Research

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

Sci Immunol. 2016 Dec 16;1(6):

Authors: Lee YN, Frugoni F, Dobbs K, Tirosh I, Du L, Ververs FA, Ru H, de Bruin LO, Adeli M, Bleesing JH, Buchbinder D, Butte MJ, Cancrini C, Chen K, Choo S, Elfeky RA, Finocchi A, Fuleihan RL, Gennery AR, El-Ghoneimy DH, Henderson LA, Al-Herz W, Hossny E, Nelson RP, Pai SY, Patel NC, Reda SM, Soler-Palacin P, Somech R, Palma P, Wu H, Giliani S, Walter JE, Notarangelo LD

Abstract
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.

PMID: 28783691 [PubMed]

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Plasma cell deficiency in humans with heterozygous mutations in SEC61A1.

J Allergy Clin Immunol. 2017 Aug 03;:

Authors: Schubert D, Klein MC, Hassdenteufel S, Caballero-Oteyza A, Yang L, Proietti M, Bulashevska A, Kemming J, Kühn J, Winzer S, Rusch S, Fliegauf M, Schäffer AA, Pfeffer S, Geiger R, Cavalié A, Cao H, Yang F, Li Y, Rizzi M, Eibel H, Kobbe R, Marks AL, Peppers BP, Hostoffer RW, Puck JM, Zimmermann R, Grimbacher B

Abstract
BACKGROUND: Primary antibody deficiencies (PAD) are the most frequent primary immunodeficiencies in humans. The genetic causes for PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum (ER) membrane. SEC61A1 is a target gene of XBP1s and strongly induced during plasma cell differentiation.
OBJECTIVE: Characterization of a novel genetic defect and its pathological mechanism in eleven patients from two unrelated families with PAD.
METHODS: Whole exome sequencing (WES) and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B cell differentiation and survival.
RESULTS: We investigated two families with patients suffering from hypogammaglobulinemia, severe recurrent respiratory tract infections and normal peripheral B- and T cell subpopulations. Upon in vitro stimulation, B cells showed an intrinsic deficiency to develop into plasma cells (PCs). Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in co-translational protein translocation and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response (UPR) in multiple myeloma (MM) cell lines.
CONCLUSION: We describe a monogenic defect leading to a specific plasma cell deficiency in humans, expanding our knowledge about the pathogenesis of antibody deficiencies.

PMID: 28782633 [PubMed – as supplied by publisher]

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TCRαβ(+) and CD19(+) cell depleted Haploidentical and Mismatched Hematopoietic Stem Cell Transplantation in Primary Immune Deficiency.

J Allergy Clin Immunol. 2017 Aug 02;:

Authors: Shah RM, Elfeky R, Nademi Z, Qasim W, Amrolia P, Chiesa R, Rao K, Lucchini G, Silva JMF, Worth A, Barge D, Ryan D, Conn J, Cant AJ, Skinner R, Abd Hamid IJ, Flood T, Abinun M, Hambleton S, Gennery AR, Veys P, Slatter M

Abstract
BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is used as a therapeutic approach for primary immunodeficiencies (PIDs). The best outcomes have been achieved using 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 TCRαβCD3+ cells from the graft.
METHODS: CD3+TCRαβ+/CD19(+) depleted grafts were given in conditioned (except three) children with PIDs. Treosulfan (busulfan in one), fludarabine, thiotepa and ATG or alemtuzumab conditioning was used in 77% cases, and all but four received GvHD prophylaxis.
RESULTS: 25 patients with 12 types of PIDs received 26 HSCTs. Three were transplanted for refractory GvHD developing after first cord transplantation. At 20.8 months (5 month- 3.3 years) median follow up, 21/25 patients survived and were cured of underlying immunodeficiency. Overall and Event Free Survival at 3 years was 83.9% and 80.4% respectively. Cumulative incidence (CI) of Grade II-IV acute GvHD was 22±8.7%. No case of visceral or chronic GvHD was seen. CI of graft failure, CMV or/and adeno viral infections and transplant related mortality at 1 year were 4.2±4.1%, 58.8±9.8% and 16.1±7.4% respectively. Patients going into transplant with systemic viral infections had poor survival in comparison to those with absent or resolved infection (33.3% vs.100%).
CONCLUSION: CD3+TCRαβ(+) and CD19(+) depleted haploidentical or mMUD HSCT is a practical and viable alternative for children with a range of PIDs.

PMID: 28780238 [PubMed – as supplied by publisher]

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Novel nonsense gain-of-function NFKB2 mutations associated with a combined immunodeficiency phenotype.

Blood. 2017 Aug 04;:

Authors: Kuehn HS, Niemela JE, Sreedhara K, Stoddard JL, Grossman J, Wysocki CA, de la Morena MT, Garofalo M, Inlora J, Snyder MP, Lewis DB, Startakis CA, Fleisher TA, Rosenzweig SD

Abstract
NF-κB signaling through its NFKB1-dependant canonical and NFKB2-dependant non-canonical pathways play distinctive roles in a diverse range of immune processes. Recently, mutations in these two genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically-uncharacterized primary immunodeficiencies we detected two novel nonsense gain-of-function (GOF) NFKB2 mutations in three patients from two families (E418X and R635X), and a novel missense change in another patient (S866R). Their immunophenotype was assessed by flow-cytometry and protein expression; activation of canonical and non-canonical pathways was examined in PBMCs and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real time PCR, and multiplex assays. The S866R change disrupted a C-terminus NF-κB2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with ACTH deficiency, GH deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in three patients led to constitutive nuclear localization and activation of both canonical and non-canonical NF-κ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in two asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a non-fully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.

PMID: 28778864 [PubMed – as supplied by publisher]

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Spinal Abscess Caused by Salmonella Bacteremia in a Patient with Primary Myelofibrosis.

Am J Case Rep. 2017 Aug 04;18:859-864

Authors: Fareed S, Nashwan AJ, Abu Jarir S, Husain A, Suliman DS, Ibrahim F, Moustafa A, Akhter MS, Yassin MA

Abstract
BACKGROUND In Primary Myelofibrosis (PMF; a clonal disorder arising from the neoplastic transformation of early hematopoietic stem cells) patients, spinal cord compression (SCC) is a common complication or even a presentation symptom due to extramedullary hematopoiesis (EMH). However, a case of SCC caused by a spinal abscess is unusual. To the best of our knowledge, this is the first case report of this rare condition. CASE REPORT We are reporting the case of a 50-year-old male with primary myelofibrosis and long-standing splenomegaly with back pain as a presenting symptom who was found to have spinal cord compression. An MRI was performed, as EMH was suspected. The blood cultures revealed an infection with Salmonella, so the patient was placed on ceftriaxone, with no response. The patient demonstrated substantial clinical improvement after 2 weeks of neurosurgical intervention and pain management. CONCLUSIONS In PMF patients, back pain with fever or mild neurological symptoms needs to be investigated urgently because of the high risk of irreversible spinal cord damage leading to partial or complete loss of functional independence and shortened survival. The compression could be related to EMH or infections due to an immunodeficiency.

PMID: 28775247 [PubMed – in process]

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Long-Term Engraftment of Primary Bone Marrow Stromal Cells Repairs Niche Damage and Improves Hematopoietic Stem Cell Transplantation.

Cell Stem Cell. 2017 Aug 03;21(2):241-255.e6

Authors: Abbuehl JP, Tatarova Z, Held W, Huelsken J

Abstract
Hematopoietic stem cell (HSC) transplantation represents a curative treatment for various hematological disorders. However, delayed reconstitution of innate and adaptive immunity often causes fatal complications. HSC maintenance and lineage differentiation are supported by stromal niches, and we now find that bone marrow stroma cells (BMSCs) are severely and permanently damaged by the pre-conditioning irradiation required for efficient HSC transplantation. Using mouse models, we show that stromal insufficiency limits the number of donor-derived HSCs and B lymphopoiesis. Intra-bone transplantation of primary, but not cultured, BMSCs quantitatively reconstitutes stroma function in vivo, which is mediated by a multipotent NT5E(+) (CD73)(+) ENG(-) (CD105)(-) LY6A(+) (SCA1)(+) BMSC subpopulation. BMSC co-transplantation doubles the number of functional, donor-derived HSCs and significantly reduces clinically relevant side effects associated with HSC transplantation including neutropenia and humoral immunodeficiency. These data demonstrate the potential of stroma recovery to improve HSC transplantation.

PMID: 28777945 [PubMed – in process]

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T-Cell Lymphopenia Detected by Newborn Screening in Two Siblings with an Xq13.1 Duplication.

Front Pediatr. 2017;5:156

Authors: Rios X, Chinn IK, Orange JS, Hanson CI, Rider NL

Abstract
Newborn screening for severe combined immunodeficiency has proven successful in identifying infants with T-cell deficiencies before they become severely ill. Additionally, the newborn screen can detect subtle early phenotypes that may become severe later in life. We present the case of siblings with features suggestive of T-cell lymphopenia identified as having low T-cell receptor excision circles counts by newborn screening. Expanded immune testing showed robust lymphocyte mitogen and antigen responses with normal vaccine responses and immunoglobulin levels for both boys over time. Genetic analysis revealed an Xq13.1 duplication in each child not found in the mother. The variant is downstream of the IL2RG gene with potential regulatory significance, suggesting a mechanism for the T-cell lymphopenia. The newborn screen provided these patients heightened surveillance and patient-specific management, including delayed live vaccines and Pneumocystis jiroveci pneumonia prophylaxis. Fortunately, the brothers have not suffered invasive or opportunistic infections and are well at ages 3 and 4 years. In this report, we illustrate the challenges of managing seemingly asymptomatic immunodeficient patients without a definitive genetic diagnosis and show how unbiased genetic analysis can expand understanding about primary immunodeficiency phenotypes.

PMID: 28770187 [PubMed]

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Autoimmune Regulator Deficiency Results in a Decrease in STAT1 Levels in Human Monocytes.

Front Immunol. 2017;8:820

Authors: Zimmerman O, Rosen LB, Swamydas M, Ferre EMN, Natarajan M, van de Veerdonk F, Holland SM, Lionakis MS

Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by biallelic autoimmune regulator (AIRE) mutations that manifests with chronic mucocutaneous candidiasis (CMC) and autoimmunity. Patients with STAT1 gain-of-function (GOF) mutations also develop CMC and autoimmunity; they exhibit increased STAT1 protein levels at baseline and STAT1 phosphorylation (pSTAT1) upon interferon (IFN)-γ stimulation relative to healthy controls. AIRE interacts functionally with a protein that directly regulates STAT1, namely protein inhibitor of activated STAT1, which inhibits STAT1 activation. Given the common clinical features between patients with AIRE and STAT1 GOF mutations, we sought to determine whether APECED patients also exhibit increased levels of STAT1 protein and phosphorylation in CD14(+) monocytes. We obtained peripheral blood mononuclear cells from 8 APECED patients and 13 healthy controls and assessed the levels of STAT1 protein and STAT1 tyrosine phosphorylation at rest and following IFN-γ stimulation, as well as the levels of STAT1 mRNA. The mean STAT1 protein levels in CD14(+) monocytes exhibited a ~20% significant decrease in APECED patients both at rest and after IFN-γ stimulation relative to that of healthy donors. Similarly, the mean peak value of IFN-γ-induced pSTAT1 level was ~20% significantly lower in APECED patients compared to that in healthy controls. The decrease in STAT1 and peak pSTAT1 in APECED patients was not accompanied by decreased STAT1 mRNA or anti-IFN-γ autoantibodies; instead, it correlated with the presence of autoantibodies to type I IFN and decreased AIRE(-)(/)(-) monocyte surface expression of IFN-γ receptor 2. Our data show that, in contrast to patients with STAT1 GOF mutations, APECED patients show a moderate but consistent and significant decrease in total STAT1 protein levels, associated with lower peak levels of pSTAT1 molecules after IFN-γ stimulation.

PMID: 28769929 [PubMed]

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Autosomal recessive agammaglobulinemia due to defect in μ heavy chain caused by a novel mutation in the IGHM gene.

Genes Immun. 2017 Aug 03;:

Authors: Silva P, Justicia A, Regueiro A, Fariña S, Couselo JM, Loidi L

Abstract
Agammaglobulinemia is a primary immunodeficiency disorder characterized by profoundly low or absent serum antibodies and low or absent circulating B cells. The most common form is X-linked agammaglobulinemia (XLA) caused by mutations in BTK gene. The remaining cases, clinically similar to XLA, are autosomal recessive agammaglobulinemia (ARA). Nearly 30% of ARA cases present mutations in the μ heavy constant region gene IGHM. Here, we present a 7-month-old patient, born from non-consanguineous parents, who is affected by ARA due to defect in the μ heavy chain. The genetic study showed that the patient is compound heterozygous for an IGHM gene deletion and the novel nonsense mutation X57331.1:g.275C>A (p.Tyr43*) (ClinVar Accession Number: SCV000537868.1). This finding allows for an adequate genetic counseling to the family and also broadens the spectrum of already described point mutations at this locus. The IGHM gene is very complex and it is likely that yet unidentified mutations appear in other patients.Genes and Immunity advance online publication, 3 August 2017; doi:10.1038/gene.2017.14.

PMID: 28769069 [PubMed – as supplied by publisher]

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