SHP-1 localization to the activating immune synapse promotes NK cell tolerance in MHC class I deficiency.

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products.

Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8-99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells.

Autologous NK cells as consolidation therapy following stem cell transplantation in multiple myeloma.

Few approaches have been made toward exploring autologous NK cells in settings of cancer immunotherapy. Here, we demonstrate the feasibility of infusing multiple doses of ex vivo activated and expanded autologous NK cells in patients with multiple myeloma (MM) post-autologous stem cell transplantation. Infused NK cells were detected in circulation up to 4 weeks after the last infusion. Elevations in plasma granzyme B levels were observed following each consecutive NK cell infusion. Moreover, increased granzyme B levels were detected in bone marrow 4 weeks after the last infusion. All measurable patients had objective, detectable responses after NK cell infusions in terms of reduction in M-component and/or minimal residual disease. The present study demonstrates that autologous NK cell-based immunotherapy is feasible in a setting of MM consolidation therapy. It opens up the possibility for usage of autologous NK cells in clinical settings where patients are not readily eligible for allogeneic NK cell-based immunotherapies.

Liquid Biopsy in Hepatocellular Carcinoma: Opportunities and Challenges for Immunotherapy.

Hepatocellular carcinoma (HCC) is one of the deadliest cancer types worldwide. HCC is often diagnosed at a late stage when the therapeutic options are very limited. However, even at the earlier stages, the best treatment is liver transplantation, surgical resection or ablation. Surgical resection and ablation may carry a high risk of tumor recurrence. The recent introduction of immunotherapies resulted in clinical responses for a subgroup of patients, but there were still no effective predictive markers for response to immunotherapy or for recurrence after surgical therapy. The identification of biomarkers that could correlate and predict response or recurrence would require close monitoring of the patients throughout and after the completion of treatment. However, this would not be performed efficiently by repeated and invasive tissue biopsies. A better approach would be to use liquid biopsies including circulating tumor DNA (ctDNA), circulating RNA (e.g., microRNAs), circulating tumor cells (CTC) and extracellular vesicles (EVs) (e.g., exosomes) for disease monitoring in a non-invasive manner. In this review, we discuss the currently available technology that can enable the use of liquid biopsy as a diagnostic and prognostic tool. Moreover, we discuss the opportunities and challenges of the clinical application of liquid biopsy for immunotherapy of HCC.

The Effect of Mesenchymal Stromal Cells Derived From Endometriotic Lesions on Natural Killer Cell Function.

Endometriosis is an inflammatory disease that presents with ectopic endometriotic lesions. Reduced immunosurveillance of these lesions has been proposed to be playing a role in the pathology of endometriosis. Mesenchymal stromal cells (MSC) are found in ectopic lesions and may decrease immunosurveillance. In the present study, we examined if MSC contribute to reduced immunosurveillance through their immunosuppressive effects on natural killer (NK) cells. Stromal cells from endometriotic ovarian cysts (ESCcyst) and eutopic endometrium (ESCendo) of women with endometriosis and their conditioned medium were used in co-cultures with allogeneic peripheral blood NK cells. Following culture, NK cells were examined phenotypically for their expression of activating, inhibitory, maturation, and adhesion receptors and co-receptors, as well as the degranulation (CD107a) marker and the immunostimulatory (interferon-γ) and immunosuppressive (transforming growth factor beta 1 and interleukin-10) cytokines. Moreover, NK cell cytotoxicity was examined using chromium 51 release killing assays. There were no differences between ESCcyst and ESCendo regarding their effects on NK cell cytotoxicity in both conditioned medium and direct co-culture experiments. Additionally, there were no differences between ESCcyst and ESCendo regarding their impact on NK cells' phenotype and degranulation in both conditioned medium and direct co-culture experiments. Although there were no differences found for DNAX accessory molecule-1 (DNAM-1) and NKp44, we found that the expression of the NK cell ligand CD155 that binds DNAM-1 and proliferating cell nuclear antigen (PCNA) that binds NKp44 was significantly less on ESCcyst than on ESCendo. These findings were not supported by the results that the expression of the known and unknown ligands on ESCcyst for DNAM-1 and NKp44 using chimeric proteins was not significantly different compared to ESCendo. In conclusion, the results suggest that ectopic MSC may not contribute to reduced immunosurveillance in endometriosis through their inhibitory effects on NK cells. This suggests that NK cell inhibition in the pelvic cavity of women with endometriosis develops due to other factors.

Google-truthing to assess hot spots of food retail change: A repeat cross-sectional Street View of food environments in the Bronx, New York.

Google Street View (GSV) images can be used to "ground-truth" current and historical food retail data from approximately 2007 - when GSV was launched in a few US cities - to the present, facilitating analyses of food environments over time. A review of GSV images of all food retailers listed in a government database of licensed establishments in the Bronx, New York enabled records to be verified, businesses classified, and retail change quantified. The data revealed several trends likely to affect food access and health: increasing overall numbers of food retailers; the growth of dollar stores; and numerous openings, closings, and ownership changes across all food retail segments. Hot spot analysis identified statistically significant clusters of new dollar stores and bodegas, purveyors of less healthy processed foods, in lower-income neighborhoods in the South Bronx that face elevated rates of diet-related diseases. This article demonstrates the benefits and limitations of using GSV to conduct "virtual" food environment research.

Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D.

Sarcomas are malignancies of mesenchymal origin that occur in bone and soft tissues. Many are chemo- and radiotherapy resistant, thus conventional treatments fail to increase overall survival. Natural Killer (NK) cells exert anti-tumor activity upon detection of a complex array of tumor ligands, but this has not been thoroughly explored in the context of sarcoma immunotherapy. In this study, we investigated the NK cell receptor/ligand immune profile of primary human sarcoma explants. Analysis of tumors from 32 sarcoma patients identified the proliferative marker PCNA and DNAM-1 ligands CD112 and/or CD155 as commonly expressed antigens that could be efficiently targeted by genetically modified (GM) NK cells. Despite the strong expression of CD112 and CD155 on sarcoma cells, characterization of freshly dissociated sarcomas revealed a general decrease in tumor-infiltrating NK cells compared to the periphery, suggesting a defect in the endogenous NK cell response. We also applied a functional screening approach to identify relevant NK cell receptor/ligand interactions that induce efficient anti-tumor responses using a panel NK-92 cell lines GM to over-express 12 different activating receptors. Using GM NK-92 cells against primary sarcoma explants (n = 12) revealed that DNAM-1 over-expression on NK-92 cells led to efficient degranulation against all tested explants (n = 12). Additionally, NKG2D over-expression showed enhanced responses against 10 out of 12 explants. These results show that DNAM-1+ or NKG2D+ GM NK-92 cells may be an efficient approach in targeting sarcomas. The degranulation capacity of GM NK-92 cell lines was also tested against various established tumor cell lines, including neuroblastoma, Schwannoma, melanoma, myeloma, leukemia, prostate, pancreatic, colon, and lung cancer. Enhanced degranulation of DNAM-1+ or NKG2D+ GM NK-92 cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1+ or NKG2D+ GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/ß chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Infectious complications and NK cell depletion following daratumumab treatment of Multiple Myeloma.

Treatment with Daratumumab (Dara), a monoclonal anti-CD38 antibody of IgG1 subtype, is effective in patients with multiple myeloma (MM). However, Dara also impairs the cellular immunity, which in turn may lead to higher susceptibility to infections. The exact link between immune impairment and infectious complications is unclear. In this study, we report that nine out of 23 patients (39%) with progressive MM had infectious complications after Dara treatment. Five of these patients had viral infections, two developed with bacterial infections and two with both bacterial and viral infections. Two of the viral infections were exogenous, i.e. acute respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), while five consisted of reactivations, i.e. one herpes simplex (HSV), 1 varicella-zoster (VZV) and three cytomegalovirus (CMV). Infections were solely seen in patients with partial response or worse. Assessment of circulating lymphocytes indicated a selective depletion of NK cells and viral reactivation after Dara treatment, however this finding does not exclude the multiple components of viral immune-surveillance that may get disabled during this monoclonal treatment in this patient cohort. These results suggest that the use of antiviral and antibacterial prophylaxis and screening of the patients should be considered.

Functional Assessment for Clinical Use of Serum-Free Adapted NK-92 Cells.

Natural killer (NK) cells stand out as promising candidates for cellular immunotherapy due to their capacity to kill malignant cells. However, the therapeutic use of NK cells is often dependent on cell expansion and activation with considerable amounts of serum and exogenous cytokines. We aimed to develop an expansion protocol for NK-92 cells in an effort to generate a cost-efficient, xeno-free, clinical grade manufactured master cell line for therapeutic applications. By making functional assays with NK-92 cells cultured under serum-free conditions (NK-92SF) and comparing to serum-supplemented NK-92 cells (NK-92S) we did not observe significant alterations in the viability, proliferation, receptor expression levels, or in perforin and granzyme levels. Interestingly, even though NK-92SF cells displayed decreased degranulation and cytotoxicity against tumor cells in vitro, the degranulation capacity was recovered after overnight incubation with 20% serum in the medium. Moreover, lentiviral vector-based genetic modification efficiency of NK-92SF cells was comparable with NK-92S cells. The application of similar strategies can be useful in reducing the costs of manufacturing cells for clinical use and can help us understand and implement strategies towards chemically defined expansion and genetic modification protocols.

Natural Killer Cell-Based Therapies Targeting Cancer: Possible Strategies to Gain and Sustain Anti-Tumor Activity.

Natural killer (NK) cells were discovered 40 years ago, by their ability to recognize and kill tumor cells without the requirement of prior antigen exposure. Since then, NK cells have been seen as promising agents for cell-based cancer therapies. However, NK cells represent only a minor fraction of the human lymphocyte population. Their skewed phenotype and impaired functionality during cancer progression necessitates the development of clinical protocols to activate and expand to high numbers ex vivo to be able to infuse sufficient numbers of functional NK cells to the cancer patients. Initial NK cell-based clinical trials suggested that NK cell-infusion is safe and feasible with almost no NK cell-related toxicity, including graft-versus-host disease. Complete remission and increased disease-free survival is shown in a small number of patients with hematological malignances. Furthermore, successful adoptive NK cell-based therapies from haploidentical donors have been demonstrated. Disappointingly, only limited anti-tumor effects have been demonstrated following NK cell infusion in patients with solid tumors. While NK cells have great potential in targeting tumor cells, the efficiency of NK cell functions in the tumor microenvironment is yet unclear. The failure of immune surveillance may in part be due to sustained immunological pressure on tumor cells resulting in the development of tumor escape variants that are invisible to the immune system. Alternatively, this could be due to the complex network of immune-suppressive compartments in the tumor microenvironment, including myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Although the negative effect of the tumor microenvironment on NK cells can be transiently reverted by ex vivo expansion and long-term activation, the aforementioned NK cell/tumor microenvironment interactions upon reinfusion are not fully elucidated. Within this context, genetic modification of NK cells may provide new possibilities for developing effective cancer immunotherapies by improving NK cell responses and making them less susceptible to the tumor microenvironment. Within this review, we will discuss clinical trials using NK cells with a specific reflection on novel potential strategies, such as genetic modification of NK cells and complementary therapies aimed at improving the clinical outcome of NK cell-based immune therapies.

Autologous Peripheral Blood Mononuclear Cells as Treatment in Refractory Acute Respiratory Distress Syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a devastating disorder. Despite enormous efforts in clinical research, effective treatment options are lacking, and mortality rates remain unacceptably high. OBJECTIVES: A male patient with severe ARDS showed no clinical improvement with conventional therapies. Hence, an emergent experimental intervention was performed. METHODS: We performed intratracheal administration of autologous peripheral blood-derived mononuclear cells (PBMCs) and erythropoietin (EPO). RESULTS: We found that after 2 days of initial PBMC/EPO application, lung function improved and extracorporeal membrane oxygenation (ECMO) support was reduced. Bronchoscopy and serum inflammatory markers revealed reduced inflammation. Additionally, serum concentration of miR-449a, b, c and miR-34a, a transient upregulation of E-cadherin and associated chromatin marks in PBMCs indicated airway epithelial differentiation. Extracellular vesicles from PBMCs demonstrated anti-inflammatory capacity in a TNF-α-mediated nuclear factor-x03BA;B in vitro assay. Despite improving respiratory function, the patient died of multisystem organ failure on day 38 of ECMO treatment. CONCLUSIONS: This case report provides initial encouraging evidence to use locally instilled PBMC/EPO for treatment of severe refractory ARDS. The observed clinical improvement may partially be due to the anti-inflammatory effects of PBMC/EPO to promote tissue regeneration. Further studies are needed for more in-depth understanding of the underlying mechanisms of in vivo regeneration.