Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D(high) or NK30(high) donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities

High incidence of Noonan syndrome features including short stature and pulmonic stenosis in patients carrying NF1 missense mutations affecting p.Arg1809: genotype-phenotype correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P<0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients

De novo and biallelic DEAF1 variants cause a phenotypic spectrum.

PURPOSE: To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients

Congenital thrombocytopenia associated with a heterozygous variant in the MEIS1 gene encoding a transcription factor essential for megakaryopoiesis

The transcription factor MEIS1 (myeloid ectotrophic insertion site 1) is crucial for the maintenance of hematopoietic stem cells and for megakaryopoiesis. Germline variants in MEIS1 are associated with restless-leg syndrome, but were not previously shown to cause cytopenias. This is the first report of a patient with congenital thrombocytopenia associated with a sequence variant in MEIS1, presenting with early onset severe thrombocytopenia and mild signs of bone marrow stress. Whole exome sequencing revealed a de novo monoallelic splice site variant in MEIS1, NM_002398.3:exon4:c.432 + 5 G > C, leading to a premature stop codon. We propose that heterozygous mutations in MEIS1 may cause congenital thrombocytopenia

HLA haplotype of melanoma and NK donors.

<p>Table shows the full high resolution HLA typing of Mel008 cells, as well as the healthy NK donors (NK#1 and NK#2) and melanoma NK donors (NKmel#1 and NKmel#2).</p

Correlation between NKLR expression prior to and after rapid expansion.

<p>Each NKLR was tested on gated NK cells and is presented in a different panel, as indicated. NK cultures from different donors (N = 7) were tested at day 14 (black squares) and at day 21 (X's) of rapid expansion protocol (REP). The NKLR expression prior to rapid expansion was tested on un-manipulated peripheral blood NK cells. Each dot represents the average of at least three staining experiments. FAB - fold above background.</p

Pattern of NKLR-ligands on primary melanoma cells.

<p>Figure shows the median fluorescence intensity (MFI) of the binding of various NKLR-Ig fusion proteins to primary melanoma cells, as indicated in the figure. Figure shows a representative experiment.</p

NKLR profile analysis on gated NK cells.

<p>Figure shows the NKLR analysis of 3 different donors, as indicated in the left. NK cells were defined as CD56⁺CD3⁻ cells, as depicted in the left vertical panel. Gated NK cells were further co-stained for NKG2D and CD16 (central vertical panel) or for NKp46 and NKp30 (right vertical panel). Circles highlight various subpopulations.</p

NK NKLR expression profile in healthy donors vs. melanoma patients.

<p>The expression profile of each NKLR is presented in an independent panel, as indicated. Each dot represents either a healthy donor or a melanoma patient. Blood specimens were collected from 30 healthy donors and 40 stage IV melanoma patients with clinical evidence of disease. Y-axis denotes the percent of NKLR-positive cells out of gated NK cell. Horizontal lines represent median values. P value under each panel was calculated with an aparametric t-test and represents the statistical significance of the difference between median values.</p