Reem Elfeky

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

T-cell receptor αβ+ and CD19+ cell–depleted haploidentical and mismatched hematopoietic stem cell transplantation in primary immune deficiency

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is used as a therapeutic approach for primary immunodeficiencies (PIDs). The best outcomes have been achieved with HLA‐matched donors, but when a matched donor is not available, a haploidentical or mismatched unrelated donor (mMUD) can be useful. Various strategies are used to mitigate the risk of graft‐versus‐host disease (GvHD) and rejection associated with such transplants. Objective: We sought to evaluate the outcomes of haploidentical or mMUD HSCT after depleting GvHD‐causing T‐cell receptor (TCR) &agr;&bgr; CD3+ cells from the graft. Methods: CD3+TCR&agr;&bgr;+/CD19+ depleted grafts were given in conditioned (except 3) children with PIDs. Treosulfan (busulfan in 1 patient), fludarabine, thiotepa, and anti‐thymocyte globulin or alemtuzumab conditioning were used in 77% of cases, and all but 4 received GvHD prophylaxis. Results: Twenty‐five patients with 12 types of PIDs received 26 HSCTs. Three underwent transplantation for refractory GvHD that developed after the first cord transplantation. At a median follow‐up of 20.8 months (range, 5 month‐3.3 years), 21 of 25 patients survived and were cured of underlying immunodeficiency. Overall and event‐free survival at 3 years were 83.9% and 80.4%, respectively. Cumulative incidence of grade II to IV acute GvHD was 22% ± 8.7%. No case of visceral or chronic GvHD was seen. Cumulative incidences of graft failure, cytomegalovirus, and/or adenoviral infections and transplant‐related mortality at 1 year were 4.2% ± 4.1%, 58.8% ± 9.8%, and 16.1% ± 7.4%, respectively. Patients undergoing transplantation with systemic viral infections had poor survival in comparison with those with absent or resolved infections (33.3% vs 100%). Conclusion: CD3+TCR&agr;&bgr;+ and CD19+ cell–depleted haploidentical or mMUD HSCT is a practical and viable alternative for children with a range of PIDs.

Treosulfan and Fludarabine Conditioning for Hematopoietic Stem Cell Transplantation in Children with Primary Immunodeficiency: UK Experience

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.

Non-posttransplant lymphoproliferative disorder malignancy after hematopoietic stem cell transplantation in patients with primary immunodeficiency: UK experience

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.

Could recurrent otitis media predict primary antibody deficiencies in Egyptian children

Background Recurrent ear infection is a significant warning sign of primary immunodeficiency diseases. Objective To estimate the frequency of IgA deficiency among children presenting to the outpatient clinic with recurrent otitis media (ROM > 4 times/year) and identify other possible risk factors of ROM in our community. Materials and methods Three hundred children (154 males and 146 females), who presented to the outpatient clinic of Childrens Hospital, Ain Shams University with ROM, were consecutively enrolled in the study over a 1-year period. According to the age of enrollment, children were classified into two groups: group A (1-6 years) and group B (>6-12 years). The demographic features of both groups were evaluated together with assessment of serum IgA level. Results Of all patients studied, only two (0.7%) had a low serum IgA level for normal age-reference values. None of the patients had neutropenia or lymphopenia. Iron-deficiency anemia was diagnosed in 76 cases, with higher rates among the patients in group A than group B. All patients received several courses of various empirical broad-spectrum antibiotics, but with either an incomplete course (n = 192) or a poor response (n = 49). Conclusion The current study showed a relatively low incidence of IgA deficiency among children with ROM and indicated other environmental risk factors that participated in the occurrence of OM in our community.

Haematopoietic stem cell transplantation for RAG1/2 severe combined immunodeficiency or Omenn syndrome: a single centre experience

Introduction: Initial trough Cyclosporine A (CsA) blood level can infl uence incidence of GVHD and relapse in patients with acute leukemia. We sought to evaluate the impact of initial CsA level (CSA-1) on the incidence of acute and chronic GVHD, relapse and survival and also explore factors that may aff ect CSA-1. Materials (or patients) and Methods: All patients who underwent allogeneic stem cell transplant (ASCT) for acute leukemia from January 2008 to March 2013 were included in this retrospective study. GVHD prophylaxis used was CsA (starting dose 1.5 mg/kg twice daily) from day -1 in combination with either methotrexate (MTX) or mycophenolate mofetil (MMF). CSA-1 was measured on day 4 or day 5 of starting CsA. Dose of CsA was modifi ed depending on CSA-1 to achieve therapeutic level of 150-200 ng/ml. For analysis, patients were divided into three groups based on modifi cation of CsA dose Group A (dose escalated), Group B (dose de-escalated) and Group C (dose unchanged). Comparisons were done between 3 groups for baseline characteristics, incidence of acute and chronic GVHD, incidence of relapse, transplant related mortality (TRM), relapse free survival (RFS) and overall survival (OS). Comparison between 3 groups was done by using chi-square test and survival outcomes by Kaplan Meier method. Multivariate analysis to determine factors predicting CsA dose escalation or de-escalation was done using logistic regression. Results: Seventy-four patients underwent 77 transplants; AML52, ALL-23, biphenotypic-2; 42 in CR1, 20 in CR2, 15 in relapsed/ refractory state; 65 from matched related, 10 from matched unrelated and 2 from haploidentical donors. Source of stem cells was peripheral blood in 70, bone marrow in 5 and cord blood in 2 transplants. The median age was 30 years (range 6-51). Conditioning regimen was full intensity (FI) i.e. TBI-Cy or Bu-Cy in 42 and reduced intensity (RI) i.e. fl udarabine based in 35 transplants. Total body irradiation (TBI) was used in 32 patients. GVHD prophylaxis was CsA+MTX in 53 and CsA+MMF in 24 patients. There were 27 patients in group A, 13 in group B and 37 in group C. On univariate analysis, use of FI regimen, cyclophosphamide and TBI; and Body Mass Index (BMI) <22 kg/m2 were associated with lower CSA-1 while use of fl udarabine, RI regimen and BMI>22 kg/m2 were associated with higher CSA-1. On multivariate analysis, fl udarabine use and BMI>22 kg/m2 predicted for higher CSA-1 requiring CsA dose de-escalation (P=0.038 and 0.034 respectively). Incidence of all grade and grade II-IV acute GVHD was 48% and 11% in group A, 38% and 31% in group B, and 43% and 19% in group C respectively (P= NS). Incidence of chronic GVHD was 52% in group A, 38% in group B and 57% in group C (P=NS). Incidence of relapse was 41% in group A, 23% in group B and 32% in group C (P= NS). TRM was 7% in group A, 38% in group B and 11% in group C (P= NS). OS and RFS at 4 years was 33% and 29% in group A, 43% and 46% in group B, and 53% and 45% in group C respectively (P=NS). Discussion: BMI <22 kg/m2 is associated with lower CSA-1 requiring CsA dose escalation. Use of fl udarabine based conditioning and BMI>22 kg/m2 is associated with higher CSA-1 requiring CsA dose de-escalation. Transplant outcomes including rates of acute and chronic GVHD, TRM, relapse incidence and overall survival are not signifi cantly aff ected by initial CsA level. Disclosure of Interest: None Declared.