Successful Milk Oral Immunotherapy Promotes Generation of Casein-Specific CD137+ FOXP3+ Regulatory T Cells Detectable in Peripheral Blood.

Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (TREG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT. Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4+ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4+ T cell phenotypes were quantified by flow cytometry. Results: Our culture system induced activated casein-specific FOXP3+Helios+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG cells negatively correlated with the time required to reach the maintenance phase of desensitization. Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.

Enhanced interleukin-8 production in mononuclear cells in severe pediatric obstructive sleep apnea.

BACKGROUND: Obstructive sleep apnea (OSA) is a risk factor for cardiovascular disease, metabolic disorders, and cognitive dysfunction. Current thinking links chronic intermittent hypoxia (CIH) with oxidative stress and systemic inflammation. However, the sequence of events leading to the morbidities associated with OSA is poorly understood in children. Monocytes are known to be altered by chronic hypoxia. Thus in this prospective study, we investigated inflammatory cytokine profiles from cultures of peripheral blood mononuclear cells (PBMC) obtained from children with severe OSA and sleep-related CIH. METHODS: Ten children with OSA (cases) and 5 age-matched children without OSA (controls) were recruited for study. Samples of plasma and PBMC were obtained before and after adenotonsillectomy. The levels of the inflammatory cytokines, interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor-α (TNFα), were measured in both plasma and ex vivo culture supernatants of PBMC incubated with lipopolysaccharide (LPS) using the cytometric bead assay. RESULTS: Upon activation of PBMC by LPS, the levels of IL-8 in the culture supernatants from cases were threefold higher than in controls. The levels of the other cytokines including IL-1ß, IL-6, and TNFα, in culture supernatant of PBMC from cases showed no difference from controls; nor were there significant differences in plasma cytokine levels. CONCLUSION: We speculate that in young children with sleep-related CIH, an enhanced production capacity of IL-8 precedes the development of systemic inflammatory markers. Future work should evaluate IL-8 production capacity as a potential biomarker for OSA severity.

Modulation of the Interleukin-21 Pathway with Interleukin-4 Distinguishes Common Variable Immunodeficiency Patients with More Non-infectious Clinical Complications.

PURPOSE: Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and clinical manifestations such as infections, autoimmunity, and malignancy. We sought to determine if responsiveness to interleukin-21 (IL-21), a key cytokine for B cell differentiation, correlates with distinct clinical phenotypes in CVID. METHODS: CVID subjects were recruited through the Canadian Primary Immunodeficiency Evaluative Survey registry. Peripheral blood mononuclear cells were cultured with anti-CD40 ± interferon-gamma, interleukin-4 (IL-4), IL-21, and/or IL-4+IL-21. B cell subpopulations and IgG production were determined at baseline and day 7 by flow cytometry and ELISA. Clinical complications were compared using contingency tables. RESULTS: CVID subjects exhibited decreased CD27+ B cells and IgG production after 7 days of stimulation with anti-CD40+IL-21 (p < 0.05). In a subset of subjects [CVID responders (R)], the addition of IL-4 led to significant increases in CD27+ B cells and IgG (p < 0.05). In CVID non-responders (NR), CD27+ B cells and IgG remained lower despite the addition of IL-4. CVID NR experienced significantly more non-infectious clinical complications of CVID than R [OR 8.8, 95% confidence interval (CI) 1.6 to 48.13]. Previous studies reported that CVID subjects with ≤ 2% class-switched memory B cells were more at risk of these complications, but no significant association was found among this cohort of subjects [OR 3.5, CI 0.9 to 13.3]. In fact, 34.6% of CVID NR had > 2% class-switched memory B cells at baseline. CONCLUSIONS: The IL-4 and IL-21 in vitro assays distinguish two groups of CVID subjects and can be used with baseline B cell subpopulation phenotyping to better identify patients experiencing more vs. fewer clinical non-infectious complications and potentially to modulate therapy.

Positive CD63 Basophil Activation Tests Are Common in Children with Chronic Spontaneous Urticaria and Linked to High Disease Activity.

BACKGROUND: The basophil activation test (BAT) using CD63 expression is a sensitive and specific tool for the diagnostic workup of autoimmune chronic spontaneous urticaria (CSU). The definition of a positive BAT is directly dependent on the reference range and the cutoff values established in control populations. As of now, the pediatric reference range and cutoff values of the CD63 BAT remain to be established. METHODS: In this study, we analyzed CD63 expression in 80 children (1-17 years old) without chronic urticaria (i.e., controls) and compared the values to those of a pediatric cohort of 105 CSU patients and 23 physical urticaria (PU) patients. RESULTS: Based on the log-normal distribution of CD63 values in control subjects, the reference range and the cutoff for positive CD63 BAT values was established to be 1.2-1.8% (95% CI) and 1.8%, respectively. Children with CSU showed significantly elevated and significantly increased BAT values compared to healthy controls (Wilcoxon rank test p value <0.001). In contrast, no difference was found between BAT results in controls and PU patients. In pediatric CSU patients, a higher disease activity was associated with higher BAT values. CONCLUSIONS: Our study provides, for the first time, reference and cutoff values for the CD63 BAT in children. Our findings show that positive CD63 BAT are common in children with CSU and linked to a high disease activity.

Impaired RASGRF1/ERK-mediated GM-CSF response characterizes CARD9 deficiency in French-Canadians.

BACKGROUND: Caspase recruitment domain-containing protein 9 (CARD9) deficiency is an autosomal recessive primary immunodeficiency conferring human susceptibility to invasive fungal disease, including spontaneous central nervous system candidiasis (sCNSc). However, clinical characterization of sCNSc is variable, hindering its recognition. Furthermore, an in-depth understanding of the bases for this susceptibility has remained elusive. OBJECTIVES: We sought to comprehensively characterize sCNSc and to dissect the mechanisms by which a hypomorphic CARD9 mutation causes susceptibility to Candida species. METHODS: We describe the clinical and radiologic findings of sCNSc caused by CARD9 deficiency in a French-Canadian cohort. We performed genetic, cellular, and molecular analyses to further decipher its pathophysiology. RESULTS: In our French-Canadian series (n = 4) sCNSc had onset in adulthood (median, 38 years) and was often misinterpreted radiologically as brain malignancies; 1 patient had additional novel features (eg, endophthalmitis and osteomyelitis). CARD9 deficiency resulted from a hypomorphic p.Y91H mutation and allelic imbalance established in this population through founder effects. We demonstrate a consistent cellular phenotype of impaired GM-CSF responses. The ability of CARD9 to complex with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is intact in our series, arguing against its involvement in susceptibility to fungi. Instead, we show that the p.Y91H mutation impairs the ability of CARD9 to complex with Ras protein-specific guanine nucleotide-releasing factor 1 (RASGRF1), leading to impaired activation of nuclear factor κB and extracellular signal-regulated kinase (ERK) in monocytes and subsequent GM-CSF responses. Successful treatment of a second patient with adjunctive GM-CSF bolsters the clinical relevance of these findings. CONCLUSIONS: Hypomorphic CARD9 deficiency caused by p.Y91H results in adult-onset disease with variable penetrance and expressivity. Our findings establish the CARD9/RASGRF1/ERK/GM-CSF axis as critical to the pathophysiology of sCNSc.

CARD9 deficiency and spontaneous central nervous system candidiasis: complete clinical remission with GM-CSF therapy.

We demonstrate autosomal-recessive Caspase Recruitment Domain-containing protein 9 (CARD9) deficiency in a patient with relapsing C. albicans meningoencephalitis. We identified a novel, hypomorphic mutation with intact Th17 responses, but impaired GM-CSF responses. We report complete clinical remission with adjunctive GM-CSF therapy, suggesting that a CARD9/GM-CSF axis contributes to susceptibility to candidiasis.

Acquired Omenn-like syndrome, a novel posttransplant autoaggression syndrome reversed by rapamycin.

Graft-versus-host disease is uncommon in autologous hematopoietic cell transplantation (HCT) and is typically brief and mild. We report unusual, protracted, and severe Omenn syndrome-like autoaggression following autologous HCT. We identified a profound FOXP3(+) regulatory T cell defect that coincided with hyperinflammatory T cell responses which were reversible with rapamycin in vitro.

Natural killer cells and B lymphocytes in L-selectin and Mac-1/LFA-1 knockout mice: marker-dependent, but not cell lineage-dependent changes in the spleen and bone marrow.

The majority of B lymphocytes, virgin T lymphocytes and a subpopulation of memory T cells express the addressin, L-selectin. Natural killer (NK) cells in rodents and humans also express L-selectin. We have shown that a similar proportion (40%) of NK cells in mouse spleen also express the integrin, CD18Mac-1, and moreover, that NK cells express both the addressin and the integrin constitutively. It was the aim of the present study to quantify, in knock-out mice deficient for either the L-selectin addressin, or the CD18:Mac-1/LFA-1 integrins, NK cells and B cells in both the spleen and their bone marrow birth site. These cells, in both organs, were immunophenotypically stained with FITC-conjugated anti-NK1.1 (to identify NK cells), and FITC-conjugated anti-mouse B220 (to identify B lymphocytes) and subjected to flow-cytometric analysis using a FACScan equipped with a doublet discrimination module. From the known total organ (spleen, femurs) cellularity, obtained by means of an electronic cell counter, at the time of extraction of each organ, the absolute numbers of NK cells and B lymphocytes from each mouse were obtained. The results revealed that there are significantly more NK cells and B lymphocytes in the spleens of CD18:Mac-1/LFA-1 knockout mice than in control (same strain) mice. Moreover, in L-selectin knockout mice spleens, NK cells and B lymphocytes were elevated by 26.2% and 17.8% respectively. NK cells and B lymphocytes in the bone marrow of the integrin knockout showed no difference from control, however, both cell types in the bone marrow of the L-selectin knockout mice fell to only 3/4 their control levels. Collectively, the results demonstrated that there are organ-specific, but not cell lineage-specific differences in the absolute numbers of NK cells and B lymphocytes, in integrin-deficient (CD18:Mac-1/LFA-1 knockout) mice and addressin-deficient (L-selectin knockout) mice.