Hélène Decaluwe

Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia

Background Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. Methods We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. Results Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. Conclusions Based on our genetic results, TTC7A is the likely causal gene for MIA.

Gamma(c) deficiency precludes CD8+ T cell memory despite formation of potent T cell effectors.

Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing the common γ chain (γc) are critical for the generation and peripheral homeostasis of naive and memory T cells. Recently, we demonstrated that effector functions fail to develop in CD4+ T cells that differentiate in the absence of γc. To assess the role of γc cytokines in cell-fate decisions that condition effector versus memory CD8+ T cell generation, we compared the response of CD8+ T cells from γc+ or γc− P14 TCR transgenic mice after challenge with lymphocytic choriomeningitis virus. The intrinsic IL-7-dependent survival defect of γc− naive CD8+ T cells was corrected by transgenic expression of human Bcl-2. We demonstrated that although γc-dependent signals are dispensable for the initial expansion and the acquisition of cytotoxic functions following antigenic stimulation, they condition the terminal proliferation and differentiation of CD8+ effector T cells (i.e., KLRG1high CD127low short-lived effector T cells) via the transcription factor, T-bet. Moreover, the γc-dependent signals that are critical for memory T cell formation are not rescued by Bcl2 overexpression. Together, these data reveal an unexpected divergence in the requirement for γc cytokines in the differentiation of CD4+ versus CD8+ cytotoxic T lymphocytes.

Epitope Specificity and Relative Clonal Abundance Do Not Affect CD8 Differentiation Patterns during Lymphocytic Choriomeningitis Virus Infection

ABSTRACT To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional properties of dominant and subdominant populations in the response to lymphocytic choriomeningitis virus (LCMV). To improve functional discrimination, in addition to the usual tests of phenotype and function, we used a sensitive technique that allows the screening of all CD8 effector genes simultaneously in single cells. Surprisingly, these methods failed to reveal a major impact of clonal dominance in CD8 properties throughout the response. Aiming to increase clonal dominance, we examined high-frequency transferred P14 T-cell receptor transgenic (TCR Tg) cells. Under these conditions LCMV is cleared faster, and accordingly we found an accelerated response. However, when Tg and endogenous cells were studied in the same mice, where they should be subjected to the same antigen load, they showed overlapping properties, and the presence of P14 cells did not modify endogenous responses to other LCMV epitopes or a perturbed immunodominance hierarchy in the memory phase. Using allotype-labeled Tg cells, we found that during acute infection up to 80% downregulated their TCR and were undetectable by tetramer binding, and that tetramer-negative and tetramer-positive cells had very different features. Since Tg cells are not available to evaluate immune responses in humans and, in many cases, are not available from the mouse, the tetramer-based evaluation of early immune responses in most situations of high viremia may be incomplete and biased.

Cytokines and persistent viral infections

Intracellular pathogens such as the human immunodeficiency virus, hepatitis C and B or Epstein-Barr virus often cause chronic viral infections in humans. Persistence of these viruses in the host is associated with a dramatic loss of T-cell immune response due to functional T-cell exhaustion. Developing efficient immunotherapeutic approaches to prevent viral persistence and/or to restore a highly functional T-cell mediated immunity remains a major challenge. During the last two decades, numerous studies aimed to identify relevant host-derived factors that could be modulated to achieve this goal. In this review, we focus on recent advances in our understanding of the role of cytokines in preventing or facilitating viral persistence. We concentrate on the impact of multiple relevant cytokines in T-cell dependent immune response to chronic viral infection and the potential for using cytokines as therapeutic agents in mice and humans.

Procalcitonin in children with Escherichia coli O157:H7 associated hemolytic uremic syndrome.

Shiga toxin producing Escherichia coli (STEC) are noninvasive enteric pathogens that may cause hemorrhagic colitis (HC) and diarrhea-associated hemolytic uremic syndrome (D+ HUS). We hypothesized that development of D+ HUS is associated with increased serum procalcitonin (PCT) levels. PCT was measured by an immunoluminometric assay in 113 patients. Concentrations of PCT were different in normal controls, disease control groups (rotavirus enteritis, HC due to non-STEC pathogens, chronic renal failure), and children with uncomplicated O157:H7 HC or D+ HUS. Children with D+ HUS showed higher PCT levels than those with uncomplicated O157:H7 HC, and increased concentrations were noted in cases requiring peritoneal dialysis. Severely increased concentrations were observed in children with D+ HUS on d 5 or 6 after the onset of enteritis, whereas serial measurements in those with uncomplicated O157:H7 HC remained within the normal range throughout the first week of illness. PCT was correlated with serum concentrations of lipopolysaccharide (LPS)-binding protein and serum levels of alanine aminotransferase. Using two separate sets of real-time PCR primers, we were unable to detect elevated PCT mRNA transcripts in nonadherent undifferentiated (monocytic) or differentiated (macrophage-like) THP-1 cells stimulated with purified Shiga toxin-1 and/or LPS. Our data show that serum levels of PCT are associated with the severity of illness in children with D+ HUS. Further studies are needed to determine the role of PCT in the pathogenesis of thrombotic microangiopathy associated to childhood D+ HUS.

IL-2 and IL-15 regulate CD8+ memory T-cell differentiation but are dispensable for protective recall responses.

The ability to mount effective secondary responses is a cardinal feature of memory CD8+ T cells. An understanding of the factors that regulate the generation and recall capacities of memory T cells remains to be ascertained. Several cues indicate that two highly related cytokines, IL‐2 and IL‐15, share redundant functions in this process. To establish their combined roles in memory CD8+ T‐cell development, maintenance, and secondary responses, we compared the outcome of adoptively transferred IL2Rβ+/− or IL2Rβ−/− CD8+ T cells after an acute viral infection in mice. Our results demonstrate that both IL‐2 and IL‐15 signals condition the differentiation of primary and secondary short‐lived effector cells by altering the transcriptional network governing lineage choices. These two cytokines also regulate the homeostasis of the memory T‐cell pool, with effector memory CD8+ T cells being the most sensitive to these two interleukins. Noticeably, the inability to respond to both cytokines limits the proliferation and survival of primary and secondary effectors cells, whereas it does not preclude potent cytotoxic functions and viral control either initially or upon rechallenge. Globally, these results indicate that lack of IL‐2 and IL‐15 signaling modulates the CD8+ T‐cell differentiation program but does not impede adequate effector functions.

SCID genotype and 6-month posttransplant CD4 count predict survival and immune recovery

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.

Very Early-Onset Inflammatory Manifestations of X-Linked Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare primary immune deficiency caused by mutations in genes coding for components of the nicotinamide adenine dinucleotide phosphate oxidase, characterized by severe and recurrent bacterial and fungal infections, together with inflammatory complications. Dysregulation of inflammatory responses are often present in this disease and may lead to granulomatous lesions, most often affecting the gastrointestinal (GI) and urinary tracts. Treatment of inflammatory complications usually includes corticosteroids, whereas antimicrobial prophylaxis is used for infection prevention. Curative treatment of both infectious susceptibility and inflammatory disease can be achieved by hematopoietic stem cell transplantation. We report herein three patients with the same mutation of the CYBB gene who presented with very early-onset and severe GI manifestations of X-linked CGD. The most severely affected patient had evidence of antenatal inflammatory involvement of the GI and urinary tracts. Extreme hyperleukocytosis with eosinophilia and high inflammatory markers were observed in all three patients. A Mycobacterium avium lung infection and an unidentified fungal lung infection occurred in two patients both during their first year of life, which is indicative of the severity of the disease. All three patients underwent bone marrow transplantation and recovered fully from their initial symptoms. To our knowledge, these are the first reports of patients with such an early-onset and severe inflammatory manifestations of CGD.

IL2Rβ-dependent signals drive terminal exhaustion and suppress memory development during chronic viral infection.

Significance During chronic viral infection, CD8+ T cells are gradually deprived of their principal effector functions and irreversibly loose their plasticity to develop into memory populations, precluding the establishment of long-lasting protective immunity. Relevant host-derived factors directing this T-cell exhaustion process have remained elusive. Growing evidence suggests that the cytokine milieu dramatically impacts the outcome of chronic viral infection. However, it is unclear if cytokines directly promote CD8+ T-cell exhaustion and, if so, which specific cytokines are involved in this process. Here we demonstrate a critical role for two highly related cytokines, IL-2 and IL-15, in the terminal differentiation of highly exhausted CD8+ T cells and the lack of immunological memory observed during chronic viral infection. Exhaustion of CD8+ T cells severely impedes the adaptive immune response to chronic viral infections. Despite major advances in our understanding of the molecular regulation of exhaustion, the cytokines that directly control this process during chronicity remain unknown. We demonstrate a direct impact of IL-2 and IL-15, two common gamma-chain–dependent cytokines, on CD8+ T-cell exhaustion. Common to both cytokine receptors, the IL-2 receptor β (IL2Rβ) chain is selectively maintained on CD8+ T cells during chronic lymphocytic choriomeningitis virus and hepatitis C virus infections. Its expression correlates with exhaustion severity and identifies terminally exhausted CD8+ T cells both in mice and humans. Genetic ablation of the IL2Rβ chain on CD8+ T cells restrains inhibitory receptor induction, in particular 2B4 and Tim-3; precludes terminal differentiation of highly defective PD-1hi effectors; and rescues memory T-cell development and responsiveness to IL-7–dependent signals. Together, we ascribe a previously unexpected role to IL-2 and IL-15 as instigators of CD8+ T-cell exhaustion during chronic viral infection.

Experience with subcutaneous immunoglobulin therapy in two pediatric cases of immune thrombocytopenia purpura

Background Immune thrombocytopenia purpura (ITP) can co-exist with primary immunodeficiencies. Intravenous immunoglobulin (IGIV) therapy is an effective treatment. Subcutaneous immunoglobulin (SCIG) formulations that can be home-delivered have recently been developed. We describe 2 cases of pediatric ITP associated with hypogammaglobulinemia, treated with SCIG. Case description Case 1 A 14-year old male presented with a symptomatic thrombopenia. Infusions of IGIV led to an immediate improvement in platelet count. However, he experienced post-infusion intractable headaches, nausea and vomiting, which recurred after subsequent infusions. Intravenous anti-D therapy resulted in a severe allergic reaction. Short course prednisone protocol was implemented for symptomatic episodes. Preliminary blood work for splenectomy revealed low IgG level. The patient was put on SCIG replacement therapy (116 mg/kg/week). He experienced only one relapse since, which remained corticoresponsive. Case 2