This is the third report for this project. The two main protocols for this project have been approved and are in recruitment and accrual stages now. We continue to enroll a cohort of patients with severe atopic dermatitis (AD) alone, and those with AD in the context of immune deficiency. We have also begun accruing patients with genetic disorders which include atopy as a part of the syndrome -- now numbering over 100 patients total. Three main findings have occurred in this project within the past year: 1) Because of our interest in patients with symptoms of atopy in the context of unidentified syndromes, we have been studying a series of families with cold urticaria, antibody deficiency, autoimmunity and granulomatous disease segregating in an autosomal dominant pattern. Patients had low IgM and IgA, low NK cells, normal T cells and low to absent class-switched B cells. Three patients are now on IVIG for common variable immune deficiency. In collaboration with the Kastner lab in the NHGRI, we performed linkage analysis, identifying a disease associated locus on chromosome 16q. We performed candidate gene sequencing on genes within the locus which led to the identification of 3 independent genomic deletions in PLCG2. The deletions lead to gain of PLCG2 enzymatic function but loss of distal signaling function, resulting in defective calcium flux and ERK phosphorylation in response to surface receptor cross-linking in B and NK cells, and poor in vitro expansion and switched immunoglobulin secretion in response to SAC and CpG and abnormal B-cell receptor editing in B-cells. Cytosolic calcium levels in unstimulated, purified primary B cells from PLAID (PLCG2 associated Antibody deficiency and Immune Dysregulation) patients steadily rose with decreasing temperature, but were unchanged with cooling in wild type B cells. Similarly, BCR-stimulated PLAID B cells exhibited increased ERK phosphorylation with decreasing temperatures, peaking at 25C, whereas wild type B cells had diminished ERK phosphorylation with decreasing temperatures. Taken together, these data provide evidence that the capacity for receptor-mediated cellular activation is diminished in PLAID B cells at physiologic temperatures, but is enhanced at subphysiologic temperatures. A paper describing these results is currently under revision at a major journal. 2) We have been able to study the role of STAT3 in lymphocyte homeostasis and atopy. STAT3 disruption leads to elevated IgE and atopic dermatitis, amongst a variety of other infectious and non-immunologic phenotypes in the Hyper-IgE syndrome (HIES). In order to measure the impact of STAT3 mutations on lymphocytes memory, we performed detailed surface staining of naive and memory markers on PBMC samples from HIES patients compared to age matched controls. We found that there was a significant decrease within the central memory T cell compartment of both CD4 and CD8 T cells, as well as a decrease in the B cell memory compartment. We also noticed that naive cells were actually increased in HIES patients. In collaboration with the NIAID LCID, Dr. Hollands lab, we were able to validate our findings in a patient mosaic for a mutant STAT3 allele. The mutant allele was found in 30% of sorted naive T cells, however it could not be found in sorted memory cells, arguing for a cell-intrinsic competitive disadvantage for memory CD4 and CD8 T cells bearing mutant STAT3. To gain a mechanistic understanding of the effect of STAT3 mutation on memory T-cells, we looked for evidence of increased in vivo death or decreased turnover of these memory cells. In fact there was no difference in ex vivo apoptotic cells in any of the lymphocyte subsets, and the turnover was normal to elevated. The turnover may in part have been due to elevated levels of serum IL-15 which we measured in HIES patients. We then turned to an in vitro model of primary proliferation and differentiation of naive T-cells into central and effector memory T-cells and were surprised to find that there was actually a defect in TCR-driven proliferation of naive CD4 and CD8 T cells from AD-HIES patients. Those fewer cells which were able to divide had more effector-like surface marker expression as compared to controls which acquired more of a central memory-like phenotype. This primary defect in the capacity for naive T cells to divide and differentiate into central memory T cells may account for the increased naive but decreased central memory T cell populations in HIES. We then asked whether these T-cell defects might translate into clinical phenotypes. First, we measured VZV-specific memory T cells and found that they were markedly reduced in HIES patients. We then surveyed the cohort of HIES patients followed at the NIH and found a 20-70 fold increase in incidence over that of the aged matched general population, depending on the decade measured. Interestingly, primary varicella infection is almost universally uncomplicated in HIES patients, as is the case with most viral infections in these patients. We then examined patient PBMC for reactivity to another chronic latent herpesvirus, EBV, and found diminished central memory responses in AD-HIES. EBV viremia, detectable at low levels normally in less than 10% of the population, was present, and in some cases at high levels, in >50% of patients examined. It is important to note that HIES patients have an increased risk for lymphoma, both EBV+ and EBV- and this may be in part due to the lack of adequate surveillance by central memory T cells. It should be noted that the increased prevalence of shingles and EBV viremia are both previously unrecognized phenotypes within this patient population, as most infectious phenotypes were thought to be restricted to extracellular bacteria or fungi. In summary, it appears that STAT3 mutations lead to difficulties in the differentiation and maintenance of long-lived T cell memory. This may contribute to the failure to control certain chronic viruses. A manuscript describing these findings is currently under revision in a major immunology journal. 3) In order to assess the effects of STAT3 mutations on allergic disease, we performed detailed allergic histories on the majority of the NIH AD-HIES cohort and compared them to patients with marked IgE elevations but no STAT3 mutation. Patients with HIES had a substantially lower incidence of food allergy, asthma and anaphylaxis compared to those with similarly elevated IgE levels. Allergen-specific IgE was measured using ImmunoCap RAST and prominent allergen-specific IgE was measured in most patients, arguing against a defect in antibody specificity. Foxp3+ regulatory T cells (Tregs) have been shown to directly inhibit mast cell function, and many models of experimental atopy can be suppressed with regulatory T cells. We enumerated Foxp3+ T cells in HIES patients and find that within the diminished central memory compartment, there is a marked enrichment of Tregs compared to control. In summary we have shown that specific atopy and anaphylaxis are found at lower rates than expected in HIES patients given their marked elevation in serum IgE. We find that specific IgE appears to be intact, and that two potential mechanisms for this decrease in allergic reactivity in patients are a mast-cell intrinsic defect, as well as an enrichment for Foxp3+ Tregs.

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