SBDS Expression and Localization at the Mitotic Spindle in Human Myeloid Progenitors

BACKGROUND: Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle. CONCLUSIONS/SIGNIFICANCE: SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34(+) hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained

Extensive Ethnic Variation and Linkage Disequilibrium at the FCGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C-ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C-ORF haplotype showed strong LD with, among others, rs201218628 (FCGR2A-Q27W, r 2 = 0.63). LD between these two variants was weaker (r 2 = 0.17) in Africans, whereas the FCGR2C-ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C-ORF haplotype and rs1801274 (FCGR2A-H131R) were in weak LD (r 2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C-ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation

Extensive Ethnic Variation and Linkage Disequilibrium at the FCGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in &gt;4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C-ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C-ORF haplotype showed strong LD with, among others, rs201218628 (FCGR2A-Q27W, r2 = 0.63). LD between these two variants was weaker (r2 = 0.17) in Africans, whereas the FCGR2C-ORF haplotype was nearly absent in Asians (minor allele frequency &lt;0.005%). The FCGR2C-ORF haplotype and rs1801274 (FCGR2A-H131R) were in weak LD (r2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C-ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Polymorphisms in the mannose-binding lectin gene as determinants of age-defined risk of coronary artery lesions in Kawasaki disease

OBJECTIVE: To evaluate the relationship between polymorphisms in the gene coding for mannose-binding lectin (MBL) and the occurrence of coronary artery lesions (CALs) among different age groups of patients with Kawasaki disease. METHODS: The frequencies of the genotypes, defined as mutations in codons 52, 54, and 57, and the functional promoter variants of the MBL2 gene were determined in 88 patients with acute Kawasaki disease (median age at onset 1.9 years). The possible influence of the MBL2 genotype on the development and progression of CALs in Kawasaki disease was assessed according to age categories and MBL genotypes in univariate and multivariate analyses. RESULTS: In patients younger than age 1 year, we found an increased risk of developing CALs in the presence of a variant MBL2 genotype (P = 0.008). In contrast, in patients older than age 1 year, we found an increased risk of CALs in those patients with the wild-type genotype (P = 0.005). CONCLUSION: Our findings indicate that MBL has an ambiguous role in Kawasaki disease and contributes differently to the pathophysiologic development of CALs, being protective in infants but potentially harmful in patients of older age. The data also imply that the standard treatment of intravenous immunoglobulins to reduce the development of lesions may not be as effective in the very young as it is in the older patients. For the very young, alternative or adjuvant treatment may be indicated, particularly in infants who are MBL-deficien

Toll-like receptor-induced reactivity and strongly potentiated IL-8 production in granulocytes mobilized for transfusion purposes

Transfusion of granulocytes from granulocyte-colony stimulating factor (G-CSF)/dexamethasone (dexa)-treated donors can be beneficial for neutropenic recipients that are refractory to antimicrobial therapy. G-CSF/dexa treatment not only increases the number of circulating neutrophils but also affects their gene expression. Because of the intended transfusion of these granulocytes into patients who are severely ill, it is of importance to establish to what extent mobilization affects the cellular behavior of neutrophils. Here, we studied the effects of mobilization on Toll-like receptor (TLR)-mediated responses. Mobilized granulocytes displayed increased gene and protein expression of TLR2, TLR4, TLR5, and TLR8. Although mobilized granulocytes displayed normal priming of nicotinamide adenine dinucleotide phosphate oxidase activity and a slight increase in adhesion in response to TLR stimulation, these cells produced massive amounts of interleukin-8 (IL-8), in particular to TLR2 and TLR8 stimulation. The increase in IL-8 release occurred despite reduced IL-8 mRNA levels in the donor granulocytes after in vivo G-CSF/dexa treatment, indicating that the enhanced TLR-induced IL-8 production was largely determined by posttranscriptional regulation. In summary, granulocytes mobilized for transfusion purposes show enhanced TLR responsiveness in cytokine production, which is anticipated to be beneficial for the function of these cells on transfusion into patients

Association of mannose-binding lectin genotype with cardiovascular abnormalities in Kawasaki disease

Kawasaki disease is an acute vasculitis of possible infectious cause, which in particular affects the coronary arteries. Young children rely mostly on their innate immune system for protection against invading microorganisms, of which mannose-binding lectin is an important component. We aimed to investigate the possible role of the gene for this molecule (MBL) in white Dutch patients with Kawasaki disease. In 90 patients, frequency of mutations in the MBL gene was higher than in healthy children. In children younger than I year, those with mutations were at higher risk of development of coronary artery lesions than were those without (odds ratio 15-7, 95% CI 1.4-176.5, p=0.026). Our findings suggest that the innate immune system contributes differently to pathophysiology of Kawasaki disease at various age

Haplotypes of FcγRIIa and FcγRIIIb polymorphic variants influence IgG-mediated responses in neutrophils

Human blood neutrophils normally express two FcγRs (FcγRIIa and FcγRIIIb) that, upon multivalent binding of IgG in immune complexes or on opsonized targets, mediate responses such as phagocytosis, Ab-dependent cellular cytotoxicity, and respiratory burst. Allelic variants have been described for both FcγRIIa (131H/R) and FcγRIIIb (NA1/NA2/SH), with different binding affinity for IgG subclasses. Because neither of these variants acts alone, we have set out to systematically analyze in a large cohort of healthy FCGR2/3-genotyped volunteers how the different haplotypes of neutrophil FcγRs functionally interact. Maximal IgG-induced H2O2 production by neutrophils from individuals with different (homozygous) haplotypes was observed in the following order: 131HH-NA2NA2 > 131RR-NA1NA1 > 131HH-NA1NA1 > 131RR-NA2NA2. Although FcγRIIa 131H is known to bind IgG1 and IgG2 more avidly, no such differences in affinity are known for FcγRIIIb variants. Nonetheless, a remarkable impact of the FcγRIIIb variants on IgG-mediated neutrophil activity was thus demonstrated, which was not explained by differences in FcγR surface expression. The FcγR expression profile was changed by overnight G-CSF/IFN-γ activation of the neutrophils and eliminated any haplotypic impact on the respiratory burst. To our knowledge, our results are the first to provide an integrated functional analysis of neutrophil FcγR haplotypes and suggest that particularly the early phase of IgG-mediated neutrophil reactivity is influenced by FCGR2/3 genotypic variatio

Phenotypic Variation in IgG Receptors by Nonclassical FCGR2C Alleles

The balance between activating and inhibitory signals from the different Fc gamma Rs for IgG ensures homeostasis of many inflammatory responses. FCGR2C is the product of an unequal crossover of the FCGR2A and FCGR2B genes encoding the activating Fc gamma RIIa (CD32a) and inhibitory Fc gamma RIIb (CD32b), respectively. A single nucleotide polymorphism (SNP) in exon 3 of FCGR2C results in either expression of the activating Fc gamma RIIc (CD32c) (FCGR2C-open reading frame [ORF]) or its absence because of a stop codon (FCGR2C-Stop). Two additional variations in Fc gamma RIIb/c expression on leukocytes have now been identified. In case of "nonclassical" FCGR2C-ORF alleles, Fc gamma RIIc expression was unexpectedly absent, because of novel splice site mutations near exon 7 leading to another stop codon. In some individuals with FCGR2C-Stop alleles Fc gamma RIIb was detected on NK cells, which normally are devoid of this protein. Individuals with these nonclassical FCGR2C-Stop alleles carried a deletion of FCGR2C-FCGR3B that extends into the promoter region of the adjacent FCGR2B gene and probably deletes a negative regulatory element in the FCGR2B promoter in NK cells. Fc gamma RIIb expression on NK cells effectively inhibited killing mediated by Fc gamma RIIIa (CD16a) in Ab-dependent cytotoxicity tests. Our findings demonstrate a more extensive and previously unnoticed variation in Fc gamma R expression with relevance to immunity and inflammation. The Journal of Immunology, 2012, 188: 1318-132