Immunodeficiency Canada Satellite Symposium at 2014 Canadian Society of Allergy and Clinical Immunology Annual Scientific Meeting
October 23, 2014
Delta Ottawa Centre
Ottawa, ON

Chronic Granulomatous Disease (CGD) is a primary immunodeficiency caused by mutations that disable the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex. This dysfunction subsequently leads to decreased or absent production of reactive oxygen species required for phagocytic killing of certain pathogens. CGD can be inherited in an X-linked or autosomal recessive fashion. The different genetic forms of CGD vary in their residual NADPH oxidase function and can thus be differentiated based on this quantification. Importantly, residual reactive oxygen species production has been correlated with long-term prognosis for patients with CGD (Kuhns et al. 2010).

Dihydrorhodamine 123 is a non-fluorescent probe that oxidizes in the presence of reactive oxygen species to fluorescent Rhodamine 123. The Dihydrorhodamine 123 assay uses flow cytometry techniques to quantify the level of fluorescence in activated neutrophils which reflects the levels of reactive oxygen species. This assay allows both the diagnosis of patients with CGD and the differentiation between X-linked and autosomal recessive forms of the disease. This assay can also be used to identify X-linked carriers and predict which specific molecular defect a patient might carry.

Using a case of X-linked CGD, this presentation highlights the diagnostic and prognostic utility of the Dihydrorhodamine 123 assay.

Signal transducer and activator of transcription 1 (STAT1) is a DNA binding transcription factor that has an important role in the signaling pathways of several cytokines and growth factors. The STAT1 gene encodes for two isoforms, STAT1α and STAT1β. STAT1β largely acts as a dominant negative inhibitor (Zakharova et al. 2003). STAT1α is involved in the signaling pathways of a wide variety of cytokines and growth factors including IFN γ, IFN α / β, IL2, IL3, IL6, IL9, IL10, IL11, IL12, IL15, IL21, growth hormone, fibroblast growth factor and epidermal growth factor (Najjar and Fagard 2010). Following binding of the cytokine or growth factor to its respective receptor, there is phosphorylation of the intracellular domains of the receptors, typically by the JAK tyrosine kinase family, which in turn provides the docking site for cytoplasmic STAT1. STAT1 is then phosphorylated forming homodimers or heterodimers, allowing for translocation into the nucleus where it binds to target DNA sequences leading to transcription of target genes (Zakharova et al. 2003; Najjar and Fagard 2010; Boisson-Dupuis et al. 2012; Casanova et al. 2012). After activation, STAT1 is dephosphorylated and transferred back to the cytosol.

In recent years, the broad genetic and clinical heterogeneity of STAT1 mutations have been well described (Boisson-Dupuis et al. 2012; Casanova et al. 2012). Patients with an autosomal recessive complete loss of function STAT1 mutation present at an early age with overwhelming mycobacterial and viral infections such as herpes simplex virus, due to lack of cell response to IFN γ and IFN α / β (Dupuis et al. 2003; Chapgier et al. 2006; Vairo et al. 2011). This patient population represents one of the most severe forms of STAT1 mutations, which is fatal without correction with hematopoietic stem cell transplant (HSCT). Partial autosomal recessive STAT1 mutations caused by hypomorphic alleles, with an impaired but not abolished STAT1 dependent response to IFN γ and IFN α / β, results in a milder phenotype with treatable mycobacterial and viral infections (Chapgier et al. 2009; Kong et al. 2010; Kristensen et al. 2011). Autosomal dominant loss of function STAT1 mutations are characterized by susceptibility to weakly pathogenic mycobacterial infections (Dupius et al. 2001; Chapgier et al. 2006). Autosomal dominant gain of function STAT1 mutations predisposes patients to chronic mucocutaneous candidiasis (CMCC) and autoimmunity (van de Veerdonk et al. 2011). Recently described, patients with de novo heterozygous mutations in or near the DNA binding domain encoding T385M, I294T, or C284R amino acid substitutions, can present with progressive combined immunodeficiency with loss of T and B cell numbers and function over time (Sharfe et al. 2014). STAT1 expression in this patient population appeared to be significantly reduced but not completely absent (Sharfe et al. 2014). Over a number of years, the loss of effective immunity resulted in the emergence of the autoimmune disease, and, ultimately, 3 of 5 patients having fatal overwhelming infections. Two additional patients with heterozygous T385M STAT1 are reported with one patient dying at age 14 years old with disseminated intravascular coagulation and pulmonary insufficiency of unknown etiology (Takezaki et al. 2012). Here we present a case report of a 7 year old male patient with a de novo heterozygous T385M STAT1 mutation managed with HSCT.

We present here a 7 year old male patient born to non consanguineous parents of English descent with an unremarkable family history. He presented at age 4 years old with oral thrush following antibiotic use. In addition, he developed fungal lesions on his face and lips, which were managed with fluconazole. He had several episodes of acute otitis media with one documented pneumonia. Genetic analysis confirmed a G > A substitution (c1154T) in STAT1 causing substitution of amino acid threonine to methionine (T385M). The mutation was determined to be de novo and not present in either parent. Two additional patients identified with the same mutation demonstrated a progressive combined immunodeficiency and died at ages 7 years old and 10 years old of overwhelming infections to CMV and EBV (Sharfe et al. 2014). Based on the apparent life threatening mutation, our patient was offered the option of restorative HSCT.

The patient received a 10/10 HLA matched sibling donor transplant. While engraftment was rapid and remains robust, the post transplant course was complicated largely by infections and graft versus host disease (GvHD).

Given the apparent fatal nature of the de novo heterozygous STAT1 mutations, HSCT may be considered as a treatment option. There are two early reports of patients with severe CMCC managed with HSCT, however it is unknown if these patients had an underlying STAT1 mutation. These reports include a 7 year old female with CMCC and aplastic anemia (Deeg et al. 1986) and a 12 year old male with CMCC, Coomb's positive hemolytic anemia and recurrent pneumonia managed with a HLA matched sibling donor HSCT (Hoh et al. 1996). Vairo et al. 2011 reported a successful HSCT in a 3 year old patient with complete autosomal recessive STAT1 deficiency (Vairo et al. 2011). To date there is one report of a HLA matched sibling donor HSCT in a patient with a de novo STAT1 mutation in the DNA binding domain (Aldave et al. 2013). This patient received reduced intensity conditioning with fludarabine, melphalan and antithymocyte globulin. There was a progressive loss of donor cells starting at day + 107 and the patient ultimately died of fulminant interstitial lung disease 10 months after the HSCT. HSCT was offered to our patient based on knowledge that two patients with the same de novo heterozygous T385M STAT1 mutation had progressive combined immunodeficiency and died of overwhelming infections (Sharfe et al. 2014). Prior to HSCT, our patient appeared to be in the early stages of the disease with normal lymphocyte numbers but impaired functioning with poor mitogen response and non protective specific antibodies. The patient continues to have full donor chimerism, but is still receiving immunosuppression for GvHD. Overall, patients with recurrent infections with or without CMCC and evidence of progressive combined immunodeficiency should be investigated for a possible STAT1 mutation. Patients with apparent fatal mutations may be considered as candidate for restorative HSCT.

The complement system is an essential part of the innate immune system, comprising of the classical, alternative, and lectin pathways. Activation of these pathways results in clearance of pathogens and altered self-cells via opsonisation, chemotaxis, and direct lysis. The mannose-binding lectin (MBL) is secreted by the liver and binds to specific carbohydrate moieties on the surface of pathogens, thus activating the lectin pathway through the MBL-associated serine proteases (MASPs). MBL deficiency has been associated with respiratory infections, neonatal sepsis, bronchiectasis, inflammatory diseases, disease progression in cystic fibrosis and common variable immunodeficiency (CVID), as well as susceptibility to infection in pediatric oncology patients. MBL deficiency is common in the general population: 10–30% exhibit low MBL levels, depending on the definition used. The genetic mutations known in humans are single nucleotide polymorphisms (SNPs) in the MBL2 gene. It has been suggested that specific SNPs may explain the large inter-individual susceptibility to infections.

This is a case of three siblings with the same four heterogeneous polymorphic mutations in the MBL2 gene. The youngest boy, aged 2 years, has multiple otitis media and perianal abscesses. The eldest boy, aged 7 years, has a history of recurrent perianal abscesses, sinusitis, and a pneumonia requiring intravenous antibiotics. The middle child, a 3 year old girl, has been asymptomatic aside from having nasal polyps and a normal sweat chloride. An immunodeficiency workup revealed normal neutrophil count, immunoglobulin levels and function, normal B cell and T cell enumeration, normal lymphocyte proliferation, and normal neutrophil oxidative burst index (NOBI) in the two boys. The MBL assay was undetectable (<1%) on multiple occasions in the two boys and extremely low (2–4%) in the girl.

MBL deficiency has been associated with many diseases. It appears to be a common immunodeficiency that has been defined by different approaches and needs a unifying classification. MBL genetic polymorphisms have been shown to increase susceptibility to infections and neonatal sepsis. This case illustrates the varying clinical presentations in three siblings, two of them having recurrent skin abscesses, with the same heterozygous polymorphic mutations; thus necessitating a high level of suspicion and close follow-up. Furthermore, there is currently no consensus on the management of patients with MBL deficiency.

Introduction: Bare lymphocyte syndrome or MHC class II deficiency is a rare autosomal recessive combined immunodeficiency characterized by recurrent infections and autoimmune phenomena. Patients usually present with a (S)CID phenotype. Immunologically they have CD4 lymphopenia and lack antigen-specific responses.

Patients Characteristics and Results: We present a cohort of 19 patients with MHC class II deficiency with a spectrum of clinical presentation. The patients are from 5 different nationalities and presented at the mean age of 9 months (0–76) with a diagnostic delay of 17 months (0–184). Following myeloablative conditioning regimens, patients were transplanted from matched siblings (n = 3), matched family donors (n = 2), MUD (n = 7) and haploidentical donors (n = 7). Acute GvHD occurred in 8, chronic GvHD in 6 patients. 11 patients survive following treatment with hematopoietic stem cell transplantation. Four patients died within 3 months after hematopoietic stem cell transplantation (HSCT), 4 patients between 4 months to 3 years following HSCT. Causes of death were GvHD or infectious complications (sepsis, CMV, aspergillosis). Age at last follow-up ranges between 6 months and 16 years (mean 26 months).

Conclusion: Clinical presentation in patients with MHC class II deficiency may or may not parallel the (S)CID phenotype. Clinical variability may be explained by residual and/or inducible expression of MHCII on thymic epithelium and antigen-presenting cells as well as on environmental or epigenetic factors. Patients have been transplanted successfully from HLA-identical and non-identical donors. The challenges of HSCT are both non-engraftment and severe GvHD.

Background: CVID is characterized by hypogammaglobulinemia and increased susceptibility to infections. We previously described that CVID have impaired in vitro capacity to undergo isotype class-switching in response to interleukin-21(IL-21)(Desjardins and Mazer 2013a, 2013b). The aim is to present our recent findings and the characterization of 2 subgroups of CVID subjects.

Methods: We recruited CVID patients at McGill University Health Centre with age/sex matched healthy controls. Peripheral blood B-cell subpopulations were determined at baseline by flow cytometry. Peripheral blood mononuclear cells were isolated with Ficoll-Hypaque centrifugation and cultured in complete media with anti-CD40 (1 µg/mL) ± interferon-gamma (10 ng/mL), interleukin-4 (IL-4, 200 U/µL), IL-21 (50 ng/mL) and/or IL-4+IL-21. Percentage of memory B-cells and IgG production at day 7 were determined by flow cytometry and ELISA. Statistical analyses were performed by one-way ANOVA and Student t test.

Results: From the 22 CVID subjects recruited, all exhibited decreased percentages of CD19⁺CD27⁺memory B-cells (p < 0.01) and IgG production (p = 0.01) compared to controls following aCD40/IL-21 stimulation. In a subset of CVID (CVID group 2), the addition of IL-4 to the culture media (aCD40/IL-4/IL-21) allowed significant increases in the percentage of memory B-cells (1.8 → 5.9%) and IgG production (0.5 → 3.0 mg/L). These levels were comparable to controls. In CVID group 1, the percentage of memory B-cells and IgG remained low. The 2 subgroups had different baseline B-cell characteristics: CVID group 1 had significantly more naïve CD19⁺ CD27^–IgM⁺B-cells (▵ 19.2%, p < 0.001) and less CD19⁺CD27⁺IgM^–memory B-cells (d = –11.6%, p < 0.0001) and CD19⁺CD27⁺CD38⁺IgM^–plasmablasts (▵ = –2.6%, p < 0.01) compared to controls. CVID group 2 demonstrated an increase in CD19⁺ CD27⁺IgM⁺B-cells (▵ = 7.4%, p = 0.02).

Conclusions: CVID subjects have impaired in vitro responses to aCD40/IL-21. In a subset of CVID subjects, the percentage of memory B-cells and IgG production can be increased to control levels through the IL-21 pathway (aCD40/IL-4/IL-21). The 2 subgroups of CVID subjects had different baseline B-cell characteristics suggesting possibly different underlying pathology.

Background: Severe combined immune deficiency (SCID) is caused by an array of genetic disorders resulting in an impaired adaptive immune system. It usually presents within the first few months of life with multiple or chronic infections as the production and function of T lymphocytes and/or B lymphocytes are extremely impaired or absent. To prevent transmission of infections in SCID patients, standardized infection control precautions and guidelines should be implemented. Although infection control policies have been described for other vulnerable pediatric populations such as hematopoietic stem cell transplant and oncology patients, SCID-specific infection control policies have not yet been described, and we have noted SCID-specific policies vary significantly between centers. This study aims to describe SCID-specific infection control protocols internationally, and from this descriptive data, future evidence-based research into effective SCID infection control practices can be proposed.

Methods: A survey regarding SCID-specific isolation protocols was disseminated online by Immunodeficiency Canada and the Clinical Immunology Society. Descriptive data was collected and analyzed. This research was approved by our local Research and Ethics Board, and participant consent was discussed at the beginning of the survey.

Results: A total of 78 responses were obtained. The majority of respondents were from the United States (53%), followed by 10% from Canada, and the remainder were from 18 other countries. Most respondents were HSCT specialists (43%) or physicians working with HSCT patients (33%).

There was significant variability with regard to indications for discharge of a clinically well child with SCID. Nearly one-third of respondents (29%) would not discharge a SCID patient home regardless of clinical status. The remainder would allow clinically well SCID babies to be home under various conditions, such as requiring that the family live close by (29%) and no visitors are allowed (29%). Ten percent of respondents had no restrictions for discharge of clinically well SCID patients.

For inpatients, the vast majority are admitted to one bedroom rooms (89%) with HEPA filters present in 51% of rooms, positive pressure air flow in 41%, laminar air flow in 19%, and anterooms in 55%. Required personal protective equipment (PPE) for staff included non-sterile gowns (61%), a surgical mask (67%) and non-sterile gloves (57%). Some centers required sterile gloves (8%) and gowns (13%) as well as hair (20%) and shoe covers (16%). Nearly half (47%) of respondents do not require caregivers to wear any PPE. Most centers (62%) allow visitors, though generally with restrictions such as number of visitors and age of children.

Guidelines for breastfeeding varied: 16% of respondents do not allow breastfeeding or expressed breast milk at all, nearly half (47%) allow it if the mother is CMV negative, and some centers process expressed milk with irradiation, pasteurization or freezing it.

Only 51% of respondents noted that their center had a SCID-specific infection control protocol. Most doctors (80%) received training on these protocols, but only 47% of nurse practitioners and 41% of nurses received training. Less than a third reported training other professionals such as other allied health workers and housekeepers (29% and 27% respectively). Only 28% of respondents reported a formal compliance assessment program for these protocols.

Conclusions: Significant variability was found with regard to SCID-specific infection control precautions at different treatment centers. There was disagreement as to whether a well child with SCID should be cared for as an inpatient, or if it is safe for them to go home. For inpatients, the type of PPE required by staff and caregivers, and whether visitors are allowed varied. Conditions for allowing breastfeeding were discordant between centers. And finally, many centers did not have a SCID-specific infection control protocol at all, and most had no formal compliance program. There is little evidence-based data regarding what environment is safest to prevent infection in a child with SCID, and it must be considered that some of the protocols that are instituted may have significant impact on infection risk, family well-being, the child's development and cost of care. Further multi-center research is needed in this area to determine the safest and healthiest environment for these children.

ZAP70 (zeta-associated protein of 70 KDa) deficiency is a fatal form of combined immunodeficiency that can be cured with hematopoietic stem cell transplantation (HSCT). The normal number of thymocytes, polyclonal CD3⁺ T cells and B cells may require special considerations in choosing the most effective regimen of conditioning and the type of HSCT. We analyzed presentation and outcome of patients with ZAP70 deficiency who received various modalities of stem cell transplantation.

Data on 19 patients with ZAP70 deficiency were examined, of whom 16 underwent HSCT in 7 different counties. HSCT was performed by using different sources of donor stem cells as well as various conditioning regimens.

Most patients presented with typical repeated microbial and fungal infections; one patient presented with lymphoma. Close family members appear to have a high frequency of autoimmunity. Sixteen of nineteen patients were treated with HSCT. Nine patients received a MRD and 8/9 survived, four patients had MUD transplants and all survived and 3 had a MMRD transplant of which only one survived. Immune reconstitution was complete and durable in patients who received myeloablative conditioning. Lack of conditioning failed in two patients and resulted in partial immune reconstitution in the other patients.

Based on these data, we conclude that carriers of monoallelic mutations in ZAP70 may be susceptible to autoimmunity. HLA matched donor and myeloablative conditioning result in superior outcome and long-term robust immune reconstitution.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life threatening inflammatory process which is characterized by uncontrolled activation of macrophages and histiocytes. Traditionally, HLH has been divided into two forms, the first is the genetic form which usually presents at young age and caused by defect in genes that are responsible for the cytotoxic function of T cells and NK cells. The second is the acquired form which is associated with infection, malignancy or rheumatologic disorder but with no known genetic abnormality. Multiple genes related to immune function have been associated with the development of HLH, but the association between mutation in the transcription factor Signal Transducer and Activator of Transcription 1 (STAT1) and HLH has not been described in detail. STAT1 is a key element in many of the signaling cascades involved in immune system function. Different mutations in STAT1 are associated with heterogeneous clinical phenotypes that range from early fatality due to overwhelming infection to limited involvement of the mucus membrane with recurrent candida infections. We report here an occurrence of fatal HLH in a patient with chronic mucocutaneous candidiasis (CMC) due to STAT1 mutation.

The patient, a female from non-consanguineous East Indian origin, was diagnosed with CMC at early infancy and found to have a mutation in the STAT1 DNA binding domain. In addition to recurrent episodes of oral thrush and skin infections, she developed hypothyroidism, autoimmune hemolytic anemia and bronchiectasis until she presented, at the age of ten years, with full blown HLH. Her immune work up that was normal initially, declined over the years with declining numbers of CD4 and NK cells, a finding that was documented in other patients with a similar STAT1 mutation. During her hospitalization the patient suffered from recurrent episodes of fever and seizures and from persistent elevated markers of HLH such as ferritin, soluble CD25 and CD163. The patient was treated according to the HLH 2004 protocol with steroids, etoposide, cyclosporine and intra-thecal methotrexate but failed to get into remission. Eventually, after 8 weeks of treatment, the patient received bone marrow transplant from matched unrelated donor but developed severe complications of gastrointestinal bleeding, pulmonary hemorrhage, toxic epidermal necrosis and renal failure with relapse of the disease. Salvage therapy with Alemtuzumab that was added after the transplantation, did not improve her symptoms and the patient succumb to uncontrolled HLH and multi organ failure.

The novelty of this case is the association of chronic mucocutaneous candidiasis and STAT1 mutation with HLH, an association that was suspected but not described previously. As dysregulation of the immune system plays a critical part in the development of HLH, we believe that the association between STAT1 mutation and HLH in not a coincidence but rather represent a novel correlation between the two. This case emphasizes the need for thorough immunologic work up that includes STAT1 genetic analysis in severe and refractory cases of HLH and the need for more studies that will help to decide what is the best treatment in patients with similar pathology.