Analysis of natural killer–cell function in familial hemophagocytic lymphohistiocytosis (FHL): defective CD107a surface expression heralds Munc13-4 defect and discriminates between genetic subtypes of the disease

Abstract Natural killer (NK) cells from patients with familial hemophagocytic lymphohistiocytosis because of PRF1 (FHL2, n = 5) or MUNC13-4 (FHL3, n = 8) mutations were cultured in IL-2 prior to their use in various functional assays. Here, we report on the surface CD107a expression as a novel rapid tool for identification of patients with Munc13-4 defect. On target interaction and degranulation, FHL3 NK cells displayed low levels of surface CD107a staining, in contrast to healthy control subjects or perforin-deficient NK cells. B-EBV cell lines and dendritic cell targets reveal the FHL3 NK-cell defect, whereas highly susceptible tumor targets were partially lysed by FHL3 NK cells expressing only trace amounts of Munc13-4 protein. Perforin-deficient NK cells were completely devoid of any ability to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was comparable in patients and healthy control subjects. However, when cytokine production was induced by coculture with 721.221 B-EBV cells, FHL NK cells resulted in high producers, whereas control cells were almost ineffective. This could reflect survival versus elimination of B-EBV cells (ie, the source of NK-cell stimulation) in patients versus healthy control subjects, thus mimicking the pathophysiologic scenario of FHL

Moderate hypofractionated radiotherapy after prostatectomy for cancer patients: toxicity and clinical outcome

Background: After radical prostatectomy (RP) radiotherapy (RT) plays a role, both as adjuvant or salvage treatment. If negative features are present such as extracapsular extension, seminal vesicle invasion, lymph invasion, and positive surgical margins, RT after RP reduces the risk of recurrence, although it is associated with an increased risk of acute and late toxicities. An intensified RT delivered in a shortened time could improve clinical outcome and be safely combined with hormonal therapy (HT). The aim of this study was to determine the acute and late toxicities associated with hypofractionated RT and to assess the impact of the addition of HT to RT in high-risk prostate cancer (PC) patients on overall response and toxicity. Materials and methods: Sixty-four PC patients undergoing RP were included in this retrospective study. All patients were recommended to receive adjuvant or salvage RT. Prescription doses were 62.5 Gy in 25 fractions to prostate bed, 56.25 Gy in 25 fractions to seminal vesicles bed, and 50 Gy in 25 fractions to pelvis if indicated. HT was administered to patients with additional adverse pathologic features including Gleason score >7, prostate-specific antigen >20 ng/mL before surgery, or prostate-specific antigen with rapid doubling time after relapse or nodal involvement. After completion of RT, patients were observed after 4 weeks, and then followed-up every 3\u20136 months. Acute and late toxicities were assessed using Common Terminology Criteria for Adverse Events v4 and Radiation Therapy Oncology Group scale, respectively. Results: For acute toxicity, only grade 1 gastrointestinal and genitourinary toxicities were detected in 17% and 11% of patients, respectively. As regards late toxicity, only 5% of the patients developed grade 1 gastrointestinal adverse event; grade 1, grade 2, and grade 3 genitourinary toxicity was recorded in 5%, 3.3%, and 3.3% of patients, respectively. Two and 5 years overall survival were 98% and 96%, respectively. The curves stratified for treatment show a slight difference between patients receiving RT or RT+HT, but the differences did not reach statistical significance (p=0.133). Conclusion: In patients with PC undergoing RP, hypofractionated RT may contribute to achieve a high overall survival with an acceptable toxicity profile. Combination of RT and HT is also well tolerated and efficacious

PD-1 in human NK cells: evidence of cytoplasmic mRNA and protein expression

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1 + NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1 12 , NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56 dim than in CD56 bright NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli

Identification of PVR (CD155) and Nectin-2 (CD112) as Cell Surface Ligands for the Human DNAM-1 (CD226) Activating Molecule

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells. As revealed by biochemical analysis, three of such mAbs recognized molecules of ∼70 kD. The other mAb reacted with two distinct molecules of ∼65 and 60 kD, respectively. Protein purification followed by tryptic digestion and mass spectra analysis, allowed the identification of the 70 kD and the 65/60 kD molecules as PVR (CD155) and Nectin-2 δ/α (CD112), respectively. PVR-Fc and Nectin-2-Fc soluble hybrid molecules brightly stained COS-7 cells transfected with the DNAM-1 (CD226) construct, thus providing direct evidence that both PVR and Nectin-2 represent specific ligands for the DNAM-1 triggering receptor. Finally, the surface expression of PVR or Nectin-2 in cell transfectants resulted in DNAM-1–dependent enhancement of NK-mediated lysis of these target cells. This lysis was inhibited or even virtually abrogated upon mAb-mediated masking of DNAM-1 (on NK cells) or PVR or Nectin-2 ligands (on cell transfectants)

The early expansion of anergic NKG2Apos/CD56dim/CD16neg natural killer cells represents a therapeutic target in haploidentical haematopoietic stem cell transplantation

Natural Killer cells are the first lymphocyte population to reconstitute early after non myelo-ablative and T cell-replete haploidentical hematopoietic stem cell transplantation with post-transplant infusion of cyclophosphamide. The present study characterizes the transient and predominant expansion starting from the 2nd week after haploidentical hematopoietic stem cell transplantation of a donor-derived unconventional subset of NKp46neg-low/CD56dim/CD16neg natural killer cells expressing remarkable high levels of CD94/NKG2A. Both transcription and phenotypic profiles indicated that unconventional NKp46neg-low/CD56dim/CD16neg natural killer cells are a distinct natural killer cell subpopulation with features of late stage differentiation, yet retaining proliferative capability and functional plasticity to generate conventional NKp46pos/CD56bright/CD16pos natural killer cells in response to interleukin-15 plus interleukin-18. While present at low frequency in healthy donors, unconventional NKp46neg-low/CD56dim/CD16neg natural killer cells are greatly expanded in the following 7 weeks after haploidentical hematopoietic stem cell transplantation and express high levels of the activating receptors NKGD and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, NKp46neg-low/CD56dim/CD16neg natural killer cells displayed a markedly defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new important perspectives to better understand the ontogenesis/homeostasis of human natural killer cells and to develop a novel immune-therapeutic approach that targets the inhibitory NKG2A check point, thus unleashing natural killer cell alloreactivity early after haploidentical hematopoietic stem cell transplantation

NKp46-expressing human gut-resident intraepithelial V\u3b41 T cell subpopulation exhibits high anti-tumor activity against colorectal cancer

\u3b3\u3b4 T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent anti-tumor activities. However, little is known about their origin, phenotype and clinical relevance in colorectal cancer (CRC). To determine \u3b3\u3b4 IEL gut-specificity, homing and functions, \u3b3\u3b4 T cells were purified from human healthy blood, lymph nodes, liver, skin, intestine either disease-free or affected by CRC or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a new subset of cytotoxic V\u3b41 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46pos/V\u3b41 IELs depends both on distinctive features of V\u3b41 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident V\u3b41 intestinal IELs or its induced-expression on IL-2/IL-15 activated V\u3b41 thymocytes are associated with anti-tumor functions. Higher frequencies of NKp46pos/V\u3b41 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor-microenvironment inhibited the expansion of NKp46pos/V\u3b41 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46pos/V\u3b41 IELs able to infiltrate CRC, thus providing new insights to either follow-up cancer progression or develop novel adoptive cellular therapies

2B4 dysfunction in XLP1 NK cells: More than inability to control EBV infection

X-linked lymphoproliferative disease 1 (XLP1) is a monogenic disorder caused by mutations in SH2D1A, resulting in the absence/dysfunction of the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). Consequently, SLAM receptors as 2B4 (CD244) and NTB-A (SLAMF6), upon ligand engagement, exert inhibitory instead of activating function. This causes an immune dysfunction that is worsened by the selective inability of NK and T cells to kill EBV-infected B cells with dramatic clinical sequelae (e.g. fulminant mononucleosis, hyperinflammation, lymphoma). Here we outline recent findings on the interplay between inhibitory 2B4 and the various activating receptors in NK cells. 2B4 engagement selectively blocks ITAM-dependent activating receptors as NCR and CD16, while it does not affect NKG2D and DNAM-1. Furthermore, inhibitory 2B4 participates to NK cell education, as highlighted by the existence in XLP1 patients of a large subset of fully functional NK cells that lack self-HLA specific inhibitory receptors and exert autoreactivity against mature dendritic cells

Excellent survival regardless of disease stage in patients with advanced nasopharyngeal cancer

Background: We present our experience in assessing the feasibility and efficacy outcomes of intensified intensitymodulated radiation therapy (IMRT) with simultaneous integrated boost (SIB) delivered to patients with nasopharyngeal carcinoma (NPC). Methods: Between March 2009 and December 2014, 35 patients affected by advanced NPC with a median age of 53 years (range 11-77) were treated with definitive radiotherapy. Radiotherapy was delivered by helical tomotherapy with the SIB technique. The prescribed doses were 66 Gy to macroscopic disease, 60 Gy to high-risk subclinical disease, and 54 Gy to low-risk disease in 30 fractions. The daily SIB dose was 2.2 Gy to macroscopic disease. Results: At the end of treatment 33 (94%) patients had obtained complete clearance of disease and 2 patients had died (1 of persistent disease after 3 months and 1 of cancer-unrelated causes after 4 months). At a median follow-up of 40 months (range 5-69), locoregional control rates at 2 and 4 years were 92.9% and 88.2%, respectively, and the overall survival after 4 years was 93.9%. The most significant acute toxicities were grade 2 and 3 mucositis (43%). No grade 3 and 4 late toxicities were observed; grade 2 xerostomia after 6 months from the end of treatment was reported in 11 patients; xerostomia toxicity decreased to grade 1 in 6/11 patients within 12 months. Conclusions: These results show that intensified IMRT with SIB is an excellent strategy offering high local control rates for NPC patients with mild acute and late toxicity