Abstract: Introduction: Pigeon fanciers’ lung (PFL) is one of the most common forms of hypersensitivity pneumonitis (HP) in the UK. Generally it is considered that PFL is caused by immune complexes, however, this does not explain why some fanciers are asymptomatic despite the presence of high levels of anti-avian antigen antibodies in their serum. Pigeon intestinal mucin (PIM) is considered to be an important antigen in PFL. Thus this study was designed in order to understand the role of specific antibodies and T cells in the pathogenesis of PFL. Methods: Anti-avian IgG and IgG subclass responses among 50 symptomatic and 50 asymptomatic pigeon fanciers were determined by ELISA and the functional affinity of IgG1 and IgG2 against a range of pigeon antigens was determined by inhibition ELISA and microcalimetry. Mucin-specific T cell clones were also generated from pigeon fanciers and T cell phenotypes and cytokine profile of these cells were identified. Results: The median titres of IgG1 and IgG2 against all the pigeon antigens tested was always higher in asymptomatic than symptomatic fanciers and these differences were significant for anti-PS IgG1 (P=0.04), anti-PDF IgG2 (P=0.028), anti-PDO IgG2 (P=0.04) and anti-PIS IgG2 (P=0.03). The functional affinity of IgG1 and IgG2 against PDO was higher in symptomatic individuals as compared to asymptomatic fanciers (P=0.006 and P=0.002, respectively) whilst the functional affinity of anti-PDF IgG2 was also significantly higher in these patients (P?0.001). Symptomatic fanciers were also significantly more likely to have high ?H and thus had higher avidity antibodies against PDO (P=0.044). 12 T cell clones specific for t mucin also were generated from an asymptomatic fancier and 90-96% of clone 04, 22, 23 were CD4-CD8- double negative (DN). Conclusion: The data suggests that the magnitude of the serum antibody response cannot determine the development of the disease and as symptomatic fanciers had higher IgG antibody avidities and therefore immune complexes in individuals with PFL may have a stronger composition and bonds. In addition, this is the first demonstration of the use of ITC to measure antibody avidity in a clinical situation. This is a rapid and simple method of measuring antibody avidity and has a diagnostic potential in PFL. Finally t mucin-specific T cell clones with double negative phenotype may have a crucial role in immune regulation in asymptomatic fanciers and can be one of the reasons why these individuals do not have any symptoms in spite of having high antibody responses.
We previously published results for 70 children who received conditioning with treosulfan and cyclophosphamide (n = 30) or fludarabine (n = 40) before undergoing hematopoietic stem cell transplantation (HSCT) for primary immunodeficiency (PID). Toxicity was lower and T cell chimerism was better in the patients receiving fludarabine, but cohort numbers were relatively small and follow-up was short. Here we report outcomes of 160 children who received homogeneous conditioning with treosulfan, fludarabine, and, in most cases, alemtuzumab (n = 124). The median age at transplantation was 1.36 years (range, .09 to 18.25 years). Donors included 73 matched unrelated, 54 1 to 3 antigen-mismatched unrelated, 12 matched sibling, 17 other matched family, and 4 haploidentical donors. Stem cell source was peripheral blood stem cells (PBSCs) in 70, bone marrow in 49, and cord blood in 41. Median duration of follow-up was 4.3 years (range, .8 to 9.4 years). Overall survival was 83%. No patients had veno-occlusive disease. Seventy-four patients (46%) had acute GVHD, but only 14 (9%) greater than grade II. Four patients underwent successful retransplantation for graft loss or poor immune reconstitution. Another patient experienced graft rejection and died. There was no association between T cell chimerism >95% and stem cell source, but a significant association was seen between myeloid chimerism >95% and use of PBSCs without an increased risk of significant GVHD compared with other sources. All 11 patients with severe combined immunodeficiency diagnosed at birth were alive at up to 8.7 years of follow-up. Long-term studies are needed to determine late gonadotoxic effects, and pharmacokinetic studies are needed to identify whether specific targeting is advantageous. The combination of treosulfan, fludarabine, and alemtuzumab is associated with excellent results in HSCT for PID.
Hematopoietic cell transplantation (HCT) has become standard-of-care for an increasing number of inborn errors of immunity (IEI). This report is the first to compare transplant outcomes according to T-cell-replete (ie, T-replete) HLA-matched grafts using alemtuzumab (n = 117) and T-cell-depleted (ie, T-depleted) HLA-mismatched grafts using T-cell receptor-αβ (TCRαβ)/CD19 depletion (n = 47) in children with IEI who underwent first HCT between 2014 and 2019. All patients received treosulfan-based conditioning except patients with DNA repair disorders. For T-replete grafts, the stem cell source was marrow in 25 (21%) patients, peripheral blood stem cell (PBSC) in 85 (73%), and cord blood in 7 (6%). TCRαβ/CD19 depletion was performed on PBSCs from 45 haploidentical parental donors and 2 mismatched unrelated donors. The 3-year overall survival (OS) and event-free survival for the entire cohort were 85% (77%-90%) and 79% (69%-86%), respectively. Analysis according to age at transplant revealed a comparable 3-year OS between T-replete grafts (88%; 76%-94%) and T-depleted grafts (87%; 64%-96%) in younger patients (aged <5 years at HCT). For older patients (aged >5 years), the OS was significantly lower in T-depleted grafts (55%; 23%-78%) compared with T-replete grafts (87%; 68%-95%) (P = .03). Grade III to IV acute graft-versus-host disease was observed in 8% of T-replete marrow, 7% of T-replete PBSC, 14% of T-replete cord blood, and 2% of T-depleted PBSC (P = .73). Higher incidence of viremia (P < .001) and delayed CD3 reconstitution (P = .003) were observed after T-depleted graft HCT. These data indicate that mismatched donor transplant after TCRαβ/CD19 depletion represents an excellent alternative for younger children with IEI in need of an allograft.
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is an inherited syndrome of early-onset systemic autoimmunity and the prototype of immune dysregulatory disorders. It is caused by mutations of forkhead box p3 (FOXP3) gene (Xp11.23), encoding a key transcription factor for natural regulatory T (nTreg) cells.1Fontenot J.D. Gavin M.A. Rudensky A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (5949) Google Scholar Treg cell dysfunction leads to severe multi-organ autoimmune phenomena including enteropathy, dermatitis, endocrinopathy, and other organ-specific diseases.2Gambineri E. Torgerson T.R. Ochs H.D. Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis.Curr Opin Rheumatol. 2003; 15: 430-435Crossref PubMed Scopus (496) Google Scholar Patients often present early in infancy and, without treatment, usually die in the first years of life. The only effective cure is hematopoietic stem cell transplantation (HSCT). However, the outcome is variable. Early HSCT with non-myeloablative conditioning provides the best outcome, before organs are damaged by autoimmunity and/or adverse effects of therapy.3Rao A. Kamani N. Filipovich A. Lee S.M. Davies S.M. Dalal J. et al.Successful bone marrow transplantation for IPEX syndrome after reduced-intensity conditioning.Blood. 2007; 109: 383-385Crossref PubMed Scopus (130) Google Scholar Complete donor engraftment in all hematopoietic lineages may not be necessary, because the preferential engraftment of donor Treg cells seems to be sufficient to control the disease.4Seidel M.G. Fritsch G. Lion T. Jurgens B. Heitger A. Bacchetta R. et al.Selective engraftment of donor CD4+25high FOXP3-positive T cells in IPEX syndrome after nonmyeloablative hematopoietic stem cell transplantation.Blood. 2009; 113: 5689-5691Crossref PubMed Scopus (57) Google Scholar, 5Barzaghi F. Passerini L. Bacchetta R. Immune dysregulation, polyendocrinopathy, enteropathy, x-linked syndrome: a paradigm of immunodeficiency with autoimmunity.Front Immunol. 2012; 3: 211Crossref PubMed Scopus (228) Google Scholar With parental informed consent and Newcastle upon Tyne Hospitals National Health Service Foundation Trust approval, we report the case of an IPEX patient presenting with severe enteropathy, dermatitis, and other signs of autoimmunity since 3 to 4 weeks of age. The identified FOXP3 gene mutation was associated with a reduced protein expression in Treg cells (Fig E1, Fig E2 available in this article's Online Repository at www.jacionline.org). Therefore, at 6 months of age, he received an unmanipulated, unrelated donor cord blood stem cell transplant (1 DP mismatch) with sub-myeloablative conditioning and graft-versus-host disease (GvHD) prophylaxis (see this article's Methods section and Fig E3 in the Online Repository at www.jacionline.org). He had an uneventful engraftment and a reasonable immune reconstitution (Fig E4 in this article's Online Repository at www.jacionline.org). Chimerism analysis in peripheral blood showed 90% donor T lymphocytes during the first 6 months after the transplant, with a decrease and stabilization at 70% 1 year post-transplant (Fig E5 in this article's Online Repository at www.jacionline.org). Consistent with the sub-myeloablative conditioning regimen the patient has received, mixed myeloid chimerism was also observed; however, as previously reported,4Seidel M.G. Fritsch G. Lion T. Jurgens B. Heitger A. Bacchetta R. et al.Selective engraftment of donor CD4+25high FOXP3-positive T cells in IPEX syndrome after nonmyeloablative hematopoietic stem cell transplantation.Blood. 2009; 113: 5689-5691Crossref PubMed Scopus (57) Google Scholar, 5Barzaghi F. Passerini L. Bacchetta R. Immune dysregulation, polyendocrinopathy, enteropathy, x-linked syndrome: a paradigm of immunodeficiency with autoimmunity.Front Immunol. 2012; 3: 211Crossref PubMed Scopus (228) Google Scholar, 6Slatter M.A. Cant A.J. Hematopoietic stem cell transplantation for primary immunodeficiency diseases.Ann N Y Acad Sci. 2011; 1238: 122-131Crossref PubMed Scopus (20) Google Scholar donor myeloid chimerism is not necessary to control the disease. Moreover, FOXP3 protein expression by Treg cells increased over time (Fig E2). Despite good post-transplant immune reconstitution, the patient continued to suffer from diarrhea and malabsorption and was dependent on parenteral nutrition. He developed episodes of upper intestinal obstruction and, despite anti-inflammatory therapy with an anti-TNF-α agent, required jejunal resections at 3, 4, and 6 months. Histopathology of resected areas revealed severe chronic mucosal injury without histological signs of GvHD, with an improvement of the architecture over time (see Fig E6 in this article's Online Repository at www.jacionline.org).7Patey-Mariaud de Serre N. Canioni D. Ganousse S. Rieux-Laucat F. Goulet O. Ruemmele F. et al.Digestive histopathological presentation of IPEX syndrome.Mod Pathol. 2009; 22: 95-102Crossref PubMed Scopus (102) Google Scholar The gut dysfunction improved progressively from month 6 to 9, and at 1 year post-transplant, the patient was independent of parenteral nutrition and thriving on enteral feeding. The intestine has a major interface with the external environment, and its integrity is important in the maintenance of immune homeostasis.8Nenci A. Becker C. Wullaert A. Gareus R. van Loo G. Danese S. et al.Epithelial NEMO links innate immunity to chronic intestinal inflammation.Nature. 2007; 446: 557-561Crossref PubMed Scopus (824) Google Scholar The intestinal mucosa contains an extensive network of secondary lymphoid tissues and is home to several lymphocyte subsets, including intestine-specific subpopulations. The beta-7 integrins (α4β7 and αEβ7) are selective mediators of lymphocyte homing to the gut-associated lymphoid tissue. In particular, α4β7 is expressed at low levels on naive T and B cells and at high levels on effector and memory T (mainly CD4+) cells.9Williams M.B. Butcher E.C. Homing of naive and memory T lymphocyte subsets to Peyer's patches, lymph nodes, and spleen.J Immunol. 1997; 159: 1746-1752PubMed Google Scholar Because the persistence of enteropathy in the patient was inconsistent with the transplant outcome, we explored the hypothesis that intestinal immune reconstitution proceeded at a different pace to that in the peripheral blood with a delay in the re-establishment of homeostasis within the gut immune system. We therefore investigated the engraftment of donor lymphocytes in the gut mucosa to evaluate any differences between peripheral blood and gut immune reconstitution that might explain the clinical course. To study gut immune reconstitution, we probed for FOXP3+ and CD4+ T cells on tissue sections of small bowel mucosa at different times after transplant (3, 6, and 9 months). We observed the presence of lymphoid nodules numerically decreasing over time, with an increased proportion of FOXP3+ cells both within nodules and the mucosal area (Fig 1), suggesting a reduction of the small bowel inflammatory state. We isolated CD4+ cells from small bowel tissue sections obtained at 3 months post-transplant and investigated their origin by genotyping FOXP3 (donor or recipient) to evaluate the donor chimerism within the relevant cellular compartment. Both wild-type and mutated nucleotides were present at the c.1037 position on the FOXP3 gene, suggesting a mixed population of lymphocytes in the gut. This was confirmed by chimerism analysis of polymorphic markers on the same cell population showing 60% donor, 40% recipient origin (Fig 2, A), while donor chimerism in the blood CD3+T cells was 91% (Fig E5). We set out to study circulating gut-homing lymphocytes in a patient blood sample, which was available at only 9 months after HSCT. We recovered CD4+CD31+α4/β7low naive and CD4+CD31−α4/β7high memory T cells by cell sorting. Sequence analysis showed wild-type FOXP3 sequence and 90% donor chimerism on CD4+CD31−α4β7high gut-homing lymphocytes, whereas wild-type and mutated FOXP3 genes along with a 50% donor chimerism were found on CD4+CD31+α4/β7low naive T cells not specifically committed to the intestine (Fig 2, B and C). Our results suggest that, in this patient, the gut immune system took longer to recover and function compared with the peripheral immune system (Fig E3). FOXP3 expression assessed in small bowel biopsies taken at different times post-transplant was found to correlate with the patient's clinical condition. Gut function and tolerance of enteral nutrition progressively improved in parallel with the increased FOXP3 expression within the gut mucosa and the appearance of donor CD4+CD31−α4/β7high cells. Furthermore, cells of donor origin were mostly present in the periphery rather than in the gut early after transplant, possibly explaining poor gut function. The increase in donor chimerism within gut-homing lymphocytes was associated with a progressive rise of FOXP3+ cells within the small bowel later on, possibly suggesting that a preferential homing of donor Treg cells to the gut is associated with disease recovery. Further studies in additional patients will be required to determine if this is applicable to other patients with IPEX. To our knowledge, this is the first study of gut immune reconstitution in a patient affected with an inherited disorder of immune tolerance, and we believe it could be a unique case study that sheds light on the role of the intestine in reconstituting the immune system after HSCT. Further investigation of function of external factors (such as microbiota) in influencing the imprinting of mucosal immunity will help to identify new therapeutic approaches. We thank the patient and his family for their support and cooperation. We greatly appreciated the collaboration with Pamela Pinzani of the Department of Biomedical, Experimental, and Clinical Science, University of Florence, Florence, Italy, for her help in microdissection experiments, and with colleagues at the Institute of Cellular Medicine of Newcastle University and at the Flow Cytometry Core Facility Laboratory, Centre for Life, Newcastle Upon Tyne, United Kingdom, for technical support in cell sorting. The male child included in this study presented at 3 to 4 weeks of life with severe enteropathy characterized by watery diarrhea that did not respond to different types of enteral feeding. He then developed eczematous-like skin changes. Moreover, he had been noted to be hypotonic at birth, with dysmorphic facial characteristics. Small bowel biopsies evidenced an eosinophilic infiltrate and villous atrophy. Investigations showed a positive Coombs test, positive islet cell antibodies and high IgE (1278 UI/L). The above-mentioned findings were suggestive for IPEX syndrome. Genetic testing confirmed IPEX with a homozygous missense mutation in exon 9 of FOXP3 (c.1037T>C), causing an amino acid substitution (p.Iso346Thr) in the forkhead domain of the protein.E1Passerini L. Olek S. Di Nunzio S. Barzaghi F. Hambleton S. Abinun M. et al.Forkhead box protein 3 (FOXP3) mutations lead to increased TH17 cell numbers and regulatory T-cell instability. J. Allergy Clin.Immunol. 2011; 128: 1376-1379.e1Scopus (45) Google Scholar Analysis of FOXP3 protein on peripheral lymphocytes showed that its expression was reduced and was lower in the patient's Treg cells compared with those of the normal control (37% of FOXP3 expressing CD4+CD25bright cells in the patient vs 82% in the control; Fig E2). Accordingly, immunohistochemistry of small bowel biopsies revealed the presence of FOXP3 protein, although presumably it was not functional (Fig E1). To cure his disease, at 6 months of age, the patient received an unmanipulated, unrelated donor cord blood stem cell transplant (1 DP mismatch) with sub-myeloablative conditioning (Alemtuzumab 0.3 mg/kg, Fludarabine 150 mg/m2, and Treosulfan 36 g/m2) and GvHD prophylaxis (tacrolimus, mycophenolate mofetil).E2Nademi Z. Slatter M. Gambineri E. Mannurita S.C. Barge D. Hodges S. et al.Single centre experience of haematopoietic SCT for patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.Bone Marrow Transplant. 2013; 49: 310-312Crossref PubMed Scopus (24) Google Scholar Written informed consent was obtained from the patient's parents, and the study was approved by the local ethics committee. T, B, and natural killer cells were analyzed on peripheral blood at different times after the transplant (Day 0 is considered the day of graft infusion). Monoclonal antibodies specific for CD45RA, CD3, CD4, CD8, CD19, CD15, CD16/56, and CD45 (BD Biosciences, San Jose, Calif) were used to define percentage of cell subset on gated lymphocytes. Absolute numbers of CD8+ cells, total CD4+ and CD4+ naive T cells, B cells, and natural killer cells were calculated. For identification of Treg cells, PBMCs were stained with the surface molecules CD4 and CD25 and then fixed, permeabilized, and stained with intracellular FOXP3 (eBioscience, San Diego, Calif). Cell fluorescence was evaluated with FACSCanto II flow cytometry and analyzed with FACSDiva software (BD, San Jose, Calif). Immunohistochemistry to detect FOXP3 was carried out on tissue sections received from the histopathology archives of Newcastle upon Tyne Teaching Hospitals National Health Service Trust. All tissue was used in accordance with Newcastle and North Tyneside Local Research Ethics Committee approval. Immunohistochemistry methods were as previously published, with minor modifications.E3Robertson H. Kirby J.A. Yip W.W. Jones D.E. Burt A.D. Biliary epithelial-mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis.Hepatology. 2007; 45: 977-981Crossref PubMed Scopus (131) Google Scholar Four μm paraffin sections on positively charged slides were deparaffinized and partially rehydrated to 95% ethanol. Endogenous peroxidase was blocked in methanol/hydrogen peroxide for 10 minutes at room temperature (RT) followed by washing in standard TRIS-buffered saline (TBS) (pH 7.6). Antigen retrieval was achieved at low temperature (65°C) in EDTA buffer (pH 8.0) for approximately 21 hours in a hot air oven. Following a wash in TBS, endogenous biotin was blocked (Vector, Hartfield, United Kingdom), and nonspecific binding of antibodies was minimized by incubation of sections with 20% normal swine serum (NSS) in TBS. Overnight incubation with primary antibody (anti-FOXP3 at 1/10 in NSS) supernatant received from Dr Alison Banham, Oxford, at 4°C was followed by washing in TBS, then incubation at RT for 1 hour with biotinylated goat anti-mouse immunoglobulins (Vector: 1/200 in NSS). Subsequently, the antibody complex was detected with avidin-biotin peroxidase (Vector: 30 minutes at RT) and nickel diaminobenzidine substrate (Sigma-Aldrich, St Louis, Mo) at RT. The counterstain was Mayer's hematoxylin, and slides were dehydrated, cleared, and mounted in distrene plasticizer xylene. Sections were scanned using an Aperio ScanScope Digital Scanner (Leica Biosystems, Nussloch, Germany). Analysis of digital images was carried out with Imagescope software, and a nuclear algorithm was used to enumerate black FOXP3+ nuclei. Submucosal tissue was excluded from the analysis. Identification of CD4+ cell populations for laser-assisted microdissection was carried out according to Gjerdrum et al.E4Gjerdrum L.M. Lielpetere I. Rasmussen L.M. Bendix K. Hamilton-Dutoit S. Laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization.J Mol Diagn. 2001; 3: 105-110Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Serial 5 μm paraffin sections, cut with a fresh blade, were placed on APES-coated PEN-membrane slides and dried at 60°C for 2 hours. Deparaffinization was carried out in fresh xylene for at least 2 × 10 minutes, followed by prolonged washing and partial rehydration in absolute then 95% ethanol. Subsequent steps to detect CD4+ cells were as for FOXP3+ cells above. Primary anti-CD4 antibody was used at 1/50 in NSS (Leica Biosystems, Novocastra). No counterstain was applied, and slides were dehydrated in fresh ethanol (graded) then stored at RT. Groups of CD4+ T cells were subsequently microdissected and collected by gravity into the lid of a 0.5-mL reaction tube using Leica LMD6500 laser microdissector (Leica Microsystems) or isolated by laser capture and pressure catapulting with the PALM Laser MicroBeam system (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany). Gut-homing T cells were isolated with cell sorting (FACSAriaII, BD Biosciences). Briefly, 500 uL of whole blood was stained with specific monoclonal antibodies CD3-PerCp-Cy5.5, CD4-PE-Cy7, CD31-PE, CD49d-FITC, beta7-APC (BD Biosciences), and incubated for 15 minutes at room temperature in the dark. Red cells were lysed, and the sample was then washed with PBS. CD4+CD31+α4/β7low naive and CD4+CD31−α4/β7high memory T cells were collected in PBS and stored as a pellet at −80°C. About 50,000 μm2 of microdissected CD4 T cells and 100,000 sorted CD4+CD31−α4/β7high memory and CD4+CD31+α4/β7low naive T cells were lysed in proteinase K buffer (Qiagen, Germantown, Md). The nucleic acids were purified with Chelex 100 (Bio-Rad, Hercules, Calif) and concentrated with Amicon Ultra 0.5 mL filters (Merck KGaA, Millipore, Darmstadt, Germany). Genomic DNA (gDNA) was isolated from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen). The FOXP3 coding sequence, including the exon-intron junction and the poly-A region were amplified from gDNA by PCR with specific primer pairs flanking the regions of interest, using a 2700 thermal cycler (Thermo Fisher Scientific, Life Technologies, Waltham, Mass). Primer pair sequences were already reported in the literature.E5Gambineri E. Perroni L. Passerini L. Bianchi L. Doglioni C. Meschi F. et al.Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: inconsistent correlation between forkhead box protein 3 expression and disease severity.J Allergy Clin Immunol. 2008; 122: 1105-1112.e1Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Approximately 50 ng of genomic DNA were amplified with the following conditions: 94°C 4 minutes, 35 cycles at 94°C 30 seconds, 30 seconds at a primer-specific melting temperature, and 68°C for 30 seconds, with a final extension at 68°C for 5 minutes. PCR products were purified using the ExoSAP-IT reagent (Affymetrix, USB, Santa Clara, Calif) and sequenced by using the BigDye Terminator Cycle Sequencing Kit (Thermo Fisher Scientific, Life Technologies) on an automated 3130 × l Genetic Analyzer (Thermo Fisher Scientific, Life Technologies). Sequencing analysis was carried out with BioEdit sequence alignment software (Ibis Therapeutics, Carlsbad, Calif). Chimerism was evaluated by multiple fluorescent short tandem repeat analysis using the AmpFlSTR Identifiler amplification kit (Thermo Fisher Scientific, Life Technologies) according to the manufacturer's instructions. The tetranucleotide short tandem repeats loci amplified in this reaction included D8S1179, D21S11, D7S820, and CSF1PO (all labeled with 6-FAM blue dye); D3S1358, TH01, D13S317, D16S539, and D2S1338 (all labeled with VIC green dye); D19S3433, vWA, TPOX, and D18S51 (all labeled with NED yellow dye); and D5S818 and FGA (all labeled with PET red dye). In addition, the amelogenin locus was analyzed to differentiate the X and Y chromosomes (labeled with PET red dye). The PCR products were analyzed with an ABI PRISM 310 DNA Genetic Analyzer (Thermo Fisher Scientific, Life Technologies). Fragment size and peak area data were determined by GeneScan software (Thermo Fisher Scientific, Life Technologies). The degree of chimerism was calculated using the method described elsewhere.E6Thiede C. Florek M. Bornhäuser M. Ritter M. Mohr B. Brendel C. et al.Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection.Bone Marrow Transplant. 1999; 23: 1055-1060Crossref PubMed Scopus (241) Google ScholarFig E2Intracytoplasmic FOXP3 expression in peripheral blood cells before and at indicated time points after transplant. Gates for analysis were set on CD4+CD25+bright T cells. x axis/arrow, Fluorescence intensity; y axis, cell counts.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Timeline representing experiments and analysis performed during this study. HE, Hematoxylin-eosin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Absolute count of T, B, and natural killer (NK) cells (per uL blood) in peripheral blood pre- and post-HSCT (3, 6, 9, and 12 months).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Donor cell chimerism in peripheral blood T cells (CD3+), B cells (CD19+), and monocytes (CD15+) during 1 year post-transplant, shown in months (m).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Histopathological findings of the resected small bowel specimens. Hematoxylin-eosin (HE) staining showed loss of mucosal epithelium with total villous atrophy at both 3 months (A) and 4 months (B) post-transplant and long slender villi with normal brush borders at 6 months post-transplant (C). No histologic evidence of GvHD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
