Search

Pediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor β chain (TCR-β) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4(+) and CD4(+)CD45RA(+) cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4(+) cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Primary hemophagocytic lymphohistiocytosis (HLH) can be caused by biallelic mutations in PRF1, encoding perforin, or UNC13D, STXBP2, STX11, RAB27A, LYST, and AP3B1, encoding proteins involved in cytotoxic lymphocyte degranulation. Natural killer (NK)-cell cytotoxicity assays can quickly screen for all of these genetic diseases, facilitating treatment, but combining NK-cell perforin expression and CD107a upregulation tests can as well. To determine the relative diagnostic accuracies for each approach, we retrospectively reviewed screening test performance in 1614 patients referred for HLH evaluation. For each test, we generated a receiver operating characteristic (ROC) curve, and calculated area under the curve (AUC) and diagnostic parameters at optimal threshold. We generated an AUC for combining perforin and CD107a tests by creating a logistic regression model and applying model-generated coefficients to patient values. Sensitivities of NK-cell function, perforin mean channel fluorescence (MCF), and CD107a MCF to detect biallelic mutations were 59.5%, 96.6%, and 93.8%, with specificities of 72.0%, 99.5%, and 73%. AUCs for NK-cell cytotoxicity, perforin MCF, CD107a MCF, and combined perforin and CD107a MCFs were 0.690, 0.971, 0.860, and 0.838. Perforin and CD107a tests are more sensitive and no less specific compared with NK cytotoxicity testing for screening for genetic HLH and should be considered for addition to current HLH criteria.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients 95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.)

Background: Autoimmunity is a fundamental consequence of impaired tolerance checkpoints in patients with hypomorphic mutations in recombination activating genes (RAG1 and 2), the major player in V(D)J recombination of antigen receptors. We recently published a cohort of 63 patients with partial RAG deficiency with autoimmune and/or hyperinflammatory manifestations. Autoimmune hemolytic anemia (AIHA) was most common, occurring in (60.3%) of patients at a median age of 1.9 years. The episodes of AIHA were often severe and refractory to first or second-line therapy. Despite the severity of early-onset disease, a large fraction of patients ware diagnosed later for an underlying genetic cause. This may be due to deceased awareness and lack of routine clinical biomarkers for underlying RAG deficiency. The natural course of AIHA episodes, diversity of antibodies targeting red blood cells (RBC) and the underlying mechanisms that promote AIHA remain elusive. Our previous study on RAG-deficient patients has revealed that autoreactive B cells are present early in B cell development, and autoantibodies are produced to a broad selection of self-antigens, including those on RBCs. A prevailing theory for the development of anti-RBC antibodies involves dysregulation of somatic hypermutation (SHM). There are specific regions of the immunoglobulin (Ig) that are naturally reactive to self-antigen in the germline state, and mutation away from self-reactivity occurs through SHM. On example is the germline Ig VH4-34 AVY segment that has been known to bind to the i/I antigen on RBCs. Increased awareness for detection and mechanistic understanding of the generation of anti-RBC antibodies are needed to promote targeted therapy in this highly vulnerable young patient population with AIHA and immune deficiency. Methods: Through retrospective chart review, we collected detailed information on the episodes of AIHA. This included clinical evidence of hemolysis, recurrence and severity of episodes, specifics on red blood cell reactivity, treatment and outcome. In addition, We performed B-cell repertoire studies to assess rate of SHM and "mutation away" from RBC reactivity. Results In our 42 patients with RAG deficiency and AIHA, we accumulated data on 20 patients. Many of the patients experienced the onset of AIHA after viral infections and often recurred (up to 8 times in lifetime). Most of the patients had warm AIHA with broad reactivity (direct, indirect, IgG, IgM, C3). In parallel, patients developed specific antibodies to cytokines targeting IFNa, IFNw and IL12. Second or third-line therapy was required in 50% of cases. One patient underwent HSCT due to severe refractory AIHA. The B-cell repertoire in selected RAG patients with AIHA contained more unmutated Ig VH4-34 segments (including the AVY segment), and demonstrate less evidence of SHM when compared to healthy donors. Conclusions Patients with partial RAG deficiency may develop severe, recurrent and treatment-refractory AIHA at early ages. Diagnosis may be expedited with increased awareness and serological evidence of broadly reactive antibodies to RBC and cytokines. Mechanistically, decreased SHM may promote the survival of naturally occurring antibodies to RBC. Studies should continue to advance understanding for targeted approach to serve as bridge therapy before HSCT is considered or indicated. Disclosures No relevant conflicts of interest to declare.

Lentivector mediated gene transfer into hematopoietic stem/progenitor cells and T cells has resulted in long-term stable integration of transgenes and significant clinical benefits in many diseases. We previously reported (De Ravin et al Sci Transl Med. 2016) early outcome data for 5 patients with X-linked severe combined immunodeficiency (X-SCID) enrolled in a first-in-human lentivector gene therapy clinical trial (NCT03315078) as salvage therapy for older children and young adults who had received prior haplo-identical hematopoietic stem cell transplantation (HSCT) as infants without chemotherapy-based conditioning. Lymphocyte-depleted haplo-identical stem cell transplant in X-SCID infants without conditioning generally results in only a T-cell engraftment that may also decline over time which, combined with B- and NK-cell dysfunction, may result in the progression of multi-system clinical problems (bronchiectasis, infections, gastrointestinal malabsorption, failure to grow, immune dysregulation). By 2016 three additional patients were treated and the cohort of 8 patients have now been followed for 3 to 7 years (Cohort A), where we observed gradual clinical benefit in the clearance of chronic norovirus and associated improved abdominal complaints, malabsorption, and growth and IgG production. Four patients were able to cease immunoglobulin replacement therapy. Despite these encouraging results, the relatively inefficient transduction of hematopoietic stem/progenitor cells (HSPCs) required large quantities of vector, and resulted in relatively low vector copy number in myeloid cells in some patients, with delayed immune cell recovery and persistent clinical disease especially in the last patient treated, P8. To address this, we developed a refined enhanced transduction (ET) procedure consisting of a single overnight transduction after 48hours pre-stimulation in cytokines (Stem cell factor, Thrombopoietin, Flt3-ligand; 100ng/mL) and incorporated transduction enhancers LentiBoost 1:100 and dimethyl prostaglandin 2 (dmPGE2; 1mM) and recently treat 6 patients, including re-treatment of P8 (Cohort B). Here we compared the early outcomes from Cohort B patients who received autologous CD34+ HSPCs processed with the enhanced transduction (ET) procedure. These patients (aged 12 to 36 yo) had significant problems with donor T cell infiltration of liver, bone marrow and kidneys, and near absent B and NK cells. Cryopreserved G-CSF/plerixafor mobilized peripheral blood CD34+ HSPCs (5x106/kg to 28x106/kg) were transduced by the ET process at 5% vector concentration, compared to previous method without transduction enhancers of two daily transductions at 20-30% final vector concentration, representing a 10-12 fold reduction in vector used. Colony forming unit assays showed 78%-92% versus 17%-58% vector-positive clones in Cohort B compared with Cohort A, with VCN of 1.5-12 copies (Cohort B), compared with 0.06 to 0.5 copies in Cohort A. At one month following infusion of gene corrected cell product, we isolated peripheral blood CD14+ myeloid cells as indication of early marrow output from engrafted HSPCs, and observed VCN of 1 to 4 copies (P9-12 and re-treat P8), a ≥10x increase from Cohort A (0.04 to 0.23) at same early time point after infusion (Fig 1). In addition to earlier clinical improvement (abdominal complaints, appetite, growth), there was also an early appearance of B and NK cells (Fig. 2) at much higher levels in Cohort B than previously observed even at years after treatment in Cohort A. In conclusion, we observed significantly improved measures of early clinical outcomes from lentivector gene therapy of older children and young adults with X-SCID using enhanced transduction procedure with addition of LentiBoost and dmPGE2, that achieves much greater transduction efficiencies with >10 fold less vector, and results in faster immune reconstitution and more significant clinical benefit by 3 months. The patients are monitored closely for potential risks from higher VCNs that may be balanced by reduced selection pressure due to the greater numbers of gene corrected clones. Figure Disclosures Puck: NIAID: Research Funding; Pfeizer: Other: spouse serves on Rare Disease Advisory Board; Invitae: Other: spouse employment. Cowan:bluebird bio: Consultancy; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; California Institute Of Regenerative Medicine: Research Funding; NIH NIAD: Research Funding; Rocket Pharma: Consultancy; Leadiant: Consultancy. Mamcarz:ASSISI Foundation of Memphis: Research Funding; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; California Institute of Regenerative Medicine: Research Funding; NHLBI: Research Funding; UpToDate: Honoraria; American Lebanese Syrian Associated Charities: Research Funding. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; California Institute for Regenerative Medicine: Research Funding; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; EMD Serono: Honoraria; ASSISI fundation of Memphis: Research Funding; Inmatics: Membership on an entity's Board of Directors or advisory committees. Meagher:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy.

To the editor: Mutations in PRF1 , which encodes perforin, were discovered to cause familial hemophagocytic lymphohistiocytosis (FHL) in 1999 and account for 20% to 50% of all FHL cases.[1][1][⇓][2][⇓][3][⇓][4][⇓][5]-[6][6] Flow cytometric detection of perforin in peripheral blood natural

Lymphadenopathy in children for which no infectious or malignant cause can be ascertained constitutes a challenging diagnostic dilemma. Autoimmune lymphoproliferative syndrome (ALPS) is a human genetic disorder of lymphocyte apoptosis resulting in an accumulation of lymphocytes and childhood onset chronic lymphadenopathy, splenomegaly, multilineage cytopenias, and an increased risk of B-cell lymphoma. In 1999, investigators at the National Institutes of Health (NIH) suggested criteria to establish the diagnosis of ALPS. Since then, with approximately 500 patients with ALPS studied worldwide, significant advances in our understanding of the disease have prompted the need for revisions to the existing diagnostic criteria and classification scheme. The rationale and recommendations outlined here stem from an international workshop held at NIH on September 21 and 22, 2009, attended by investigators from the United States, Europe, and Australia engaged in clinical and basic science research on ALPS and related disorders. It is hoped that harmonizing the diagnosis and classification of ALPS will foster collaborative research and better understanding of the pathogenesis of autoimmune cytopenias and B-cell lymphomas.

Patients with autoimmune multilineage cytopenias are often refractory to standard therapies requiring chronic immunosuppression with medications with limited efficacy and high toxicity. We present data on 30 patients treated on a multicenter prospective clinical trial using sirolimus as monotherapy. All children (N = 12) with autoimmune lymphoproliferative syndrome (ALPS) achieved a durable complete response (CR), including rapid improvement in autoimmune disease, lymphadenopathy, and splenomegaly within 1 to 3 months of starting sirolimus. Double-negative T cells were no longer detectable in most, yet other lymphocyte populations were spared, suggesting a targeted effect of sirolimus. We also treated 12 patients with multilineage cytopenias secondary to common variable immunodeficiency (CVID), Evans syndrome (ES), or systemic lupus erythematosus (SLE), and most achieved a CR (N = 8), although the time to CR was often slower than was seen in ALPS. Six children with single-lineage autoimmune cytopenias were treated and only 2 responded. Sirolimus was well tolerated with very few side effects. All of the responding patients have remained on therapy for over 1 year (median, 2 years; range, 1 to 4.5 years). In summary, sirolimus led to CR and durable responses in a majority of children with refractory multilineage autoimmune cytopenias. The responses seen in ALPS patients were profound, suggesting that sirolimus should be considered as a first-line, steroid-sparing treatment of patients needing chronic therapy. The results in other multilineage autoimmune cytopenia cohorts were encouraging, and sirolimus should be considered in children with SLE, ES, and CVID. This trial was registered at www.clinicaltrials.gov as #NCT00392951.

Autoimmune lymphoproliferative syndrome (ALPS) type Ia is caused by inherited defects in apoptosis and is characterized by nonmalignant lymphoaccumulation, autoimmunity, and increased alpha/beta(+) double-negative T cells (alpha/beta(+)-DNT cells). This study reports immunophenotypic findings in 166 members of 31 families with ALPS type Ia, associated with genetic mutations in the TNFRSF6 gene encoding Fas. The ALPS type Ia probands (n = 31) and relatives having both a Fas mutation and clinically proven ALPS (n = 28) showed significant expansion of CD8(+) T cells, alpha/beta(+)-DNT cells, gamma/delta(+)-DNT cells, CD3(+)/ HLA-DR(+) T cells, CD8(+)/CD57(+) T cells, and CD5(+) B cells. Relatives with Fas mutations, but without all the required criteria for ALPS (n = 42), had expansions of CD8(+) T cells, alpha/beta(+)-DNT cells, and gamma/delta(+)-DNT cells. Interestingly, relatives without a Fas mutation and with no features of ALPS (n = 65) demonstrated a small but significant expansion of CD8(+) T cells, both DNT cell subsets, and CD5(+) B cells. As compared to unrelated healthy controls, lymphocyte subset alterations were greatest in the probands, followed by the relatives with mutations and ALPS. Probands and relatives with mutations and ALPS also showed a lower number of CD4(+)/CD25(+) T cells that, in combination with an independent increase in HLA-DR(+) T cells, provided a profile predictive of the presence of clinical ALPS. Because quantitative defects in apoptosis were similar in mutation-positive relatives regardless of the presence of clinical ALPS, factors, other than modifiers of the Fas apoptosis pathway, leading to these distinctive immunophenotypic profiles most likely contribute to disease penetrance in ALPS.

Recent experience suggests that reduced-intensity conditioning (RIC) regimens can improve the outcomes of patients with hemophagocytic lymphohistiocytosis (HLH) undergoing allogeneic hematopoietic cell transplantation (HCT). However, studies directly comparing RIC to myeloablative conditioning (MAC) regimens are lacking. Forty patients with HLH underwent allogeneic HCT between 2003-2009 at Cincinnati Children's Hospital. Fourteen patients received MAC consisting of busulfan, cyclophosphamide, and antithymocyte globulin plus or minus etoposide. Twenty-six patients received RIC consisting of fludarabine, melphalan, and alemtuzumab. All patients engrafted. Acute graft-versus-host disease grades II to III occurred in 14% of MAC patients and 8% of RIC patients (P = .3171). Posttransplantation mixed donor/recipient chimerism developed in 18% of MAC patients and 65% of RIC patients (P = .0110). The majority of patients with mixed chimerism received intervention with reduction of immune suppression plus or minus donor lymphocyte infusion or stem cell boost, which stabilized or increased donor contribution to hematopoiesis and prevented relapse of HLH in all but 1 patient. Grade II to III graft-versus-host disease occurred in 5 of 14 RIC patients after donor lymphocyte infusion. The overall estimated 3-year survival after HCT was 43% (confidence interval = ± 26%) for MAC patients and 92% (confidence interval = ± 11%) for RIC patients (P = .0001). We conclude that RIC significantly improves the outcome of patients with HLH undergoing allogeneic HCT.

X-linked inhibitor of apoptosis (XIAP) deficiency, caused by BIRC4 mutations, is described to cause X-linked lymphoproliferative disease (XLP) phenotypes. However, compared with XLP caused by SLAM-Associated Protein deficiency (SH2D1A mutation), XIAP deficiency was originally observed to be associated with a high incidence of hemophagocytic lymphohistiocytosis (HLH) and a lack of lymphoma, suggesting that classification of XIAP deficiency as a cause of XLP may not be entirely accurate. To further characterize XIAP deficiency, we reviewed our experience with 10 patients from 8 unrelated families with BIRC4 mutations. Nine of 10 patients developed HLH by 8 years of age. Most patients presented in infancy, and recurrent HLH was common. There were no cases of lymphoma. Lymphocyte defects thought to contribute to HLH development in SLAM-Associated Protein deficiency were not observed in XIAP deficiency. We conclude that XIAP deficiency is a unique primary immunodeficiency that is more appropriately classified as X-linked familial hemophagocytic lymphohistiocytosis.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder in which genetic defects in proteins that mediate lymphocyte apoptosis, most often Fas, are associated with enlargement of lymph nodes and the spleen and a variety of autoimmune manifestations. Some patients with ALPS have relatives with these same apoptotic defects, however, who are clinically well. This study showed that the circulating levels of interleukin 10 (IL-10) were significantly higher (P

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of abnormal lymphocyte survival caused by defective Fas-mediated apoptosis. Patients with ALPS develop lymphadenopathy, hepatosplenomegaly, and increased number of a T cell population normally found in low numbers in peripheral blood called double negative T cells (DNTs, T cell phenotype CD3+/4−/8−, TCRalpha/beta +). Patients frequently develop severe autoimmune disease, primarily manifested as autoimmune cytopenias. Some patients with ALPS need long-term treatment and these patients have limited therapeutic options. Sirolimus, an mTOR inhibitor, has been shown to induce apoptosis in normal and malignant lymphocytes. Because ALPS is caused by defective lymphocyte apoptosis, we hypothesized that sirolimus would be effective by inducing apoptosis in these abnormal cells, controlling the lymphoproliferation that is the hallmark of the disease. We previously tested this hypothesis using murine models of ALPS, and have now opened a phase I/II clinical trial testing sirolimus in patients with ALPS. Six children with ALPS (2 type IA; 4 type III) were started on treatment with sirolimus, targeting a serum trough level of 5–15ng/ml. 4 patients were treated for clinically significant autoimmune cytopenias that either failed standard therapies or in whom these therapies, including high dose corticosteroids, were not tolerated. The two other patients also had autoimmune cytopenias; however, the indication for treatment was autoimmune arthritis and colitis with steroid intolerance. Four of the patients had previously failed treatment with mycophenolate mofetil and not all six could tolerate corticosteroids. One patient had failed treatment with rituximab, methotrexate, cyclosporine, tacrolimus, anti-TNFalpha agents, and cyclophosphamide. Another patient had failed treatment with mercaptopurine and plaquenil. Five of 6 patients had complete resolution of autoimmune cytopenias and normalization of blood counts within 4 weeks of initiating therapy with sirolimus. One of six patients had persistent Grade 1 thrombocytopenia (plt count >100,000/mm3 but less than 150,000/mm3). Four patients had resolution of lymphadenopathy and splenomegaly (3 complete; 1 partial with a greater than 90% reduction). Patients have been treated for 3, 3, 4, 15, 26, and 36 months, respectively. The two patients with co-morbid autoimmune arthritis and colitis showed response in these symptoms as well. All six patients had a greater than 50% reduction in DNTs. Serial PET/CTs were performed on one patient that demonstrated a complete resolution of diffuse PET-avid disease after 3 months. No patient developed any significant toxicity. One patient had developed EBV reactivation while being treated with a combination of high dose steroids, mycophenolate mofetil, and plaquenil-the EBV load went to zero after conversion to sirolimus, which may represent the effect of an mTOR inhibitor on EBV-transformed B cells. We found sirolimus significantly reduced the lymphoproliferative state and improved autoimmunity in patients with ALPS who had failed other therapies. We will continue to test sirolimus in a clinical trial; however, based on the significant improvement we found with this series we would argue that sirolimus be considered as second line therapy for patients with steroid-refractory or steroid-intolerant disease.