APCs) and seem to be involved in thymocyte selection and lineage decision. Consistent with previous results,6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 7Isaacson P.G. Norton A.J. Addis B.J. The human thymus contains a novel population of B lymphocytes.Lancet. 1987; 2: 1488-1491Abstract PubMed Scopus (201) Google Scholar we found that B cells in the thymus of young children represented less than 1% of total lymphocytes (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org) and were located exclusively in the medulla (Fig 1, A). Although their distribution and frequency were similar to those of the medullary thymic epithelial cells (Fig 1, B and C), the latter showed a 10 times larger surface area (Fig 1, D). The thymic B cells displayed a nuclear expression pattern of AIRE that was similar to that of medullary thymic epithelial cells, supported by previous findings showing that thymic B cells express AIRE6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar (Fig 1, E). B cells in early stages express CD10 and low levels of CD21. Unexpectedly, we found that half (43% ± 5%) of the thymic B cells were CD21–/low, and mainly negative for CD10. This mature phenotype is found in the adult PB, but in contrast the 10% (9.5% ± 3%) CD21–/low B cells in PB from children were mainly CD10+ (Fig 1, F and G). The proportions of early B-cell stages in the thymus were low, with less than 1% pro-B (CD19+CD10+CD34+CD24hiCD38hiIg–) and pre-B (CD19+CD10+CD34–CD24hiCD38hiIg–) and 15% immature B (CD19+CD10+) cells (Fig E1, C-E). To confirm and extend previous analyses,4Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 5Perera J. Zheng Z. Li S. Gudjonson H. Kalinina O. Benichou J.I.C. et al.Self-antigen-driven thymic B cell class switching promotes T cell central tolerance.Cell Rep. 2016; 17: 387-398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar we investigated the presence of immunoglobulin-switched B cells in the thymus and found IgG- and IgA-expressing cells (Fig 1, H). Most CD21+ B cells were unswitched IgM+IgD+ (87% ± 10%), whereas a third of the CD21–/low cells were switched IgG+ (22% ± 11%) or IgA+ (9% ± 4%). Although similar to CD21–/low B cells in adult PB, this finding was in sharp contrast to PB from the same children in whom switched cells were nearly absent (Fig 1, I, and Fig E1, F and G). Moreover, a few of the switched thymic B cells were positive for IgE or IgG4 (Fig E1, H), which suggests that all immunoglobulin isotypes are present on the thymic B cells. To exclude the possibility of cell microchimerism as the origin of the switched B cells, we analyzed the sex chromosomes of the thymic B cells from 2 male children. All B cells contained 1 X and 1 Y chromosome (Fig 1, J; see Table E3 in this article's Online Repository at www.jacionline.org), disproving the hypothesis of maternal origin. Furthermore, except for a few classical memory (CD27+CD38–) and plasma (CD27highCD38high) cells, the thymic B cells were mainly negative for the CD27 memory marker (Fig E1, I and J). Thus, in contrast to infant PB, almost a quarter in the thymus are switched CD27–CD21–/low B cells. Both CD21+ and CD21–/low B cells from adult and child PB were small in cellular size and expressed low levels of the activation markers CD69, CD95, and CD86, except for a bimodal expression of CD95 in adult CD21–/low B cells (Fig 2, A). In contrast, thymic CD21+ and CD21–/low B cells showed a bimodal expression of all markers, with the highest percentage of positive cells among the latter. MHC class II (HLA-DR) and CD40 levels were similar in CD21+ and CD21–/low B cells independent of origin. Comparing CD21–/low B cells from thymus and adult PB confirmed a larger cellular size as well as elevated levels of CD69, CD95, CD86, and CD40 in the former, whereas those of HLA-DR were similar (Fig 2, B). These results suggest that thymic CD21–/low B cells are activated and may function as APCs. CD21–/low B cells have been found to express an unusual pattern of inhibitory and homing receptors, such as CD11c, T-bet, CXCR3, and FcRL4.2Ehrhardt G.R. Hsu J.T. Gartland L. Leu C.M. Zhang S. Davis R.S. et al.Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells.J Exp Med. 2005; 202: 783-791Crossref PubMed Scopus (248) Google Scholar, 3Thorarinsdottir K. Camponeschi A. Gjertsson I. Mårtensson I.L. CD21−/low B cells: a snapshot of a unique B cell subset in health and disease.Scand J Immunol. 2015; 82: 254-261Crossref PubMed Scopus (56) Google Scholar, 8Rubtsova K. Rubtsov A.V. Cancro M.P. Marrack P. Age-associated B cells: a T-bet-dependent effector with roles in protective and pathogenic immunity.J Immunol. 2015; 195: 1933-1937Crossref PubMed Scopus (148) Google Scholar In adult PB, almost half of the CD21–/low B cells expressed CD11c, T-bet, and CXCR3, whereas only a small proportion expressed FcRL4. Of the thymic CD21–/low B cells, a quarter were positive for CD11c, CXCR3, and FcRL4, whereas less than 10% expressed T-bet (Fig 2, C; see Fig E2, A, in this article's Online Repository at www.jacionline.org). As a comparison, only low frequencies of the CD21+ B cells from PB and thymus expressed these markers. Finally, in support of a role in T-cell selection, the thymic CD21–/low B cells showed the highest levels of AIRE (Fig 2, D). To corroborate these findings, we sought to test the functionality of the thymic B cells as APCs in a thymic environment. We isolated and cocultured the CD3+ thymocytes with CD21+ or CD21–/low B cells from the same thymus, and after 24 and 72 hours assessed the expression of the activation marker CD25 on the thymocytes. As controls, we cultured part of the thymocytes without B cells in the absence or presence of phorbol 12-myristate 13-acetate. As expected, all thymocytes were positive for CD25 in the presence of phorbol 12-myristate 13-acetate, whereas in its absence around 20% were positive. After 72 hours, 30% of the thymocytes cocultured with either B-cell subset were CD25+, a percentage observed already after 24 hours but only in those cocultured with the CD21–/low B cells (Fig 2, E). This suggests that the CD21–/low B cells interact more rapidly with the thymocytes, possibly because of the higher expression of CD40 and even more so CD86 on the CD21–/low B cells than the CD21+. Under chronic immune stimulation, CD21–/low B cells exhibit reduced B-cell receptor (BCR)-induced phosphorylation of the extracellular signal-regulated kinase,9Visentini M. Marrapodi R. Conti V. Mitrevski M. Camponeschi A. Lazzeri C. et al.Dysregulated extracellular signal-regulated kinase signaling associated with impaired B-cell receptor endocytosis in patients with common variable immunodeficiency.J Allergy Clin Immunol. 2014; 134: 401-410Abstract Full Text Full Text PDF Scopus (17) Google Scholar which is important for proliferation and cell survival. We found that only half of the CD21–/low B cells from the thymus responded to BCR triggering, compared with those from adult PB of which almost all responded (Fig 2, F and G). The fact that half of the thymic CD21–/low B cells were unresponsive and expressed high levels of CD95 (Fig 2, A and B), an activation marker but also a death receptor, prompted us to investigate whether the CD21–/low cells were prone to undergo apoptosis. Fifty percent of thymic and 40% of adult PB CD21–/low B cells showed spontaneous apoptosis in vitro, as assessed by staining positive for active Caspases (CaspGLOW), whereas only a small percentage of CD21+ B cells were apoptosis prone (Fig 2, H, and Fig E2, B). The bimodal functional pattern of thymic CD21–/low B cells, with half of the cells responding efficiently to BCR stimulation together with the large size and the activated phenotype, indicated that at least part of these cells were proliferating (Fig 2, A and B). Consistent with this notion, 50% of the thymic CD21–/low B cells expressed high levels of the proliferation marker Ki67, compared with the markedly lower expression in adults and CD21+ B cells (Fig 2, I, and Fig E2, B). Collectively, these results show that around half of the thymic CD21−/low B cells are cycling and half are prone to undergo apoptosis. In this study, we demonstrate that half of the B cells residing in the infant thymus are mature CD21–/low cells that are large in size and express high levels of AIRE and typical activation markers. Despite being CD27–, almost half are switched B cells that, as expected, were nearly absent in the PB from the same infants. In mice, switching is probably facilitated by cognate interactions with thymocytes.5Perera J. Zheng Z. Li S. Gudjonson H. Kalinina O. Benichou J.I.C. et al.Self-antigen-driven thymic B cell class switching promotes T cell central tolerance.Cell Rep. 2016; 17: 387-398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar The localization of the human CD21–/low B cells in the medulla suggests a role in T-cell selection, and their role as APCs is supported by their high expression of CD40, CD86, and HLA-DR, and by their ability to activate the thymocytes. These findings, together with our observations that half of the thymic CD21–/low B cells are apoptosis prone and half proliferating, suggest that this population is composed of APCs with a high turnover. Altogether, our data provide evidence that thymic B cells are heterogeneous, with subsets reminiscent of those in health and under conditions of chronic immune stimulation.1Thorarinsdottir K. Camponeschi A. Cavallini N. Grimsholm O. Jacobsson L. Gjertsson I. et al.CD21(−/low) B cells in human blood are memory cells.Clin Exp Immunol. 2016; 185: 252-262Crossref PubMed Scopus (50) Google Scholar, 3Thorarinsdottir K. Camponeschi A. Gjertsson I. Mårtensson I.L. CD21−/low B cells: a snapshot of a unique B cell subset in health and disease.Scand J Immunol. 2015; 82: 254-261Crossref PubMed Scopus (56) Google Scholar Further untangling of the functional properties of thymic B cells may lead to a better understanding of their role in central tolerance and autoimmune diseases. We thank the Clinical Genetics Department at the Sahlgrenska Hospital for performing the Fluorescence in situ Hybridization (FISH) analysis. We also acknowledge all help and assistance by the staff at Drottning Silvias Children's Hospital, and we thank the patients and their families for the contribution to the study. Thymic tissue samples and matched PB were obtained from children undergoing corrective cardiac surgery at Sahlgrenska University Hospital, Gothenburg, Sweden, where the thymus is surgically removed to gain access to the heart. Parents gave informed consent, and the study was approved by the Regional Ethical Board at the University of Gothenburg (no. 217-12, 2012-04-26). Demographic characteristics and the congenital heart defect of patients included in the study are presented in Table E1. The children did not have any known chronic infections or inflammatory conditions. Adult PB was obtained from healthy blood donors. No informed consent was needed because no personal information about the donors was recorded (Swedish law 2003: 460, paragraphs 4 and 13). The thymic tissue was collected immediately in cold PBS. Pieces of tissue were embedded in optimal cutting temperature compound (Histolab Products AB, Västra Frölunda, Sweden) for immunohistochemistry and snap frozen in isopentane precooled with liquid nitrogen. Thymic lymphocytes were extracted by injecting tissue with PBS, cutting the tissue into small pieces (1 mm3), and gently pressing against a 40-μm cell strainer. PBMCs were obtained after separation on Ficoll (GE Healthcare, Little Chalfont, UK), according to the manufacturer's protocol. Cells were filtered using a 40-μm filter and stained immediately or frozen for later analysis. Thymus tissue was enzymatically (DNase I, Worthington, Lakewood, NJ, and Liberase TH, Roche, Risch-Rotkreuz, Switzerland) and mechanically digested with a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). For ImageStream analysis, the single-cell suspension was density centrifuged with Percoll 1.07 g/mL (GE Healthcare Life Sciences, Chicago, Ill) to enrich thymic epithelial cells. Cells were stained at a concentration of (10-50) × 106 cells/mL in a volume of 100 μL. The antibodies and dilutions used are presented in Table E2. Mouse and rat sera were used to inhibit unspecific binding. For flow cytometry, the cells were acquired on a FACSVerse (BD Biosciences, San Diego, Calif), and data were analyzed using FlowJo software (TreeStar, Inc, Ashland, Ore). For ImageStream analysis, the cells were acquired and analyzed on an ImageStream X Mark II imaging flow cytometer (Amnis, Seattle, Wash). Thymic lymphocytes were negatively depleted with anti-CD3 Dynabeads (Invitrogen, Carlsbad, Calif) and sorted into 2 populations: CD3–CD19+CD21+, CD3–CD19+CD21–/low B cells. CD3+ thymocytes were sorted from nondepleted samples and cultured in RPMI 1640 supplemented with l-glutamine, nonessential amino acids, sodium pyruvate, penicillin, streptomycin, β-mercaptoethanol, and 10% FBS (complete medium), with and without phorbol 12-myristate 13-acetate 1 μg/mL (InvivoGen, Toulouse, France). Part of the CD3+ thymocytes were cultured without phorbol 12-myristate 13-acetate but with the addition of either CD21+ or CD21–/low B cells. To support thymocytes survival, IL-2 20 ng/mL (R&D Systems, Minneapolis, Minn) and IL-7 0.2 ng/mL (Peprotech, Rocky Hill, NJ) were added to the cultures. Thymocytes CD25 expression was analyzed after 24 and 72 hours. The cells were acquired with a FACSlyric (BD Biosciences) and data were analyzed using FlowJo software. Cells were sorted on a SH800Z cell sorter (Sony Biotechnology, San Jose, Calif) with postsort purities of greater than or equal to 95% (Fig E3, A and B). For this assay, freshly isolated cells from PB and thymus were used. The phosphorylation of extracellular signal-regulated kinase was studied using the BD PhosFlow Protocol for Human PBMCs (Becton-Dickinson Biosciences, Franklin Lakes, NJ). After isolation, cells were resuspended in 100 μL of complete medium at a concentration of 2 to 10 × 106 cells and then split into 2 vials and left to equilibrate at 37°C for at least 20 minutes. An equal volume of prewarmed complete medium, with and without (unstimulated control) 20 μg/mL of F(ab')2 anti-human IgM/G/A (Jackson Immunoresearch Laboratories, Bar Harbor, Me), was then added, and then the cells were incubated for 10 minutes at 37°C. The samples were then fixed by the addition of an equal volume of prewarmed BD Cytofix Fixation Buffer for 10 minutes at 37°C, washed twice in Phosflow Perm/Wash Buffer I, split into 2 vials, and stained for 60 minutes at room temperature protected from light either with anti–phosphorylated extracellular signal-regulated kinase 1/2 Alexa Fluor 647 or with a mouse IgG Alexa Fluor 647 as control. Surface stainings with other mAbs were performed as requested by the experimental design. The samples were washed and resuspended in Phosflow Perm/Wash Buffer I and finally acquired with a flow cytometer. Freshly isolated cells from PB and thymus were resuspended in complete medium supplemented with penicillin and streptomycin and then cultured in 96-well plates at 2 × 105 cells/well. The cells were then harvested after 24 hours and the apoptotic cells detected by incubation with zVAD-FMK-FITC (CaspGLOW; eBioscience, Waltham, Mass) in RPMI 1640 for 1 hour at 37°C, according to manufacturer's protocol. Surface stainings with other mAbs were performed as requested by the experimental design. The cells were then washed and analyzed by flow cytometer. Optimal cutting temperature compound–embedded thymus pieces were cut to 7-μm sections, air dried, and fixed with acetone for 5 minutes. Sections were rehydrated in PBS and blocked with Protein block (Dako, Santa Clara, Calif). The tissues were stained for 1 hour at 4°C with primary antibody. Antibodies and dilutions are listed in Table E2. The sections were incubated for 30 minutes at 4°C with secondary antibodies and Hoechst 34580 (Thermo Fisher Scientific, Waltham, Mass). If the secondary antibodies were biotinylated, the slides were incubated at 4°C for 30 minutes with Streptavidin Alexa Fluor 488 (Thermo Fisher Scientific). Sections were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and acquired using an LSM700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Image analysis was performed with ImageJ software (Rasband WS, ImageJ, US National Institutes of Health, Bethesda, Md) measuring area with threshold set to 35, 255. Immunoglobulin switched and unswitched cells were sorted from 2 thymuses from 4-day old males with a Sony SH800 cell sorter and cytospinned to a glass slide. The slides were placed in hypotone solution (0.3% NaCl) in deionized water with increased concentration of fixation solution added (99.5% ethanol and ice cold acetic acid, 3:1) stepwise to dehydrate cells. FISH was performed using probes for X chromosome centromere and Y chromosome centromere (Vyvis CEP X DXZ1 Spectrum Green, Y DYZ3 Spectrum Orange, Abott Molecular, Inc, Chicago, Ill). All switched cells were counted and compared with unswitched cells as presented in Table E3. Outliers were removed by using the Robust regression and Outlier removal (ROUT) method, with Q set to 5%. Shapiro-Wilk test was used to evaluate normality. Data were analyzed by 1-way ANOVA followed by uncorrected Fisher Least Significant Difference (LSD) multiple comparison test, using Graph-Pad Prism version 7 (La Jolla, Calif). Statistical significance was set as P < .05. *P < .05, **P < .01, ***P < .001, and ****P < .0001.Fig E2A and B, Samples from adult PB and child thymus. A, Representative histograms for expression of CD11c, T-bet, CXCR3, and FcRL4 on CD21–/low B cells. B, Representative histograms of CaspGLOW and Ki67 in CD21+ and CD21–/low B cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3A and B, Gating strategy and purity control for the in vitro stimulation assay. The gating and purity for sorting (Fig E3, A) CD21+ and CD21–/low thymic B cells and (Fig E3, B) CD3+ thymocytes. FSC-A, Forward scatter-area; SSC-A, side scatter-area.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Demographic characteristics and cause for cardiac surgery of included patientsPatient no.Congenital heart defectSexAge at time of surgery (d)Analyzed results presented in figure12E1E2E31TGA, VSDM3C, EE-HB2APWM4F, G, I, JA-CA, B, F, G, I, JA, B3TGA, VSDM4D, F, G, I, JA-CA, B, F, G, I, JA, B4TGA, VSDM4CF-H5HLHSF5C-E6VSDM8E7IAA, TAF8C-E8DORVM11C-E9PA, VSD, DORVM12C10VSDF63C-E11VS, PSM123C, D, I12VSDM152C-E13VSDF169F-IA-D, IA, B, F, G, I, JA, B14ASDF170F-H15VSDM177C-E16FallotF195C17VSDF268C-E18FallotM376H19ASD, PDA, VSDF520F, G, IA-D, IA, B, F, G, I, JA, B20AS, PS, WSM726F, G, IA-CA, B, F, G, I, JA, B21SVDF1923EA, BAPW, Aortopulmonary window; AS, aorta stenosis; ASD, atrial septal defect; DORV, double outlet right ventricle; Fallot, Tetralogy of Fallot; F, female; HLHS, hypoplastic left heart syndrome; IAA, interrupted aortic arch; M, male; PA, pulmonary atresia; PDA, patent ductus arteriosus; PS, pulmonary stenosis; SVD, sinus venosus atrial defect; TA, truncus arteriosus; TGA, transposition of the great arteries; VSD, ventricular septal defect; WS, Williams syndrome. Open table in a new tab Table E2Antibodies used in the studyAntibodyCloneCompanyDilutionFlow cytometry CD3-APC-H7SK7BD50 CD10-APCHI10aBD20 CD11c-FITCB-ly6BD20 CD19-BV510HIB19Biolegend40 CD21-PerCP-Cy5.5Bu32Biolegend50 CD24-PE-Cy7ML5BD40 CD25-APC2A3BD50 CD27-PEL128BD40 CD34-PerCP-Cy5.58G12BD10 CD38-BV421HIT2BD20 CD40-PE5C3BD20 CD69-FITCL78BD10 CD86-APC2331 (FUN-1)BD20 CD95-bioDX2BD10 CD193(CXCR3)-PE1C6BD20 EpCAM-PE9C4Biolegend25 ERK1/2-AF48820ABD20 FcRL4-PE413D12BioLegend20 HLA-DR-bioL243BD50 IgD-PEIA6-2BD40 IgD-FITCIA6-2BD50 IgM-FITCCat nr F0058DAKO200 IgG-PEG18-145BD25 IgA-FITCCat nr F0057DAKO150 T-bet-AF4884B10BioLegend20Confocal microscopy CD19SP110Invitrogen200 CD19HIB19BD100 Cytokeratin 5Cat nr PRB-160PBioLegend100 IgA6E2C1Dako20 IgECIA-E-7.12Dako100 IgGH10015Medac200 IgG4HP6025SouthernBiotech100 IgM-bioG20-127BD100 Anti-rabbit-AF488A11034Life Technologies200 Anti-mouse-AF555A21422Life Technologies400 Anti-rabbit-bioCat nr E0432Dako400 Anti-mouse bioBA-2000Vector Laboratories200ImageStream X CD19-PEHIB19BD10 EpCAM-PE9C4Biolegend25 AIRE-bioTM-724eBioscience100 Rat IgG2a-bioeBR2aeBioscience100 Open table in a new tab Table E3Chromosomal FISH analysis shows that the switched B cells in the thymus are not derived from the motherB cell stateXXXYTotal no. of counted cellsSwitched04343Unswitched0270270Switched03434Unswitched0500500 Open table in a new tab APW, Aortopulmonary window; AS, aorta stenosis; ASD, atrial septal defect; DORV, double outlet right ventricle; Fallot, Tetralogy of Fallot; F, female; HLHS, hypoplastic left heart syndrome; IAA, interrupted aortic arch; M, male; PA, pulmonary atresia; PDA, patent ductus arteriosus; PS, pulmonary stenosis; SVD, sinus venosus atrial defect; TA, truncus arteriosus; TGA, transposition of the great arteries; VSD, ventricular septal defect; WS, Williams syndrome.

B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers' children exhibit higher BAFF levels in cord blood than non-farmers' children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

Pediatric cardiac surgery due to congenital heart defects has become more common in the last decades. As the thymus obstructs the surgeon's access during surgery it is often removed, either completely or partially. In Sweden, with 10 million inhabitants, approximately 250 infants are thymectomized annually according to the Swedish Health Care Registries. In a previous study1Gudmundsdottir J. Oskarsdottir S. Skogberg G. Lindgren S. Lundberg V. Berglund M. et al.Early thymectomy leads to premature immunologic ageing: an 18-year follow-up.J Allergy Clin Immunol. 2016; 138: 1439-1443.e10Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar of 18-year-old individuals thymectomized before the age of 6 months (N = 11), we confirmed that early thymectomy was associated with T-cell lymphopenia predominantly affecting the naive T cells. T-cell receptor excision circles (TRECs), generated during genetic recombination of T-cell receptor (TR) loci, were severely decreased after thymectomy. This prominent and consistent decrease in TRECs and naive T cells reveals a reduced thymic output. A decreased diversity of the TR repertoire was apparent when analyzed with flow cytometry using mAbs recognizing 24 different defined TR variable β chain families. Furthermore, the thymectomized individuals reported infections and allergies more frequently, although the number of subjects was too small to draw any firm conclusions.1Gudmundsdottir J. Oskarsdottir S. Skogberg G. Lindgren S. Lundberg V. Berglund M. et al.Early thymectomy leads to premature immunologic ageing: an 18-year follow-up.J Allergy Clin Immunol. 2016; 138: 1439-1443.e10Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar In a separate population-based cohort study we have observed increased risks of autoimmune diseases, cancer, and infections in individuals thymectomized early in life (Gudmundsdottir et al, unpublished results, 2017). Lymphopenia leads to a diminished immune repertoire even if the total T-cell numbers are normalized by peripheral homeostatic proliferation. The relative oligoclonality of the T-cell compartment is presumed to be a risk factor for the development of both autoimmune diseases and infections.2Merayo-Chalico J. Rajme-Lopez S. Barrera-Vargas A. Alcocer-Varela J. Diaz-Zamudio M. Gomez-Martin D. Lymphopenia and autoimmunity: a double-edged sword.Hum Immunol. 2016; 77: 921-929Crossref PubMed Scopus (31) Google Scholar Even though T cells are primarily affected by thymectomy, an impact on the B-cell repertoire, due to a lack of T-cell help during the B-cell activation, cannot be excluded. The aim of this study was to evaluate the long-term effects of early thymectomy on the T- and B-cell repertoire diversity using next-generation DNA sequencing of the TR β (TRB) chain and the immunoglobulin heavy (IGH) chain. The methods of TRB and IGH chain sequencing from CD4+, CD8+, and CD19+ cells are described in this article's Online Repository at www.jacionline.org. Qualitative differences between thymectomized individuals and controls in the TRB chain were minor. For CD4+ T cells, no significant changes were found in the number of junctional deletions and N- or P-nucleotides, and no effects were demonstrated on complementarity determining region 3 lengths (Fig 1, A). For CD8+ T cells, subtle qualitative changes were detected in the thymectomized individuals. No differences were identified in N- and P-nucleotides, but only a small increase of approximately 1 nucleotide deletion in the junctional region of the productive rearrangements was found. Although this increase was statistically significant, it had no effect on the complementarity determining region 3 lengths (Fig 1, E), or on amino acid composition (see Fig E2 in this article's Online Repository at www.jacionline.org). The clonality of the repertoire was measured using a method described by Boyd et al3Boyd S.D. Marshall E.L. Merker J.D. Maniar J.M. Zhang L.N. Sahaf B. et al.Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing.Sci Transl Med. 2009; 1: 12ra23Crossref PubMed Scopus (263) Google Scholar in which the reoccurrence of a certain rearrangement in 6 replicates is measured. Coincidences tables (Fig 1, B and F) show reoccurrences of identical TRB chain rearrangements analyzed in 6 different reactions from the same individual. The increased number of reoccurrences is indicative of oligoclonality because identical sequences are presumed to originate from the same T-cell clone. The clonality index was increased in thymectomized individuals compared with healthy controls in both CD4+ (Thymectomized individuals = 3.71 × 10−5, controls = 2.04 × 10−5) and even more so in the CD8+ T-cell subset (Thymectomized individuals = 1.92 × 10−4, controls = 7.06 × 10−5) (Fig 1, C and G, respectively). These results are in agreement with previous TR variable β chain flow cytometry results from the same individuals,1Gudmundsdottir J. Oskarsdottir S. Skogberg G. Lindgren S. Lundberg V. Berglund M. et al.Early thymectomy leads to premature immunologic ageing: an 18-year follow-up.J Allergy Clin Immunol. 2016; 138: 1439-1443.e10Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar where oligoclonality was detected for both CD4+ and CD8+ T cells, but was especially prominent in the CD8+ T cells. This discrepancy between the CD4+ and CD8+ subsets is in agreement with previous studies on normal adult TR repertoire, where clonality indexes are increased more in the CD8+ subset, and can also be associated with the increased proportion of memory T cells in the thymectomized individuals (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org).4Qi Q. Liu Y. Cheng Y. Glanville J. Zhang D. Lee J.Y. et al.Diversity and clonal selection in the human T-cell repertoire.Proc Natl Acad Sci U S A. 2014; 111: 13139-13144Crossref PubMed Scopus (409) Google Scholar There was no correlation between age at thymectomy and clonality scores (Fig E1, C). The VB-JB recombination usage was clearly affected in CD8+ T cells from thymectomized individuals in which dominant VB-JB combinations could be seen (Fig 1, H), whereas more subtle differences were observed for their CD4+ T cells (Fig 1, D). Results shown are Circos plots, where the bandwidth is proportional to the usage frequency. The different VB regions are marked, and their combination with the different JB regions shown. Examples are from 1 thymectomized individual with distinct alterations and 1 control. Complete results for VB-JB, as well as VB-DB and DB-JB recombinations, for all subjects are shown in Figs E3 and E4 in this article's Online Repository at www.jacionline.org. The CD19+ B-cell IGH chain rearrangements were similarly analyzed but no differences were detected between thymectomized and controls (see Fig E5 in this article's Online Repository at www.jacionline.org). There was a negative correlation between the number of both CD4+ and CD8+ T cells in peripheral blood and the clonality of the TRB chain rearrangements; T-cell lymphopenia was associated with increased clonality index (Fig 2, A and B). Separate analyses of naive (CD45RA+) and memory (CD45RO+) T-cell subsets revealed that the observed oligoclonality correlated only with the number of naive T cells (Fig 2, C and D), and not with the number of memory T cells (Fig 2, E and F). The naive T-cell lymphopenia in thymectomized individuals is apparent in Fig 2, C and D, where thymectomized individuals are depicted by red dots, and controls are blue, whereas this difference was not observed in the memory T-cell populations (Fig 2, E and F). The results from this study clearly show that near-total thymectomy early in life is associated with reduced diversity of both CD4+ and CD8+ T-cell receptor repertoire, whereas the B-cell repertoire was found to be unaffected. Although sequencing of the TRB chain gives a much more detailed estimate of the true TR diversity than previously used methods, it has some limitations. For example, different primers have varying PCR efficacy, causing overrepresentation of certain V and J genes. Also, sequencing errors accumulate in the vast amount of sequence data produced, which impedes the distinction between sequence errors and true rare TRB chain sequence variants.5Hou X.L. Wang L. Ding Y.L. Xie Q. Diao H.Y. Current status and recent advances of next generation sequencing techniques in immunological repertoire.Genes Immun. 2016; 17: 153-164Crossref PubMed Scopus (54) Google Scholar Nonetheless, these technical limitations presumably affect the thymectomized as well as the controls in a similar way. The constricted TRB chain repertoire following thymectomy and its correlation with the degree of T-cell lymphopenia reported in the present study strengthens the connection between thymectomy and unwanted clinical consequences and could suggest a possible mechanistic link. Decreased repertoire diversity in individuals thymectomized early in life may compromise their adaptive immune functions, leading to an elevated susceptibility to infections, autoimmune diseases, and malignancies. We gratefully acknowledge all assistance provided by staff at the Queen Silvia Children's Hospital and the Department of Clinical Immunology, Sahlgrenska University Hospital. Peripheral blood samples were drawn from individuals who were approximately 18-year-old and thymectomized before the age of 6 months (n = 11) and an equal number of healthy age- and sex-matched controls as previously described.E1Gudmundsdottir J. Oskarsdottir S. Skogberg G. Lindgren S. Lundberg V. Berglund M. et al.Early thymectomy leads to premature immunologic ageing: an 18-year follow-up.J Allergy Clin Immunol. 2016; 138: 1439-1443.e10Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar None of the included patients had clinical signs or symptoms suggestive of a syndromic congenital cardiac malformation, including trisomy 21, 22q11.2 deletion syndrome, or CHARGE syndrome. PBMCs were isolated and CD4+, CD8+ T cells and CD19+ B cells were sorted using flow cytometry; followed by DNA extraction.E1Gudmundsdottir J. Oskarsdottir S. Skogberg G. Lindgren S. Lundberg V. Berglund M. et al.Early thymectomy leads to premature immunologic ageing: an 18-year follow-up.J Allergy Clin Immunol. 2016; 138: 1439-1443.e10Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar The ratio of naive versus memory CD4+ and CD8+ T cells is provided (Fig E1, A and B). IGH and TRB chain rearrangements were amplified in independent multiplex PCR reactions using either the forward VH1-6 FR1 and JH consensus reverse primer or the 23 Vβ forward and 13 Jβ reverse primers (all BIOMED-2),E2van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. et al.Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar which were adapted by adding a multiplex identifier sequence tag adaptor. Fifty nanograms of DNA were used for each reaction. The amount of DNA in a single cell is approximately 6 picograms corresponding to approximately 8300 analyzed CD4+, CD8+, and CD19+ cells in each reaction. The PCR products were purified and sequenced using gel extraction (Qiagen, Valencia, Calif) and Agencourt AMPure XP beads (Beckman Coulter, Fullerton, Calif). Subsequently, the PCR product concentrations were measured using the Quant-it Picogreen dsDNA assay (Invitrogen, Carlsbad, Calif). The purified PCR products were sequenced using the MiSeq sequencer using low-complexity 300 base-pair paired end sequencing (V3 chemistry). The reads were uploaded to IMGT HighV-Quest.E3Alamyar E. Duroux P. Lefranc M.P. Giudicelli V. IMGT((R)) tools for the nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J repertoires, polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-QUEST for NGS.Methods Mol Biol. 2012; 882: 569-604Crossref PubMed Scopus (326) Google Scholar Subsequently, the output files were analyzed using an extended version of the IGGalaxy tool.E4Moorhouse M.J. van Zessen D. IJspeert H. Hiltemann S. Horsman S. van der Spek P.J. et al.ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS.BMC Immunol. 2014; 15: 59Crossref PubMed Scopus (25) Google Scholar For the analyses we only used unique productive or unproductive rearrangements where unique is defined as rearrangements having the same amino acid sequence of the complementarity determining region 3. Table E1 shows the number of unique productive reads for CD4+, CD8+ T cells and B cells for all subjects. Information about the V, D, and J gene usage, composition of the junctional regions, and the complementarity determining region 3 was extracted from the data provided by IMGT HighV-Quest. IGH chain rearrangements were analyzed in a multiplex PCR reaction according to BIOMED-2 as previously described.E2van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. et al.Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar, E5Driessen G.J. Ijspeert H. Weemaes C.M. Haraldsson A. Trip M. Warris A. et al.Antibody deficiency in patients with ataxia telangiectasia is caused by disturbed B- and T-cell homeostasis and reduced immune repertoire diversity.J Allergy Clin Immunol. 2013; 131: 1367-1375.e9Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Statistical analyses were done with Graphpad Prism version 7.0b (Graphpad Software Inc, San Diego, Calif). Clonality score was considered the primary outcome measure, and other outcomes as secondary. To assess differences between thymectomized individuals and controls, the 2-tailed Mann-Whitney test was used. The nonparametric Spearman correlation coefficient of clonality score and T-cell numbers, as well as for clonality score and age at thymectomy, were calculated, and a simple linear regression analysis was performed.Fig E2T-cell receptor amino acid usage in complementarity determining region 3 for (A) CD4+ T cells and (B) CD8+ T cells. The positively charged amino acids are indicated in red and the negatively charged in blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3CD4+ T-cell recombination chain usage for D-J, V-D, and V-J shown as circos plots. The width of the band is proportional to the frequency. Individual values for each Tx individuals (N = 11) and controls (N = 11). Tx, Thymectomized.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4CD8+ T-cell recombination chain usage for D-J, V-D, and V-J shown as circos plots. Individual values for each Tx individuals (N = 9) and controls (N = 11). Tx, Thymectomized.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5B-cell IGH chain characteristics and clonality index. A, IGH chain junctional deletions, N- and P-nucleotide insertions (average number, individual values with median, P value). B, Frequency of IGH chain amino acid usage in CDR3. The positively charged amino acids are indicated in red and the negatively charged in blue. C, CDR3 length distribution (group values, mean with SEM, nt = nucleotides). D, B-cell clonality index (individual results with median and 95% CI, P value). E, Coincidences table as described by Boyd et alE6Boyd S.D. Marshall E.L. Merker J.D. Maniar J.M. Zhang L.N. Sahaf B. et al.Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing.Sci Transl Med. 2009; 1: 12ra23Crossref PubMed Scopus (339) Google Scholar where increased sequence recurrences indicate increased clonality. Red dots, line, and staples, Tx individuals; blue dots, line, and staples, controls. CDR3, Complementarity determining region 3; ns, not significant; Tx, thymectomized.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Number of productive unique reads per patient for CD4+, CD8+ T cells and CD19+ B cellsThymectomyNo. of unique productive readsCD4CD8CD1911553311680321542104151041522605311244457723538414483750034402516203464486150812210678701782428467811720347723241915697140921013841103941110480599235158ControlsCD4CD8CD1911517114469289402149526686369643152311079935147416411498324705516960102924026761571514544338277133931275025684819970114103387491303012240102397059262799411155611221731087 Open table in a new tab

Early thymectomy is routinely performed in infants undergoing cardiac surgery as the thymus lies anterior to the heart in the mediastinum obstructing the surgeon's access. Early cardiac surgery has become more common in the last decades and in Sweden, with close to 10 million inhabitants, approximately 250 infants are thymectomized (Tx) annually. The population of Tx individuals is thus increasing both in numbers and in age. The ultimate effect of early thymectomy is still obscure although immunologic alterations have been described. Lymphocyte subsets are affected with a T-cell lymphopenia, characterized by a decrease in naive T cells with a concomitant increase in the memory T-cell population.1van den Broek T. Delemarre E.M. Janssen W.J. Nievelstein R.A. Broen J.C. Tesselaar K. et al.Neonatal thymectomy reveals differentiation and plasticity within human naive T cells.J Clin Invest. 2016; 126: 1126-1136Crossref PubMed Scopus (62) Google Scholar, 2van Gent R. Schadenberg A.W. Otto S.A. Nievelstein R.A. Sieswerda G.T. Haas F. et al.Long-term restoration of the human T-cell compartment after thymectomy during infancy: a role for thymic regeneration?.Blood. 2011; 118: 627-634Crossref PubMed Scopus (42) Google Scholar, 3Prelog M. Keller M. Geiger R. Brandstatter A. Wurzner R. Schweigmann U. et al.Thymectomy in early childhood: significant alterations of the CD4(+)CD45RA(+)CD62L(+) T cell compartment in later life.Clin Immunol. 2009; 130: 123-132Crossref PubMed Scopus (85) Google Scholar The effects of thymectomy on the regulatory T (Treg)-cell population differ between studies. One study showed that although the absolute Treg-cell number was lower, the Treg-cell proportion was increased, albeit with a decrease in naive Treg cells.4Schadenberg A.W. van den Broek T. Siemelink M.A. Algra S.O. de Jong P.R. Jansen N.J. et al.Differential homeostatic dynamics of human regulatory T-cell subsets following neonatal thymectomy.J Allergy Clin Immunol. 2014; 133 (e1-6): 277-280Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar T-cell receptor excision circles (TRECs) have consistently been found to decrease after thymectomy although to varying degrees.2van Gent R. Schadenberg A.W. Otto S.A. Nievelstein R.A. Sieswerda G.T. Haas F. et al.Long-term restoration of the human T-cell compartment after thymectomy during infancy: a role for thymic regeneration?.Blood. 2011; 118: 627-634Crossref PubMed Scopus (42) Google Scholar, 3Prelog M. Keller M. Geiger R. Brandstatter A. Wurzner R. Schweigmann U. et al.Thymectomy in early childhood: significant alterations of the CD4(+)CD45RA(+)CD62L(+) T cell compartment in later life.Clin Immunol. 2009; 130: 123-132Crossref PubMed Scopus (85) Google Scholar Studies published to date have not shown any alteration in overall disease risk, but generally the groups are small and often heterogeneous, with a lack of information on the amount of thymic tissue that was removed and with a short follow-up time. Here, we present results from a prospective study of the immunologic impact of early thymectomy (age, <6 months, >90% thymic removal, n = 11) with blood samples collected preoperatively, at 18 months, and 18 years later. Group characteristics and clinical data are presented in Table E1, Table E2 (see this article's Online Repository at www.jacionline.org). Multivariate factor analysis (orthogonal projection to latent structures discriminant analysis) reveals a clear distinction between Tx individuals and healthy controls (Fig 1, A and B). A decrease in T-cell number was apparent and consistent over time in the Tx group (see Table E3 in this article's Online Repository at www.jacionline.org; Fig 1, C-G). Of interest is that the Tx group had a decreased number of lymphocytes, particularly T cells (Table E3; Fig 1, H), even preoperatively. At the 18-year follow-up, the number of naive helper T cells (CD45RA+) was lower in the Tx group than in controls (0.15 vs 0.49 × 109/L; P < .0001) whereas the memory helper T cells (CD45RO+) were unaffected (0.34 vs 0.30 × 109/L; P = .48), thereby showing a proportional increase in relation to the already low CD4+ T-cell number (Fig 1, I and J). In cytotoxic T cells, the same was true: absolute CD8+CD45RA+ naive T-cell number was lower in cases (0.11 vs 0.35 × 109/L; P = .0002) but the CD8+CD45RO+ memory T-cell number was unaffected (0.11 vs 0.13 × 109/L; P = .27) (Fig 1, K and L). The cell surface marker CD31 has been defined as a marker of recent thymic emigrants.5Kimmig S. Przybylski G.K. Schmidt C.A. Laurisch K. Mowes B. Radbruch A. et al.Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood.J Exp Med. 2002; 195: 789-794Crossref PubMed Scopus (379) Google Scholar Tx individuals showed a lower proportion of CD31+ helper T cells (55% vs 81%; P = .034), but with a wide range (25% to 89%), whereas the results for the control group were higher and not as wide-ranging (76% to 90%) (Fig 1, M). There was a linear correlation between naive CD4+ and CD8+ cells, indicating that Tx individuals were similarly affected in both subsets (Fig 1, N). Also, the Tx group (black circles) is clearly distinct from the control group (open circles) with a lower number of both CD4+ and CD8+ naive T cells. The total number of Treg cells was lower in the Tx group than in controls (0.035 vs 0.053 × 109/L; P = .0417) (Fig 2, A). The proportions, however, were unaffected (6.5% vs 6.1%; P = .54) (Fig 2, B). Further analysis of the Treg-cell subset reveals that the Tx group showed a decreased number of naive Treg cells but no effect on the memory subset number, resulting in a higher percentage of memory Treg cells in the Tx group (Fig 2, C-F). However, the Treg-cell subset CD4+CD45RA−CD25++ that has been shown to have the greatest suppressive potential6Miyara M. Yoshioka Y. Kitoh A. Shima T. Wing K. Niwa A. et al.Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor.Immunity. 2009; 30: 899-911Abstract Full Text Full Text PDF PubMed Scopus (1654) Google Scholar did not differ between the groups (Fig 2, G and H). Flow cytometry gating strategies are shown in Fig E1 in this article's Online Repository at www.jacionline.org. Analysis of T-cell receptor variable chain β (TCR Vβ) usage in CD4+ and CD8+ cells showed that Tx individuals had signs of oligoclonality, which was particularly striking for the CD8+ cells (Fig 2, I). Perturbations in Tx individuals defined as a TCR Vβ chain usage deviating more than ±3 SD from the mean value was noted in 8 out of 10 individuals regarding CD8+ cells and in 6 out of 11 regarding CD4+ cells, whereas only a single chain in 1 control was affected (see Table E4 in this article's Online Repository at www.jacionline.org). Individual TCR Vβ family usage is provided in Fig E2 and flow cytometry gating strategy in Fig E3 in this article's Online Repository at www.jacionline.org. TREC PCR analysis showed nondetectable values in 10 out of 11 Tx individuals (Fig 2, J), whereas the controls had a wide distribution of TREC content. The Tx individuals had shorter telomere length in their CD8+ T-cell population compared with the controls, 2.66 versus 3.32, P = .036 (Fig 2, K), as measured by a telomere/single gene ratio according to a method originally described by Cawthon in 2002.7Cawthon R.M. Telomere measurement by quantitative PCR.Nucleic Acids Res. 2002; 30: e47Crossref PubMed Google Scholar The Tx CD4+ T-cell population also had shorter telomeres, 2.73 versus 3.32, but this difference did not reach statistical significance with a P value of .068. The telomere length in CD19+ B cells was unaffected by thymectomy (3.03 vs 3.45; P = .2335).Fig 2Treg-cell number (A) and proportion (B). Naive Treg-cell (CD4+CD25+CD127lowCD45RA+) number (C) and proportion (D). Memory Treg-cell (CD4+CD25+CD127lowCD45RO+) number (E) and proportion (F). Highly suppressive Treg-cell (CD4+CD45RA-CD25++) number (G) and proportion (H). Results shown as individual values with mean and SD; P value summary indicated on each graph. I, TCR Vβ chain usage in CD8+ cells and CD4+ cells presented as a scatter graph. Thymectomized (Tx) = red dots, controls = blue dots. Difference in usage of chains Vβ3 and Vβ14 in CD8+ cells statistically significant, indicated by arrows (Holm-Sidak method for multiple t-test comparison, α = 0.05). Nomenclature according to Wei et al.9Wei S. Charmley P. Robinson M.A. Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire.Immunogenetics. 1994; 40: 27-36Crossref PubMed Scopus (146) Google Scholar J, TRECs per 106 cells. K, Telomere/single gene ratio in CD4+, CD8+ T cells, and CD19+ B cells. Fig 2, all data from 18 years follow-up. Data shown as individual values with mean and SD; P value summary indicated on graph. ns, Not significant. *P ≤ .05 and **P ≤ .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This prospective study of the immunologic impact of early thymectomy shows a quantitative defect in the T-cell compartment primarily affecting the naive T-cell population with a probable peripheral T-cell proliferation of seeded clones leading to increased proportions of memory T cells. Treg cells seem to be affected to the same extent as other T cells with diminished numbers. The T-cell receptor repertoire is skewed with signs of oligoclonality, the thymic output severely affected with a near-absence of TRECs, and the replicative potential presumably decreased as indicated by a shorter T-cell telomere length. In general, these results are indicative of a persistent and severe immunologic dysfunction even 18 years after thymectomy. To investigate whether thymectomy has any clinical consequences, a future large-scale epidemiologic study would be of value. We gratefully acknowledge all assistance provided by staff at the Queen Silvia Children's Hospital and the Department of Clinical Immunology, Sahlgrenska University Hospital. Individuals born between 1993 and 1995 with a cardiac malformation demanding surgical correction at age less than 6 months (n = 19) were identified preoperatively at the Queen Silvia Children's Hospital, Sahlgrenska University Hospital in Gothenburg, Sweden. The operating surgeon assessed that more than 90% of the thymic tissue was removed during the operation. Patients with syndromic cardiac malformation or a known genetic disorder were excluded from the study. Blood samples were drawn preoperatively and at age 18 months; at the time of blood collection, participants had no signs of infection. For comparison at age 18 months, 10 otherwise healthy children undergoing minor surgery (mainly urological) at the same hospital were recruited. At age 18 months, lymphocyte subset analysis on fresh samples was done. The participants were contacted again 18 years later (median age, 18.7 years; range, 17.2-19.9 years) and asked to participate in a follow-up study. Eleven agreed to take part and answered a questionnaire regarding their general health, vaccinations, infections, allergies, autoimmune diseases, and cancer. An equal number of healthy age-matched controls were recruited (median age, 18.4 years; range, 17.1-19.9 years). Blood was drawn from a peripheral vein at Sahlgrenska University Hospital or the nearest health care center with express delivery to the research center within 24 hours. The blood samples were analyzed for lymphocyte subsets, TCR Vβ usage, TRECs, and telomere length. Participants showed no signs of infection at the time of blood collection. Informed consent was obtained, and the Regional Ethical Review Board at University of Gothenburg, Gothenburg, Sweden, approved the study. PBMCs were isolated with Ficoll-Paque density gradient centrifugation (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, United Kingdom), and fresh cells were analyzed directly for CD3+, CD4+, CD8+, CD16/56+, CD19+, and CD45+ cell markers (Multitest 6-Color TBNK Reagent), recent thymic emigrants CD4+CD45RA+CD31+, and TCR Vβ chain usage. The remaining cells were viably frozen using 15% dimethyl sulphoxide in FCS and stored in a −80°C freezer. Analyses of naive (CD3+CD4+CD45RA+ and CD3+CD8+CD45RA+), memory (CD3+CD4+CD45RO+ and CD3+CD8+CD45RO+), and regulatory (CD3+CD4+CD25+CD127low) T cells were performed on thawed cryopreserved PBMCs incubated with a panel of mAbs to CD3-APC-H7, CD4-PerCP-Cy5.5, CD8-PE-Cy7, CD45RO-FITC, CD45RA-APC, CD25-BV421, and CD127-PE. Analysis of recent thymic emigrants was done on fresh samples using a panel of mAbs to CD4-APC, CD45RO-FITC, CD45RA-APC-H7, and CD31-PE. All antibodies were from BD Biosciences, Franklin Lakes, NJ. The multicolor analyses were performed on a FACS Canto II flow cytometer and results analyzed using FlowJo Data analysis software version 10.0.7 (FlowJo LLC, Ashland, Ore). Further information on gating strategy is provided in Fig E1 in this article's Online Repository at www.jacionline.org. To ensure optimal flow cytometer performance, setup, and reproducibility of results, CS&T research beads (BD Biosciences) were used daily and CD-Chex Plus (Streck, Omaha, Neb) weekly according to manufacturer's instructions. TCR Vβ repertoire was analyzed using IOTest Beta Mark (Beckman Coulter Inc, Brea, Calif) according to manufacturer's instructions on fresh cells directly after isolation. Cells were also stained with CD4-PE-Cy5 (Beckman Coulter Inc) and CD8-Pacific Blue (BD Biosciences). Approximately 200,000 cells were incubated in each well, giving a minimum of 10,000 CD4+ or CD8+ cell count for all but 2 cases of suboptimal CD8+ cell count thus excluded from further analysis. The samples were processed on a FACS Canto II flow cytometer and the data processed with FlowJo Data analysis software version 10.0.7 (FlowJo LLC). Genomic DNA was isolated using the Q1Amp Blood Mini Kit (Qiagen, Venlo, The Netherlands) according to instructions. Before the PCR reaction, the purified DNA concentration was determined by ultraviolet spectrophotometry and adjusted to a concentration of 30 ng/μL with dH2O. The real-time PCR analysis was performed on a Roche LightCycler instrument. The signal joint TREC primer sequences were forward (5′-CAT CCC TTT CAA CCA TGC TGA CAC CTC T-3′) and reverse (5′-CGT GAG AAC GGT GAA TGA AGA GCA GAC A-3′) (Scandinavian Gene Synthesis AB, Köping, Sweden).E1Hochberg E.P. Chillemi A.C. Wu C.J. Neuberg D. Canning C. Hartman K. et al.Quantitation of T-cell neogenesis in vivo after allogeneic bone marrow transplantation in adults.Blood. 2001; 98: 1116-1121Crossref PubMed Scopus (103) Google Scholar PCR reactions were done in triplicates with a final reaction volume of 20 μL containing 2 μL LightCycler FastStart DNA Master SYBR Green I, 2 μL sample DNA (30 ng/μL), 2 × 0.5 μL primer solution (forward and reverse; 0.5 μM), 2.4 μL MgCl2 (4 μM), and 12.6 μL dH2O. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene was done in the same plate with GAPDH primer sequences forward (5′-CAG CCC CTT CAT ACC CTC A 3′) and reverse (5′-GGA CCA TAT TGA GGG ACA CA 3′). The reaction setup was composed of step 1: 95°C for 10 minutes, step 2: 45 cycles of 10 seconds at 95°C (denaturation), 5 seconds at 62°C to 72°C “touch down” (annealing), and finally step 3: 6 seconds at 72°C (elongation), followed by 1 cycle of melting (95°C for 0 second, 72°C for 10 seconds, 95°C for 0 seconds) and a cooling step to 40°C for 30 seconds. The TREC number was estimated by extrapolating sample quantities from a standard curve acquired by serial dilutions (108, 107, 106, 105, 104, 103, 102, and 101) of a pCR2.1-human TREC and pCR2.1-GAPDH gene plasmids (Eurofins MWG Operon).E2Sottini A. Ghidini C. Zanotti C. Chiarini M. Caimi L. Lanfranchi A. et al.Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation.Clin Immunol. 2010; 136: 217-227Crossref PubMed Scopus (86) Google Scholar In every PCR reaction, the plasmids were included in 1 concentration as standards as well as a positive control consisting of human cord blood DNA (30 ng/μL, rich in TRECs) and a negative control (dH2O). The number of TRECs was estimated according to the following formula: (Mean of TRECs quantity/(Mean of GAPDH quantity/2)) × 106 = number of TREC molecules per 106 cells. The mean quantity of GAPDH was divided by 2 because of the biallelic occurrence of this gene. Frozen PBMCs (−80°C) were thawed; the cells were pelleted, resuspended, and stained with CD4-FITC, CD8-BV421, CD19-PE, CD14-APC, and CD56-APC (BD Biosciences). CD4, CD8, and CD19 cell populations were then sorted (i-Cyt Synergi cell sorter, Sony Biotechnology Inc, San Jose, Calif) with purity of more than 95% after sorting. DNA was isolated from CD4, CD8, and CD19 cell subsets (QIAamp DNA mini kit, QIAcube, Qiagen, Venlo, the Netherlands) according to manufacturer's instructions. Telomere length was estimated using a quantitative PCR method described by CawthonE3Cawthon R.M. Telomere measurement by quantitative PCR.Nucleic Acids Res. 2002; 30: e47Crossref PubMed Scopus (2535) Google Scholar that allows an estimation of the relative telomere length from a ratio of the telomere (T) repeat copy number to a single (S) gene copy number (RPLP0). The telomere primer sequences were (5′-GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T-3′) and (5′-TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA-3′) with a final reaction concentration of 270 and 900 nM, respectively. The RPLP0 primer sequences were (5′-CAG CAA GTG GGA AGG TGT AAT CC-3′) and (5′-CCC ATT CTA TCA TCA ACG GGT ACA A-3′) with a final reaction concentration of 400 nM. The genomic DNA amount was 5 ng per reaction for all samples. Two samples were excluded from analysis because of limited yield; in those samples, the DNA concentration was 4.64 (from CD8 cells, thymectomy) and 3.16 (CD19, control) ng per reaction. The quantitative PCR reaction was performed with TATAA SYBR GrandMaster Mix # TA01 (TATAA Biocenter, Gothenburg, Sweden) in a final 10 μL reaction volume in duplicate on a LightCycler 480 instrument (Roche, Basel, Switzerland). Detection was performed in the SYBR channel. All pipetting was performed by robot (EpMotion 5070, Eppendorf, Germany). The quantitative PCR temperature protocol began with an initiation step at 95°C for 180 seconds followed by an amplification step consisting of 40 cycles of (1) 95°C for 5 seconds (denaturation), (2) 54°C for 15 seconds (anneal), and (3) 72°C for 20 seconds (elongation and fluorescent measurement). Finally, gradual heating from 60°C to 95°C for 10 seconds gave a postreaction melting curve. Assay evaluation was done on a pool of DNA samples from CD4+ and CD8+ cells. An 8-point standard curve was generated with 4 replicates in each point and run in 4-fold dilution steps. The dilution series covered a template concentration between 50 ng/μL and 0.003 ng/μL. The reaction volume and components were prepared and thermal cycling and analysis were done as described above. Linear polyacrylamide carriers were added to the dilution series to avoid unspecific interactions of the target. The telomere assay receives high efficiency and linearity when excluding the 3 lowest STD (50-0.195 ng/μL used in the calculation of efficiency), whereas the RPLP0 assay receives high efficiency and linearity when excluding the lowest STD (50-0.012 ng/μL used in the calculation of efficiency). For every thymectomized individual (Tx), a healthy age- and sex-matched control was recruited. All statistical analyses were done with Graphpad Prism version 6.0b (Graphpad Software Inc, San Diego, Calif). To assess quantitative differences in cell populations between Tx and controls, the t test for unpaired data was used for all variables with a Gaussian distribution whereas the Mann-Whitney test was used for a few variables (CD8+, CD19+ at 18 m and CD56+; Treg cells and CD8+CD45RO+ absolute numbers) that differed from normality when tested using the D'Agostino and Pearson omnibus normality test. The unpaired t test was also used comparing TRECs and relative telomere lengths between groups. TCR Vβ was analyzed by defining whether each individual's Vβ-chain usage deviated more or less than 3 SD from the mean of the controls using Fisher exact test. The comparison of TCR Vβ chain usage between the 2 groups was done using the Holm Sidak multiple comparison test. Multivariate orthogonal projection to latent structures discriminant analysis was implemented to examine whether Tx individuals and healthy controls could be discriminated on the basis of various immune variables assessed (SIMCA-P+ software, Umetrics, Umeå, Sweden). All data were scaled to unit variance so that all the variables were given equal weight regardless of their absolute value. The quality of the latent structures discriminant analysis model was based on the variable R2 (ie, the goodness of fit of the model) and Q2 (ie, how well a variable can be predicted by a model).Fig E2Individual CD4+ (above) and CD8+ (below) T-cell TCR Vβ clonograms. Thymectomized (A) and controls (B); CD4+. Thymectomized (C) and controls (D); CD8+. Shown as the percentage of CD4+ or CD8+ T cells expressing each TCR Vβ family. Nomenclature of TCR Vβ families according to Wei et al.E4Wei S. Charmley P. Robinson M.A. Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire.Immunogenetics. 1994; 40: 27-36Crossref PubMed Scopus (162) Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E2Individual CD4+ (above) and CD8+ (below) T-cell TCR Vβ clonograms. Thymectomized (A) and controls (B); CD4+. Thymectomized (C) and controls (D); CD8+. Shown as the percentage of CD4+ or CD8+ T cells expressing each TCR Vβ family. Nomenclature of TCR Vβ families according to Wei et al.E4Wei S. Charmley P. Robinson M.A. Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire.Immunogenetics. 1994; 40: 27-36Crossref PubMed Scopus (162) Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Flow cytometry analysis of TCR Vβ for both CD4+ and CD8+ T cells. A representative plot from 1 individual is shown. Each vial of antibodies contains 3 mAbs, 1 PE, 1 FITC, and 1 a combination of the 2, giving a total of 24 different specificities toward the different TCR Vβ families.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Sex, type of congenital heart defect, and age at operationPatientSexType of heart defectAge at thymectomy (d)1FAS1072FCoA103FTGA644MFallot1385MVSD616MTGA67FVSD508FTGA1489FVSD13310MTGA611MTGA48AS, Aorta stenosis; CoA, coarctation of the aorta; F, female; Fallot, Fallots tetralogy; M, male; TGA, transposition of the great arteries; VSD, ventricular septal defect. Open table in a new tab Table E2Reported clinical data from a questionnaire answered by participants and parents at 18-year follow-upClinical dataThymectomized (no. 11)Controls (no. 10)Acute otitis media97 <10 per individual in total64 ≥10 per individual in total33Surgery related to recurrent otitis media13Pneumonia52Frequent respiratory infections71Severe infection (hospital admission)61Autoimmune disease (any)11Allergy (any)52 Open table in a new tab Table E3Number of lymphocytes, CD4+ and CD8+ T cells, CD19+ B cells, and CD16+/56+ natural killer cells analyzed preoperatively, at age 18 months, and at 18-year follow-up shown as mean cell number × 109/L with 95% CI and P value using either the unpaired t test or the Mann-Whitney test as appropriateStageCellThymectomized (×109/L)95% CIControls(×109/L)95% CIP valuePreopLymphocytes3.672.75-4.60NACD4+1.721.24-2.21—CD8+0.620.43-0.80—CD19+0.850.33-1.37—CD16+/56+0.400.12-0.69—18 moLymphocytes2.991.80-4.184.253.15-5.35.0925CD4+0.790.43-1.151.901.39-2.41.0014CD8+0.520.27-0.771.000.67-1.33.0144CD19+0.940.52-1.360.980.65-1.31.5870CD16+/56+0.370.08-0.660.200.11-0.30.371718 yLymphocytes1.411.10-1.731.871.62-2.13.0202CD4+0.550.45-0.660.830.67-1.00.0050CD8+0.290.18-0.400.530.40-0.67.0038CD19+0.270.16-0.380.220.17-0.27.3157CD16+/56+0.260.10-0.430.190.15-0.24.9099NA, Not analyzed. Open table in a new tab Table E4TCR Vβ usage in CD8+ and CD4+ cells in thymectomized and controlsThymectomyOligoclonality CD8+Vβ chainOligoclonality CD4+Vβ chain1YesVβ21.3No2YesVβ2,3,5.1,5.3,12,14YesVβ7.23YesVβ3,5.1,12,14No4YesVβ13.2No5NoNo6YesVβ1,12,14,23YesVβ17YesVβ5.1YesVβ13.68YesVβ1,3,5.1,5.3,8,11,13.6,23YesVβ89NoNo10YesVβ1YesVβ2211NAYesVβ5.1,17ControlsOligoclonality CD8+Vβ chainOligoclonality CD4+Vβ chain1NoNo2NANo3NoNo4NoNo5NoNo6YesVβ13.2No7NoNo8NoNo9NoNo10NoNo11NoNoNA, Not analyzed.Nomenclature according to Wei et al.E4Wei S. Charmley P. Robinson M.A. Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire.Immunogenetics. 1994; 40: 27-36Crossref PubMed Scopus (162) Google Scholar Oligoclonality is defined as Vβ usage exceeding ±3SD from the mean of the controls. Open table in a new tab AS, Aorta stenosis; CoA, coarctation of the aorta; F, female; Fallot, Fallots tetralogy; M, male; TGA, transposition of the great arteries; VSD, ventricular septal defect. NA, Not analyzed. NA, Not analyzed. Nomenclature according to Wei et al.E4Wei S. Charmley P. Robinson M.A. Concannon P. The extent of the human germline T-cell receptor V beta gene segment repertoire.Immunogenetics. 1994; 40: 27-36Crossref PubMed Scopus (162) Google Scholar Oligoclonality is defined as Vβ usage exceeding ±3SD from the mean of the controls.

Thyroid-associated ophthalmopathy (TAO) causes irreversible increase in extraocular fat volume that contributes to the risk of exophthalmos and compressive optic neuropathy. Collagen XIII is implicated in uncontrolled cell growth in some tumours, but we are not aware of any studies of collagen XIII in TAO-affected solid tissue to date. We conducted immunohistochemical staining for collagen XIII alpha 1 (COL13A1), present in both the transmembrane and cleaved forms of collagen XIII, in consecutive prospectively collected human extraocular tissue specimens from patients with TAO and controls. We identified overexpression of collagen XIII in active TAO-affected fat. We discuss how species and cell-type specific responses of collagen XIII to stressors may help explain the different phenotypes of TAO.

Extensive knowledge has been gained the last years concerning mechanisms underlying the selection of single positive thymocytes in the thymic medulla. Less is known regarding other important processes in the thymic medulla such as the regulation of late stage thymocyte maturation. We have previously reported that exosomes are abundant in the thymus with a phenotype that indicates an epithelial cell origin and immunoregulatory properties. In this study we use an in vitro system to investigate the effects of thymic exosomes on the maturation of single positive thymocytes as well as effects on nTreg formation. We show that thymic exosomes promote the maturation of single positive CD4+CD25- cells into mature thymocytes with S1P1+Qa2+ and CCR7+Qa2+ phenotypes. Furthermore, we show that thymic exosomes reduce the formation of CD4+CD25+FoxP3+ thymocytes and that these exosome effects are independent of dendritic cell co-stimulation but require intact exosomal RNA content and surface proteins. An efficient direct uptake of exosomes by both thymocytes and thymic DC's is also demonstrated. In conclusion, this study demonstrates that exosomes may represent a new route of communication within the thymus.

Thyroid-associated ophthalmopathy (TAO) has a predilection for inferior rectus muscle that has never been explained. We conducted immunohistochemical staining for the soluble cleaved form of collagen XIII alpha 1 (COL13A1) and found constitutively low expression of COL13A1 in normal human inferior rectus muscles and moderate expression of COL13A1 in normal human medial rectus muscles. COL13A1 is known to be essential to development and maintenance of neuromuscular junctions and there is some evidence to suggest it may help support normal immune function. The combination of constitutively low expression of COL13A1, high physiological and metabolic demands, and consequentially relatively high exposure to stressors via the blood stream may help explain the particular vulnerability of inferior rectus to TAO compared to other extraocular muscles.

Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

Thymocytes go through several steps of maturation and selection in the thymus in order to form a functional pool of effector T cells and regulatory T cells in the periphery. Close interactions between thymocytes, thymic epithelial cells and dendritic cells are of vital importance for the maturation, selection and lineage decision of the thymocytes. One important question that is still unanswered is how a relatively small epithelial cell population can present a vast array of self-antigens to the manifold larger population of developing thymocytes in this selection process. Here we review and discuss the literature concerning antigen transfer from epithelial cells with a focus on exosomes. Exosomes are nano-sized vesicles released from a cell into the extracellular space. These vesicles can carry proteins, micro-RNAs and mRNAs between cells and are thus able to participate in inter-cellular communication. Exosomes have been shown to be produced by thymic epithelial cells and to carry tissue restricted antigens and MHC molecules, which may enable them to participate in the thymocyte selection process.

Diagnosis of severe peanut allergy is difficult and delays in making an accurate diagnosis may place the patient at risk. Adults with a history of anaphylaxis must strictly avoid any contact with peanuts or products that may contain traces of peanuts. For these persons, conventional evaluations with skin prick testing (SPT) and IgE tests may not be sufficient to assess the risk of anaphylaxis. Therefore, we investigated whether the basophil activation test (BAT) could be used for the diagnosis of severe peanut allergy in adults. We compared the non-invasive BAT with conventional laboratory diagnostic tests, including SPT and specific IgE to allergen extracts and components, for the diagnosis of severe peanut allergy.

Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

Objetivos: El objetivo de esta investigacion fue caracterizar y comparar los infiltrados inflamatorios en pacientes con granulomatosis orofacial aislada (GOF-S) y GOF con enfermedad de Crohn (EC +GOF). Diseno del estudio: Las muestras de biopsia fueron obtenidas de pacientes con GOF-S (n = 11) y GOF+ EC (n = 11) y se inmunotineron con anticuerpos frente a CD1a, CD3, CD4, CD8, CD11c, CD20, CD68 y triptasa de los mastocitos, seguido de un analisis cuantitativo. Resultados: Los analisis del tejido conectivo revelaron un numero significativamente mayor de expresion de celulas T CD3 y celulas dendriticas CD11c en el tejido conjuntivo de pacientes con GOF-S en comparacion con los pacientes con GOF + EC. Los mastocitos mostraron un alto nivel de activacion, aunque no se detecto ninguna diferencia significativa al comparar los dos grupos. Conclusiones: Los resultados muestran una composicion diferente del infiltrado inflamatorio en pacientes con GOF-S en comparacion con los pacientes con GOF + EC. Las presentes observaciones apoyan que los mecanismos inmunes en parte divergentes estan involucrados en estas dos diferentes subcategorias de GOF

Orofacial granulomatosis (OFG) is a debilitating inflammatory disorder in the orofacial region characterized by disfiguring perioral swelling and intraoral tag and cobblestone phenomenon. The etiology is unknown. OFG may occur concomitantly with other granulomatous diseases such as Crohn disease (CD) and sarcoidosis. The aim of this investigation was to characterize the inflammatory cell infiltrate in patients with OFG solely (OFG-S) and patients with OFG and coexisting Crohn disease (OFG+CD).

OBJECTIVES:The aim of this investigation was to characterise and compare the inflammatory infiltrates in patients with orofacial granulomatosis solely (OFG-S) and OFG with coexisting Crohn's disease (OFG+CD).STUDY DESIGN:Biopsy specimens with granulomas were obtained from patients with OFG-S (n=11) and OFG+CD (n=11) and immunostained with antibodies against CD1a, CD3, CD4, CD8, CD11c, CD20, CD68 and mast cell tryptase, followed by quantitative analysis.RESULTS:Analyses of the connective tissue revealed a significantly higher number of CD3-expressing T cells and CD11c-expressing dendritic cells in the connective tissue of patients with OFG-S compared to patients with OFG+CD. Mast cells displayed a high level of activation, although no significant difference was detected when comparing the two groups.CONCLUSIONS:The results show a different composition of the inflammatory infiltrate in patients with OFG-S compared to patients with OFG+CD. The present observations support that partly-divergent immune mechanisms are involved in these two different subcategories of OFG.

Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall's corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.

Small vesicles were first described in prostatic and seminal fluids more than 30 years ago [5]. They were called prostasomes and are members of the same family now called exosomes. All cells in the body can release extracellular vesicles that function as intercellular messengers. The smallest of these, exosomes, are produced by almost all types of cells in the body and exist in all body fluids. Exosomal signalling takes place in two different ways: either with a cargo of functional proteins and/or by transfection of functional RNA molecules from one cell to the cytoplasm of another, or by ligand-receptor mediated interactions between molecules of the exosome membrane and the cellular membrane of the target cell. The importance of exosomes both in health and disease is rapidly acknowledged and clinical applications in diagnosticts and therapy are under development. The content of proteins and nucleic acids of exosomes will soon be used as bio markers for different diseases such as cancer and cardiac disease. Clinical tests are ongoing where exosomes are used as natural vectors for cancer specific peptides in the treatment of cancer. Exosomes will most probably soon be used in therapy by using them as natural vectors for new RNA based therapies and also for follow up of therapy.