| PART ONE REGULATORY MECHANISM OF IGE PRODUCTION IN DOCK8 DEFICIENCY BY FOLICULLAR HELPER T CELL AND ITS IL-21Background: Autosomal recessive hyper-immunoglobulin E syndrome(AR-HIES) caused by DOCK8 defects is characterized by recurrent elevated serum Ig E level, elevated peripheral eosinophil count, severe atopy, recurrent viral and bacterial infections, and early-onset malignancy. DOCK8 gene maps to the chromosomal locus 9p24.3,which activates the Rho-GTP, regulates the actin cytoskeleton and influences the cell migration. DOCK8 immunodeficiency syndrome is a combined immunodeficiency especially in the antibody production mediated by B cells. And the regulatory mechanism of Ig E production in DOCK8 deficiency is still unknown.Objective: To analyze the clinical, genetic, and immunologic characteristics of DOCK8 mutations in Chinese patients and investigate the regulatory mechanism of Ig E production in DOCK8 deficiency through patients and DOCK8-KO mice.Methods: Patients, who presented typical or partial clinical features of HIES but had no STAT3 mutations, were subjected to deep sequencing targeting DOCK8, flow cytometry and western blotting for the expression of DOCK8 protein. Clinical features and immune function of the patients were evaluated to confirm the diagnosis of DIDS. The count and function of Follicular helper T cells and the related molecule such as Bcl-6 and IL-21 in DOCK8 patients were evaluated by flow cytometry and real time PCR. Next we established the DOCK8-KO mice and determined the function. The count and function of Follicular helper T cells and the related molecule including ICOS, PD-1, Bcl-6 and IL-21 in DOCK8-KO mice were evaluated by flow cytometry and real time PCR compared with WT mice. We also made the OVA-induced DOCK8-KO mice for analysis of their susceptibility to allergy. Finally the DOCK8-KO mice and OVA-induced mice were treated with nasal administration of recombinant mouse IL-21.Results:1. In total, seven patients from unrelated seven families were subjected to DOCK8 analysis, with clinical HIES scores of 75, 62, 47, 49, 44, 42, and 45, respectively. The most common manifestation was recurrent virus infections, pneumonia, eosinophilia and significantly elevated serum Ig E levels. Deep sequencing analysis revealed various novel mutations in the DOCK8 of the patients, including large deletions and point mutations. A large heterozygous deletion spanning exons 19–48 of the DOCK8 gene was found in patient 1. Patient 1 also had a spontaneous homozygous deletion of the G nucleotide at position 5842 and a homozygous point mutation(C>A) at position 5843. The frame shift resulted in premature stop codon at position 1953. Patient 2 had a large homozygous deletion of exon 11 and a large heterozygous deletion of exons 12–33. Patients 3 and 4 both had a homozygous deletion of G3152 in exon 26 that led to frame shift which resulted in premature stop codon at position 1093. Patients 3 and 4 also had a homozygous point mutation(G >C) at position 5175 in exon 40 that resulted in the substitution of aspartic acid for glutamic acid at position 1725(E1725D). Patients 3 and 4 were from unrelated families. Patient 5 had a large homozygous deletion at exon 2 and large heterozygous deletions at exon 1 and exons 3–39. Patient 6 had a large homozygous deletion at exon 7 and large heterozygous deletions at exons 8–10. Patients 7 had a spontaneous homozygous deletion of the T and G nucleotide at position 1278–1279 in exon 11 that led to frame shift which resulted in premature stop codon at position 435. DOCK8 protein was not detectable neither by flow cytometry nor western blotting. The expression of TRECs was extremely low in all the patients when compared to healthy controls. The frequency of CD3+CD4+ T cells, CD27+ memory B cells and memory CD4+T were lower compared to the normal reference. The expression of CD107α in NK cells and the expression of IL-10 in CD19+ cells were lower in patients than in healthy controls. T and B lymphocyte failed to proliferate in response to in vitro stimulation, as determined by using a CFSE dilution assay. The percentages of CD3+CD4+T cells that produced IL-4, IL-17, and IFN-γ were normal, as well as the percentage of CD4+CD25+Foxp3+T cells. DOCK8 deficient patients presented decreased follicular helper T cell(Tfh cell) counts compared with the normal percentage in the peripheral blood. More importantly, IL-21 produced by Tfh cells showed a dramatical decrease.2.(1) We established DOCK8-KO mice using TALEN technology. DOCK8-KO mice showed hyper-Ig E in their serum and increased Ig E+B cells in peripheral blood and spleen as compared with WT mice. The percentage of follicular helper T cell is decreased in DOCK8-KO mice, as well as the related molecules. More importantly, IL-21 produced by Tfh cells also showed a dramatical decrease, which mirrored the same change in human.(2)We successfully established the asthmatic model with low dose OVA(1/4 of the commonly used dose) in DOCK8-KO mice. The mice presented airway hyperreactivity, increased Ig E level and eosinophilia. The frequency and count of Ig E+ B cell were elevated in OVA induced DOCK8-KO mice and WT mice, but the former had significantly higher Ig E+B cells than the latter. The serum total Ig E and OVA specific Ig E levels were significantly increased. And the number of IL-21+CXCR5+ cell was decreased dramatically in OVA induced DOCK8-KO mice. The frequency of memory Tfh cells and the related molecules were decreased dramatically in OVA induced DOCK8-KO mice.3.(1) DOCK8-KO mice were treated with recombinant mouse IL-21 and we found that the frequency of Ig E+B cells and Tfh cells nearly returned to normal level. The regulating transcription factor Bcl-6 and the related molecules such as ICOS and PD-1 were restored to normal level.(2)OVA induced mice were treated with recombinant mouse IL-21 and we also found that the frequency of Ig E+ B cells and Tfh cells returned to normal level. The regulating transcription factor Bcl-6 and the related molecules, ICOS and PD-1 were restored to normal level.Conclusion: Abnormal Tfh cell development and decreased production of IL-21 in DOCK8 deficiency appear to play the central role in resulting in hypersensitivity of B cells to allergen and production of large amount of Ig E. PART TWO INFLUENCE OF IKKβ DEFICIENCY ONNF-KB PROTEIN TRANSPORTATION TO THE NUCLEUSBackground: Deficiency of Innate and Acquired Immunity Caused by IKBKB Mutation is a special type of combined immunodeficiency disease, with clinical features of repeated bacterial, viral and fungal infection. Most patients had thrush, bronchitis, pneumonia, chronic diarrhea, growth retardation, cerebral hemorrhage, epilepsy, omphalitis, retarded shedding of the umbilical cord. IKBKB gene maps to the chromosomal locus 8 p11.2, which encodes IKKβ that serves as a protein subunit of IκB kinase, a component of the cytokine-activated intracellular signaling pathway involved in triggering immune responses. IKK’s activity causes activation of a transcription factor known as Nuclear Transcription factor kappa-B or NF-κB. Activated IKK phosphorylates a protein called the inhibitor of NF-κB, IκB(IκBα), which binds NF-κB to inhibit its function. Phosphorylated IκB is degraded via the ubiquitination pathway, freeing NF-κB, and allowing its entry into the nucleus of the cell where it activates various genes involved in inflammation and other immune responses.Objective: To analyze the genotype, clinical manifestations and immune function in a patient with IKBKB mutation and investigate the effect of phosphorylation in 395 amino acids on NF-κB protein transportation to the nucleus.Methods: A 17 years old boy was enrolled in the present study. This patient became ill at 2 month of age with severe and repeated respiratory infection, chronic diarrhea, and poor weight gain. Candidate genes were tested by next generation sequencing and confirmed by Sanger sequencing. Protein was detected by flow cytometry and western blotting. Immune function of the patient was determined by flow cytometry. The amount of NF-κB in nucleus was detected by EMSA and confirmed the phosphorylation of 395 amino acids in cell lines.Results: This patient had a homozygous point mutation(T>C) at position 1183 in exon 12 that resulted in the substitution of histidine for tyrosine at position 395(Y395H). IKKβ protein was undetectable neither by flow cytometry nor western blotting. The frequency of CD27+ memory B cells was lower as compared to the normal reference. The CD4+CD25+Foxp3+T cells were absent. The expression of IL-10 in CD19+was increased in patient, while the percentages of CD3+CD4+T cells that produced IL-4, IL-17, and IFN-γ were lower in patient than in healthy controls. T and B lymphocyte failed to proliferate in response to in vitro stimulation, as determined by using a CFSE dilution assay. The expression of CD107 a in NK cells and the TCRVβ diversity were normal. The amount of NF-κB in nucleus was lower than that in healthy controls. And we also found that the degradation of IKKβ protein was significantly faster in 395 amino acids mutant cell lines.Conclusion: The present case was the 10 th patient with IKBKB deficiency worldwide. The responsible mutation of IKBKB has not been previously reported. IKKβ protein 395 amino acids site is an important phosphorylation site. The substitution of histidine for tyrosine at position 395 amino acid leads to the increased degradation rateof IKKβ protein resulted in affecting the NF-κB transportation to nucleus. |
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