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Griscelli syndrome type 2 (GS-2) is a rare congenital immune dysfunction characterized by partial albinism and recurrent episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by a variant in the gene encoding Rab27a leading to a degranulation defect in melanocytes, natural killer (NK)- and T cells. Prognosis of patients with GS-2 is limited by repetitive episodes of life-threatening HLH with onset in early childhood. The only curative treatment is an allogeneic hematopoietic stem cell transplantation (HSCT). Here, we report on an 18 year old female patient with a homozygous missense p.Arg50Glnfs*35 variant in exon 2 of RAB27A who presented with an exceptionally late onset of severe HLH. Her phenotypically inapparent albinism complicated to correctly diagnose GS-2. Immune function assays confirmed a T- and NK cell degranulation deficiency characteristic for patients with primary HLH, while microscopic hair analysis revealed melanin clumps secondary to melanocyte functional impairment. To understand why disease onset occurred unusually late in this patient, we investigated the patient’s T cell and polymorphonuclear neutrophil (PMN) function in more detail. We could show that intracellular granzyme B storage in cytotoxic T cells was increased compared to healthy donors and that the patient’s T cells maintained some degranulation activity. Both, antigen-specific cytotoxic response and proliferation capacity of the patient’s T cells were preserved. We demonstrate for the first time that also PMN degranulation, assessed as stimulation-induced CD66b and CD11b cell membrane expression, is dysfunctional in patients with Rab27a deficiency-associated primary HLH. The patient was treated with steroids and cyclosporine A for immunosuppression to control the HLH. After two severe episodes within only a few months, she eventually received an allogeneic HSCT and has not experienced further HLH episodes for now more than 3 years after the HSCT procedure. This case should raise awareness for the possibility of initial manifestation of primary, genetically-determined HLH even in adult patients.

Bleeding tendency, a prominent feature of patients with Gaucher disease (GD), is associated with abnormal platelet function. Brain-derived neurotrophic factor (BDNF) is a protein with neuroprotective potential stored in alpha granules of circulating platelets. Here we studied BDNF levels in 50 patients with type I GD (GD1) and their correlation with platelet activity and bleeding tendency. Flow cytometry was used to test unstimulated and stimulated measurement of platelet surface-activated expression of αIIbβ3 integrin, P-selectin and lysosomal-associated membrane protein (LAMP3/CD63). Serum and plasma BDNF levels were quantified using ELISA. The bleeding history was recorded by a bleeding questionnaire. Serum BDNF levels were positively correlated with platelet count and moderately correlated with unstimulated and stimulated platelet P-selectin expression. Patients with more than one bleeding manifestation were shown to have lower serum BDNF levels, albeit similar platelet count. Plasma BDNF levels were significantly elevated in splenectomized patients and showed a moderate positive correlation with stimulated platelet CD63 expression. These observations demonstrate the first association between BDNF levels in the peripheral blood with platelet dysfunction and increased bleeding manifestation. The role of measuring serum BDNF for assessing platelet alpha degranulation defects and bleeding risk in patients with GD and the general population needs further study.

Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.

Purpose Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a genetic disorder that results in immune dysregulation. It requires prompt and accurate diagnosis. A natural killer (NK) cell degranulation assay is often used to screen for FHL3 patients. However, we recently encountered two cases of late-onset FHL3 carrying novel UNC13D missense mutations: in these cases, the degranulation assays using freshly isolated and interleukin (IL)-2-activated NK cells yielded contradictory results. Since the defective degranulation of CD57 + cytotoxic T lymphocytes (CTLs) in these cases was helpful for making the diagnosis, we assessed whether the CD57 + CTL degranulation assay more effectively identified FHL3 patients than the NK cell assays. Methods Forty additional patients with hemophagocytic lymphohistiocytosis were prospectively screened for FHL3 by measuring the perforin expression in NK cells and the expression of Munc13-4, syntaxin-11, and Munc18-2 in platelets and by performing NK cell and CTL degranulation assays. The results were confirmed by genetic analysis. Results The freshly isolated NK cell degranulation assay detected FHL3 patients with high sensitivity (100%) but low specificity (71%). The IL-2-stimulated NK cell assay had improved specificity, but 3 out of the 31 non-FHL3 patients still showed degranulation below the threshold level. The CD57 + CTL degranulation assay identified FHL3 patients with high sensitivity and specificity (both 100%). Conclusions The CD57 + CTL degranulation assay more effectively identified FHL3 patients than the NK cell-based assays.

Melanomas are aggressive skin tumors characterized by high metastatic potential. Immunotherapy is a valuable alternative for metastatic melanoma patients resistant to chemotherapy. Natural Killer (NK) cells are efficient anti-tumor cytotoxic effectors. We previously showed that blood NK cells from stage IV metastatic melanoma patients display decreased NK receptors and that chemotherapy modifies the functional status of blood NK cells. To investigate the role of NK cells along melanoma progression, we have here studied NK cells from patients at different stages of the disease. First, we showed that ex vivo NK cells from certain stage III-IV patients displayed low degranulation potential. Using a dynamic label-free assay, we found that immunoselected IL-2 activated blood NK cells from patients efficiently lysed melanoma cells through NKp46 and NKG2D receptors, independently to the clinical stage. Moreover, the ex vivo phenotype of circulating NK cells from 33 patients (stage I to IV) was extensively analyzed. NK cells from patients displayed higher variability in the percentages of Natural Cytotoxicity Receptors (NCR) and Natural Killer Group 2D (NKG2D) receptor expression compared to donor NK cells. The main defect was the decreased expression of NCR1 (NKp46) by NK cells from metastatic patients. Interestingly, we found a positive correlation between the NK cell percentages of NKp46 and the duration of stage IV in melanoma patients. Finally, we showed that NK cells infiltrated primary melanomas and displayed a predominant peritumoral distribution. These results are new arguments for the development of NK-based therapies in melanoma patients.

Although BCR–ABL+ stem cells in chronic myeloid leukemia (CML) resist elimination by targeted pharmacotherapy in most patients, immunological graft-versus-leukemia effects can cure the disease. Besides cytotoxic T cells, natural killer (NK) cells may have a role in immune control of CML. Here, we explored the functionality of NK cells in CML patients and in a transgenic inducible BCR–ABL mouse model. Compared with controls, NK-cell proportions among lymphocytes were decreased at diagnosis of CML and did not recover during imatinib-induced remission for 10–34 months. Functional experiments revealed limited in vitro expansion of NK cells from CML patients and a reduced degranulation response to K562 target cells both at diagnosis and during imatinib therapy. Consistent with the results in human CML, relative numbers of NK1.1+ NK cells were reduced following induction of BCR–ABL expression in mice, and the defects persisted after BCR–ABL reversion. Moreover, target-induced degranulation by expanded BCR–ABL+ NK cells was compromised. We conclude that CML is associated with quantitative and functional defects within the NK-cell compartment, which is reproduced by induced BCR–ABL expression in mice. Further work will aim at identifying the mechanisms of NK-cell deficiency in CML and at developing strategies to exploit NK cells for immunotherapy.

Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P<0.01) and NKp46-positive cells (26% vs. 50%, P<0.01). Decreased expression of these receptors was associated with functional defects, such as reduced NK degranulation capacity when challenged with the tumor target cell line K562 (10.3 vs. 15.8%, P<0.05). This defect could be restored in vitro by stimulating NK cells from T2D patients with IL-15 (P<0.05). NKG2D expression was found to be negatively correlated with HBA1c level (r=-0.50; P=0.009), suggesting that sustained hyperglycemia could directly influence NK cell defects. We demonstrated that endoplasmic reticulum (ER) stress, an important mediator in diabetes-associated complications, was inducible in vitro in normal NK cells and that tunicamycin treatment resulted in a significant decrease in NKG2D expression (P<0.05). Furthermore, markers of the Unfolded Protein Response (UPR) BiP, PDI and sXBP1 mRNAs were significantly increased in NK cells from T2D patients (P<0.05, P<0.01, P<0.05, respectively), indicating that ER stress is activated in vivo through both PERK and IRE1 sensors. These results demonstrate for the first time defects in NK cell-activating receptors NKG2D and NKp46 in T2D patients, and implicate the UPR pathway as a potential mechanism. These defects may contribute to susceptibility to infections and malignancies and could be targetted therapeutically.

Objectives/Goals: We hypothesized that adding cytomegalovirus (CMV) viral Fc gamma receptors (vFcγRs) to a glycoprotein B (gB) protein subunit vaccine would improve vaccine-elicited Fc mediated effector functions such as antibody dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC), over gB subunit alone. Methods/Study Population: We immunized rabbits (n = 4 per group) at Weeks 0, 4, and 8 with 20μg gB alone or with one vFcgR (gp34, gp68, or gp95) at 20μg or 40μg, adjuvanted with squalene emusion, Addavax. Plasma from immunized rabbits was analyzed for antigen-specific IgG binding via enzyme-linked immunosorbent assays (ELISAs). ADCP was measured by conjugating whole virions to a fluorescent marker (AF647), incubating the fluorescent virus with rabbit plasma, and measuring uptake of virus by THP-1 monocytes via flow cytometry. ADCC was measured by natural killer cell degranulation via flow cytometric detection of CD107a expression following co-incubation with CMV-infected fibroblasts and rabbit plasma. Results/Anticipated Results: Each vFcγR demonstrated immunogenicity, although average vFcγR-binding IgG titers were between 4- to 10-fold higher in animals receiving the 40μg dose of each vFcγR compared to the 20μg dose. We observed similar IgG binding responses against gB among all vaccine groups. Comparing groups at peak immunogenicity (Week 10), ADCP responses were improved over gB alone by approximately twofold in animals receiving 40ug of each vFcγR. This effect was maintained across several human CMV strains with variable vFcγR genes. ADCC responses were undetectable in all animals immunized with gB alone, yet those receiving 40μg gp34 or gp95 demonstrated detectable ADCC. Discussion/Significance of Impact: HCMV-specific ADCP and ADCC are associated with protection against vertical CMV transmission, so a vaccine including vFcγRs which can improve vaccine-elicited Fc-effector responses is promising toward reducing the immense global impact of congenital CMV and associated neurologic birth defects.

Functional rescue of NK-cells in solid tumors represents a central aim for new immunotherapeutic strategies. We have conducted a genomic, phenotypic and functional analysis of circulating NK-cells from patients with HCV-related liver cirrhosis and hepatocellular carcinoma. NK-cells were sorted from patients with HCC or liver cirrhosis and from healthy donors. Comparative mRNA gene expression profiling by whole-human-genome microarrays of sorted NK-cells was followed by phenotypic and functional characterization. To further identify possible mediators of NK-cell dysfunction, an in vitro model using media conditioned with patients’ and controls’ plasma was set up. Metabolic and cell motility defects were identified at the genomic level. Dysregulated gene expression profile has been translated into reduced cytokine production and degranulation despite a prevalent phenotype of terminally differentiated NK-cells. NKG2D-downregulation, high SMAD2 phosphorylation and other phenotypic and molecular alterations suggested TGF-β as possible mediator of this dysfunction. Blocking TGF-β could partially restore functional defects of NK-cells from healthy donors, exposed to TGF-β rich HCC patients’ plasma, suggesting that TGF-β among other molecules may represent a suitable target for immunotherapeutic intervention aimed at NK-cell functional restoration. By an unbiased approach, we have identified energy metabolism and cell motility defects of circulating NK-cells as main mechanisms responsible for functional NK-cell impairment in patients with hepatocellular carcinoma. This opens the way to test different approaches to restore NK-cell response in these patients.