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Hematopoietic stem cell transplantation and gene therapy are the only curative therapies for severe combined immunodeficiency (SCID). In patients lacking a matched donor, TCRαβ/CD19-depleted haploidentical family donor transplant (TCRαβ-HaploSCT) is a promising strategy. Conditioned transplant in SCID correlates to better myeloid chimerism and reduced immunoglobulin dependency. We studied transplant outcome in SCID infants according to donor type, specifically TCRαβ-HaploSCT, and conditioning, through retrospective cohort analysis of 52 consecutive infants with SCID transplanted between 2013 and 2020. Median age at transplant was 5.1 months (range, 0.8–16.6). Donors were TCRαβ-HaploSCT (n = 16, 31.4%), matched family donor (MFD, n = 15, 29.4%), matched unrelated donor (MUD, n = 9, 17.6%), and matched unrelated cord blood (CB, n = 11, 21.6%). Forty-one (80%) received fludarabine/treosulfan-based conditioning, 3 (6%) had alemtuzumab only, and 7 (14%) received unconditioned infusions. For conditioned transplants (n = 41), 3-year overall survival was 91% (95% confidence interval, 52–99%) for TCRαβ-HaploSCT, 80% (41–98%) for MFD, 87% (36–98%) for MUD, and 89% (43–98%) for CB (p = 0.89). Cumulative incidence of grade II–IV acute graft-versus-host disease was 11% (2–79%) after TCRαβ-HaploSCT, 0 after MFD, 29% (7–100%) after MUD, and 11% (2–79%) after CB (p = 0.10). 9/10 patients who received alemtuzumab-only or unconditioned transplants survived. Myeloid chimerism was higher following conditioning (median 47%, range 0–100%) versus unconditioned transplant (median 3%, 0–9%) (p n = 29/36, 81% vs n = 4/9, 54%) (p

Hematopoietic stem cell transplantation and gene therapy are the only curative therapies for severe combined immunodeficiency (SCID). In patients lacking a matched donor, TCRαβ/CD19-depleted haploidentical family donor transplant (TCRαβ-HaploSCT) is a promising strategy. Conditioned transplant in SCID correlates to better myeloid chimerism and reduced immunoglobulin dependency. We studied transplant outcome in SCID infants according to donor type, specifically TCRαβ-HaploSCT, and conditioning, through retrospective cohort analysis of 52 consecutive infants with SCID transplanted between 2013 and 2020. Median age at transplant was 5.1 months (range, 0.8-16.6). Donors were TCRαβ-HaploSCT (n = 16, 31.4%), matched family donor (MFD, n = 15, 29.4%), matched unrelated donor (MUD, n = 9, 17.6%), and matched unrelated cord blood (CB, n = 11, 21.6%). Forty-one (80%) received fludarabine/treosulfan-based conditioning, 3 (6%) had alemtuzumab only, and 7 (14%) received unconditioned infusions. For conditioned transplants (n = 41), 3-year overall survival was 91% (95% confidence interval, 52-99%) for TCRαβ-HaploSCT, 80% (41-98%) for MFD, 87% (36-98%) for MUD, and 89% (43-98%) for CB (p = 0.89). Cumulative incidence of grade II-IV acute graft-versus-host disease was 11% (2-79%) after TCRαβ-HaploSCT, 0 after MFD, 29% (7-100%) after MUD, and 11% (2-79%) after CB (p = 0.10). 9/10 patients who received alemtuzumab-only or unconditioned transplants survived. Myeloid chimerism was higher following conditioning (median 47%, range 0-100%) versus unconditioned transplant (median 3%, 0-9%) (p 0.001), as was the proportion of immunoglobulin-free long-term survivors (n = 29/36, 81% vs n = 4/9, 54%) (p 0.001). TCRαβ-HaploSCT has comparable outcome to MUD and is a promising alternative donor strategy for infants with SCID lacking MFD. This study confirms that conditioned transplant offers better myeloid chimerism and immunoglobulin freedom in long-term survivors.

Autosomal dominant hyper-IgE syndrome caused by dominant-negative loss-of-function mutations in signal transducer and activator of transcription factor 3 (STAT3) (STAT3-HIES) is a rare primary immunodeficiency with multisystem pathology. The quality of life in patients with STAT3-HIES is determined by not only the progressive, life-limiting pulmonary disease, but also significant skin disease including recurrent infections and abscesses requiring surgery. Our early report indicated that hematopoietic stem cell transplantation might not be effective in patients with STAT3-HIES, although a few subsequent reports have reported successful outcomes. We update on progress of our patient now with over 18 years of follow-up and report on an additional seven cases, all of whom have survived despite demonstrating significant disease-related pathology prior to transplant. We conclude that effective cure of the immunological aspects of the disease and stabilization of even severe lung involvement may be achieved by allogeneic hematopoietic stem cell transplantation. Recurrent skin infections and abscesses may be abolished. Donor TH17 cells may produce comparable levels of IL17A to healthy controls. The future challenge will be to determine which patients should best be offered this treatment and at what point in their disease history.

This study aimed to identify a risk profile for development of transplant-associated thrombotic microangiopathy (TA-TMA) in children undergoing hematopoietic stem cell transplantation (HSCT). Between 2013 and 2016, 439 children underwent 474 HSCTs at 2 supraregional United Kingdom centers. At a median of 153 days post-HSCT, TA-TMA occurred among 25 of 441 evaluable cases (5.6%) with no evidence of center variation. Sex, underlying disease, intensity of the conditioning, total body irradiation–based conditioning, the use of calcineurin inhibitors, venoocclusive disease, and viral reactivation did not influence the development of TA-TMA. Donor type: matched sibling donor/matched family donor vs matched unrelated donor vs mismatched unrelated donor/haplo-HSCT, showed a trend toward the development of TA-TMA in 1.8% vs 6.1% vs 8.3%, respectively. Presence of active comorbidity was associated with an increased risk for TA-TMA; 13% vs 3.7% in the absence of comorbidity. The risk of TA-TMA was threefold higher among patients who received >1 transplant. TA-TMA rates were significantly higher among patients with acute graft-versus-host disease (aGVHD) grades III to IV vs aGVHD grade 0 to II. On multivariate analysis, the presence of active comorbidity, >1 transplant, aGVHD grade III to IV were risk factors for TA-TMA (odds ratio [OR]: 5.1, 5.2, and 26.9; respectively), whereas the use of cyclosporine A/tacrolimus-based GVHD prophylaxis was not a risk factor for TA-TMA (OR: 0.3). Active comorbidity, subsequent transplant, and aGVHD grades III to IV were significant risk factors for TA-TMA. TA-TMA might represent a form of a vascular GVHD, and therefore, continuing control of aGVHD is important to prevent worsening of TA-TMA associated with GVHD.

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.

Treosulfan is given off-label in pediatric allogeneic hematopoietic stem cell transplant. This study investigated treosulfan's pharmacokinetics (PKs), efficacy, and safety in a prospective trial. Pediatric patients (n = 87) receiving treosulfan-fludarabine conditioning were followed for at least 1 year posttransplant. PKs were described with a two-compartment model. During follow-up, 11 of 87 patients died and 12 of 87 patients had low engraftment (≤ 20% myeloid chimerism). For each increase in treosulfan area under the curve from zero to infinity (AUC(0-∞) ) of 1,000 mg hour/L the hazard ratio (95% confidence interval) for mortality increase was 1.46 (1.23-1.74), and the hazard ratio for low engraftment was 0.61 (0.36-1.04). A cumulative AUC(0-∞) of 4,800 mg hour/L maximized the probability of success (> 20% engraftment and no mortality) at 82%. Probability of success with AUC(0-∞) between 80% and 125% of this target were 78% and 79%. Measuring PK at the first dose and individualizing the third dose may be required in nonmalignant disease.

Bacterial sepsis led to multiorgan failure in persons receiving immunosuppressive and antiinflammatory drugs., Severe infections are emerging as major risk factors for death among children with juvenile idiopathic arthritis (JIA). In particular, children with refractory JIA treated with long-term, multiple, and often combined immunosuppressive and antiinflammatory agents, including the new biological disease-modifying antirheumatic drugs (DMARDs), are at increased risk for severe infections and death. We investigated 4 persons with JIA who died during 1994–2013, three of overwhelming central venous catheter–related bacterial sepsis caused by coagulase-negative Staphylococus or α-hemolytic Streptococcus infection and 1 of disseminated adenovirus and Epstein-Barr virus infection). All 4 had active JIA refractory to long-term therapy with multiple and combined conventional and biological DMARDs. Two died while receiving high-dose systemic corticosteroids, methotrexate, and after recent exposure to anti–tumor necrosis factor-α biological DMARDs, and 2 during hematopoietic stem cell transplantation procedure. Reporting all cases of severe infections and especially deaths in these children is of paramount importance for accurate surveillance.

Background Congenital cytomegalovirus (cCMV) infection is an important illness that is a common cause of hearing loss in newborn infants and a major cause of disability in children. For that reason, treatment of symptomatic patients with either ganciclovir or its pro-drug valganciclovir is recommended. Treatment duration of 6 months has been shown to be more beneficial than shorter courses; however, there is uncertainty regarding emergence of resistance strains, secondary effects and long term sequelae. Case presentation Here we present a female infant with symptomatic cCMV who was treated from day 5 of life with oral valganciclovir. In spite of close monitoring of her drug levels and increments of her treatment dose according to weight gain, she developed ganciclovir resistance after 4 months of treatment, with increasing viraemia and petechiae. Adherence to treatment was assessed and felt to be good. Clinically, although she had marked developmental delay, she was making steady progress. In view of the development of resistance treatment was stopped at 5 months of age. No secondary effects of ganciclovir were noted during the whole course. Conclusions There were few cases in the literature reporting resistance to ganciclovir for cCMV before the new recommendations for a 6 months treatment course for this infection were published. As demonstrated in our patient, surveillance with periodic viral loads and drug monitoring are vital to identify emerging resistance and optimise antiviral dosing according to weight gain.

Purpose We aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions. Methods Of a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs. Results We found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions. Conclusions We show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs. Electronic supplementary material The online version of this article (doi:10.1007/s10875-016-0343-9) contains supplementary material, which is available to authorized users.

Journal of Infection - In Press.Proof corrected by the author Available online since vendredi 9 septembre 2016

Short summary Secondary malignancy post haematopoietic stem cell transplantation (HSCT) for malignant disorders is well recognized. There are very few published reports on malignancy post HSCT for Primary Immunodeficiency (PID). We report 12 cases of 944 patients, who developed non-PTLD malignancy post-HSCT for PID.

As part of collaborative multi-centre study started in 2000, 7 children in the UK fulfilled the inclusion criteria for treatment with autologous T cell depleted (TCD) haematopoietic stem cell transplantation (HSCT) for severe juvenile idiopathic arthritis (JIA). Here we report on the outcome and transplant-related complications. Outcome The initial, often dramatic clinical response in all patients was followed in 4 with sustained benefit, including the withdrawal of immunosuppressive and anti-inflammatory treatment, significant catch-up growth and immense improvement of the quality of life during 5–8 years long follow-up. Two patients relapsed within 1–12 months, and one died 4 months post transplant. Complications Adenovirus reactivation with dissemination was lethal in one patient, whilst Epstein-Barr (EBV) and cytomegalovirus (CMV) reactivation-driven haemophagocytic syndrome responded to antiviral and immunomodulatory treatment in 2 patients. Both the conditioning and the T cell depletion of the graft, leading to severe immunosuppression and prolonged immune system function reconstitution, are the main predisposing factors for potentially life-threatening transplant-related complications. Conclusions Autologous TCD HSCT for children with severe JIA results in two-phase response. The initial remission seen in all patients is due to immunosuppressive conditioning. This is followed by sustained drug-free remission in over 50% of patients, which is due to ‘immunomodulatory’ effects of TCD HSCT. The procedure carries a significant morbidity and mortality risk. However, this risk should be balanced against the risks of life-threatening infections occurring in this very selective group of patients on long-term and combined immunosuppressive and anti-inflammatory therapies. How to correctly identify and appropriately assess the patients in need for autologous TCD HSCT, particularly in relation to optimizing the timing for the procedure in regards to the newly available therapies with different biologic response modifiers, are some of the most important questions awaiting answers from this on-going study.

Chronic granulomatous disease (CGD) causes recurrent infection and inflammatory disease. Despite antimicrobial prophylaxis, patients experience frequent hospitalisations and 50% mortality by 30 years. Haematopoietic stem cell transplantation (HSCT) can cure CGD with resolution of infection and colitis. This study reports the survival and long-term outcome in 20 conditioned patients treated between 1998 and 2007, using 10 matched sibling (MSD) and 10 unrelated donors (URD). Age at HSCT, graft-versus-host disease (GvHD), growth, and outcome were analysed. Fourteen had > or = 1 invasive infection, 10 had colitis and seven had growth failure before HSCT. Median age at transplantation was 75 months (range 15 months-21 years). Eighteen (90%) were alive 4-117 months (median 61) after HSCT with normal neutrophil function. Two died from disseminated fungal infection. Two experienced significant chronic GvHD, with continuing sequelae in 1. Colitis resolved within 8 weeks of HSCT. Mean weight and height for age Z scores on recovery from HSCT rose significantly (P < 0.001). HSCT with MSD or URD gave excellent engraftment and survival, remission of colitis and catch-up growth, with low incidence of significant GvHD. Transplant-associated complications were restricted to those with pre-existing infection or inflammation, supporting the argument for early HSCT for more CGD patients with a well matched donor.

BACKGROUND Pneumocystis carinii is an important pathogen in immunodeficiency but may be an unrecognised cause of respiratory compromise. OBJECTIVES To ascertain the incidence of P carinii pneumonia (PCP) at presentation of severe combined immunodeficiency (SCID), whether it had been diagnosed, and the effect of treatment on outcome. SETTING The supraregional paediatric bone marrow transplant unit for primary immunodeficiencies at Newcastle General Hospital. METHODS Retrospective case note review of infants referred with a diagnosis of SCID from 1992 to 1998. RESULTS Ten of 50 infants had PCP at presentation; only one was diagnosed before transfer. Eight were diagnosed by bronchoalveolar lavage and two by lung biopsy. In only one was P carinii identified in nasopharyngeal secretions. Five required ventilation for respiratory failure but all were successfully treated with co-trimoxazole and methylprednisolone with or without nebulised budesonide. Nine survived to bone marrow transplantation and four are long term survivors after bone marrow transplantation; no deaths were related to PCP. CONCLUSIONS PCP is a common presenting feature of SCID but is rarely recognised. Bronchoalveolar lavage or lung biopsy are needed for diagnosis. Treatment with co-trimoxazole is highly successful.

Adenovirus causes disseminated disease following bone marrow transplantation (BMT). We report a child who underwent T-cell-depleted BMT. Adenovirus subgenus F serotype 41 was detected antemortem by PCR in cerebrospinal fluid and postmortem in other tissues. Serotypes 40 and 41, associated with gastrointestinal disease, have not previously been implicated in disseminated disease.

Sensitive imaging is needed in children with recurrent bacterial meningitis to detect cranial anatomical defects Recurrent bacterial meningitis in childhood is unusual and should prompt a search for immune deficiency. A variety of immunological defects may predispose to recurrent meningitis, including antibody or complement deficiency and hyposplenism. It is equally important to consider cranial anatomical defects such as skull fractures, particularly those affecting the base of the brain and extending to the sinuses and petrous pyramids.1 Craniospinal dermal sinuses, neurenteric or dermoid cysts, occult intranasal encephaloceles, or transethmoidmeningoceles are also potential portals of entry for pathogens into the subarachnoid space. 2 3 Encephaloceles may occur anywhere in the midline and arise from failure of closure of the embryonic neuraxis, creating a defect in the dura and cranium with or without protrusion of brain and meningeal tissue. Basal ethmoidal encephaloceles may extend into the nose and be mistaken for nasal polyps2 or into ethmoid sinuses or orbits. Sometimes there may be a delay in establishing a diagnosis owing to a failure to consider anatomical defects or the use of insufficiently sensitive imaging procedures. We describe two children with recurrent bacterial meningitis due to cranial anatomical defects in whom diagnosis was delayed. ### Case 1 A 9 year old boy presented with pneumococcal …

Background Results of treatment of severe T-lymphocyte immunodeficiencies by means of hematopoietic stem cell (HSC) transplantation have improved. T cell–depleted haploidentical transplantations are successful if there is no HLA-identical donor. Methods to remove T lymphocytes include addition of anti-CD52 antibodies and CD34 + HSC selection. Objective Assessment of long-term immune function is important after these treatments. We looked at immune reconstitution in 36 survivors for more than 2 years after HSC transplantation for severe T-lymphocyte immunodeficiencies and compared engraftment quality between the 2 T-lymphocyte depletion methods. Methods Chimerism, T- and B-lymphocyte subsets, immunoglobulin levels, and specific antibody production at last follow-up were examined. The χ 2 (Fisher exact test) and Wilcoxon rank sum analyses were used to compare the groups. Results Nineteen patients received anti-CD52–treated and 19 anti-CD34–treated HSCs. More anti-CD52–treated patients had full donor myeloid chimerism ( P = .025). All patients had full donor T-lymphocyte chimerism. There was no difference in donor B-lymphocyte chimerism, but significantly more anti-CD52–treated patients had class-switched memory B lymphocytes ( P = .024), normal IgG levels, and normal responses to tetanus and Haemophilus influenzae type B vaccination. More anti-CD52–treated patients with common γ chain or Janus-associated kinase 3 severe combined immunodeficiency had donor B lymphocytes. Conclusion Long-term T-lymphocyte function is good with either treatment method, with a low incidence of graft-versus-host disease. The results imply more incomplete donor chimerism in anti-CD34–treated patients with less B-lymphocyte function.

Patients with severe combined immunodeficiency and preexisting viral pneumonitis formally had a poor outcome from hematopoietic stem cell transplantation. With inhaled steroid and antitumor necrosis factor alpha antibody treatment, results improved. The poor outcome of patients with viral central nervous system infection prompted this retrospective single center review.Eight of 71 patients with severe combined immunodeficiency transplanted since 1987 were identified with viral central nervous system infection (adenovirus [1], cytomegalovirus [2], Epstein-Barr virus [2], parvovirus [1], varicella zoster virus [1], human herpesvirus 6 [1]). Nonspecific neurologic symptoms included drowsiness, irritability, head lag, fisting and floppiness. Later symptoms included unresponsiveness, apnea, posturing, hypotonia, hyperreflexia and seizures. All had neuroradiologic investigations. Only one had an initially normal computed tomography scan. Magnetic resonance image abnormalities included cerebral atrophy, basal ganglia changes, diffuse leukoencephalopathy, and multifocal mass lesions. Five patients had virus identified from cerebrospinal fluid by polymerase chain reaction and brain tissue examination from 3 patients identified human herpesvirus 6, adenovirus type 41 and varicella zoster virus. Three children remain alive, 2 received replete marrow and one remains untransplanted. Others who received T cell depleted marrow died of neurologic sequelae.Outcome of viral central nervous system infection after hematopoietic stem cell transplantation for severe combined immunodeficiency is poor, particularly associated with T cell depleted marrow.

To the Editor:Nijmegen breakage syndrome (NBS) shares clinical features with Fanconi anemia (FA), principally developmental delay, microcephaly, and cancer predisposition,1 but immunodeficiency is more common in NBS than in FA.2 Patients are susceptible to lymphoma, particularly B-cell lymphoma.2 Co

To the Editor: 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.1 Treg cell dysfunction leads to severe multi-organ autoimmune phenomena including enteropathy, dermatitis, endocrinopathy, and other organ-specific diseases.2 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.3 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.4,5 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 (Figs E1 and 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,4-6 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).7 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.8 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.9 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. Fig 1 FOXP3 “reactive nuclei” (arrow) in and around lymphoid follicles in small bowel mucosa and submucosa on immunohistochemically stained sections at 3 months (A), 6 months (B), and 9 months (C) post-transplant. (FOXP3 reactive nuclei ... 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). Fig 2 Exon 9 FOXP3 sequencing and chimerism analysis on laser microdissected CD4+ T cells from small bowel biopsies at 3 months post-transplant (A) and on CD31−CD4+α4β7high memory T cells (B) and CD31+CD4+α4β7low naive ... 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.