Innate Effector Cells Research Articles

The role of microRNA in the developmental and functional regulation of NK cells

Natural killer (NK) cells are important innate effector cells serving as the first line of defense against certain infections and tumors. NK cells also play a role in regulating immune responses. NK cells develop from hematopoietic stem cells in the bone marrow. The development, maturation and function of NK cells depend on the regulation of microenvironment, intracellular transcription factors and post-transcriptional regulations. MicroRNA (miRNA) is a group of small non-coding RNAs that regulate the expression of target genes at the post-transcriptional level. It also plays important roles in various life processes of NK cells, including regulating the activation and effector function of NK cells. We showed that the miRNA profiles in NK cells changed with aging, which in turn affected the development and function of NK cells. Based on the regulatory effect of miRNA on the development and function of NK cells, miRNA may serve as a potential target to protect or restore the function of NK cells in patients, thereby treating related diseases. Here, we briefly summarized recent advances on the roles of miRNA in the developmental and functional regulation of NK cells.

Selective delipidation of Mycobacterium bovis BCG retains antitumor efficacy against non-muscle invasive bladder cancer.

Repeated instillations of bacillus Calmette et Guérin (BCG) are the gold standard immunotherapeutic treatment for reducing recurrence for patients with high-grade papillary non-muscle invasive bladder cancer (NMIBC) and for eradicating bladder carcinoma-in situ. Unfortunately, some patients are unable to tolerate BCG due to treatment-associated toxicity and bladder removal is sometimes performed for BCG-intolerance. Prior studies suggest that selectively delipidated BCG (dBCG) improves tolerability of intrapulmonary delivery reducing tissue damage and increasing efficacy in preventing Mycobacterium tuberculosis infection in mice. To address the lack of treatment options for NMIBC with BCG-intolerance, we examined if selective delipidation would compromise BCG's antitumor efficacy and at the same time increase tolerability to the treatment. Murine syngeneic MB49 bladder cancer models and in vitro human innate effector cell cytotoxicity assays were used to evaluate efficacy and immune impact of selective delipidation in Tokyo and TICE BCG strains. Both dBCG-Tokyo and dBCG-TICE effectively treated subcutaneous MB49 tumors in mice and enhanced tumor-infiltrating CD8+ T and natural killer cells, similar to conventional BCG. However, when compared to conventional BCG, only dBCG-Tokyo retained a significant effect on intratumoral tumor-specific CD8+ and γδ T cells by increasing their frequencies in tumor tissue and their production of antitumoral function-related cytokines, i.e., IFN-γ and granzyme B. Further, dBCG-Tokyo but not dBCG-TICE enhanced the function and cytotoxicity of innate effector cells against human bladder cancer T24 in vitro. These data support clinical investigation of dBCG-Tokyo as a treatment for patients with BCG-intolerant NMIBC.

NK Cell Subset Redistribution and Antibody Dependent Activation after Ebola Vaccination in Africans.

Natural killer cells play an important role in the control of viral infections both by regulating acquired immune responses and as potent innate or antibody-mediated cytotoxic effector cells. NK cells have been implicated in control of Ebola virus infections and our previous studies in European trial participants have demonstrated durable activation, proliferation and antibody-dependent NK cell activation after heterologous two-dose Ebola vaccination with adenovirus type 26.ZEBOV followed by modified vaccinia Ankara-BN-Filo. Regional variation in immunity and environmental exposure to pathogens, in particular human cytomegalovirus, have profound impacts on NK cell functional capacity. We therefore assessed the NK cell phenotype and function in African trial participants with universal exposure to HCMV. We demonstrate a significant redistribution of NK cell subsets after vaccine dose two, involving the enrichment of less differentiated CD56dimCD57− and CD56dimFcεR1γ+ (canonical) cells and the increased proliferation of these subsets. Sera taken after vaccine dose two support robust antibody-dependent NK cell activation in a standard NK cell readout; these responses correlate strongly with the concentration of anti-Ebola glycoprotein specific antibodies. These sera also promote comparable IFN-γ production in autologous NK cells taken at baseline and post-vaccine dose two. However, degranulation responses of post-vaccination NK cells were reduced compared to baseline NK cells and these effects could not be directly attributed to alterations in NK cell phenotype after vaccination. These studies demonstrate consistent changes in NK cell phenotypic composition and robust antibody-dependent NK cell function and reveal novel characteristics of these responses after heterologous two dose Ebola vaccination in African individuals.

Hypoxia-inducible factor regulation of NK cell dysfunction in lupus

Abstract Natural Killer (NK) cells are innate antiviral and antitumor effector cells that have emerged as potent regulators of adaptive immune responses. However, NK cells are known to be dysfunctional in many diseases including systemic lupus erythematosus (lupus), where inability to restrain autoreactive T and B cells leads to potentially irreversible organ damage. To determine mechanisms driving NK-cell dysfunction in autoimmunity, we performed transcriptomic analysis of these cells in pediatric patients with lupus. Analysis of the differentially expressed genes revealed a putative gene regulatory node controlled by the heterodimeric pairing of the transcription factors EPAS1 and ARNT that is active in NK cells from patients with lupus, but not controls. Cytokines linked to lupus promote up-regulation of the protein encoded by EPAS1 in NK cells. Small molecule inhibitors of EPAS1 (PT2385) enhanced the capacity of NK cells to kill K562 cells. These results suggest that a hypoxia-related transcriptional program contributes to functional restraint of NK cells in lupus. Supported by T32GM063483, AR073228, AR47363 (Flow Cytometry Core), DK78392 (Flow Cytometry Core), DK90971 (Flow Cytometry Core)

Assembly and localization of extracellular matrix protein fibronectin modulates Natural killer cell infiltration in tumor spheroids.

Abstract Natural Killer cells (NK), the major innate effector cells, with their broad cytotoxicity against tumors are ideal candidates for immunotherapy. Once having entered the malignant site, NK cells encounter a complex environment composed of tumor cells, non-tumor cells along with the extra cellular matrix (ECM). As one of the integral components of the tumor microenvironment (TME), the normal regulation of the extra cellular matrix (ECM) is necessary to prevent tumor progression and metastasis. Nevertheless, the impact of the matrix architecture found in solid tumors on immune cells and especially NK cells is not well characterized. Here we investigated the role of Fibronectin, a fibrous ECM protein, and its assembly and localization on NK cell infiltration into clear cell renal cell carcinoma (ccRCC) spheroids. 3D tumor spheroids established from the ccRCC cell line 786-O (RCC-) that lacks the Von Hippel-Lindau (VHL) gene are incapable of fibronectin assembly in their ECM. However, addition of the VHL gene into the 786-O cells (RCC+) reversed this phenotype facilitating fibronectin assembly. Fibronectin in RCC-spheroids exhibited prominent peripheral localization in contrast to RCC+ spheroids where fibronectin was detected towards the center of the spheroid. Coincident with fibronectin localization, PKH26 labeled Natural killer cell line, NKL, showed significantly increased infiltration into RCC+ spheroids compared to RCC-spheroids that lacked the tumor suppressor VHL. Thus, assembly and localization of fibronectin modulates NK cell infiltration in RCC tumors. Supported by grants from NIH U54MD012388 (NR and AV) and NIH U54CA143925 (NACP-NAU) to NR.

Gut Microbiota-Derived Small Extracellular Vesicles Endorse Memory-like Inflammatory Responses in Murine Neutrophils.

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune-plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram-negative bacteria) or LTA (Gram-positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow-derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low-dose priming with gut microbiota-derived small EVs enhanced pro-inflammatory sensitivity as indicated by elevated levels of TNF-α, IL-6, ROS and MCP-1 and increased migratory and phagocytic activity. In contrast, high-dose priming resulted in a tolerant phenotype, marked by increased IL-10 and decreased transmigration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were correlated with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut–immune axis.

Siglec-F Promotes IL-33-Induced Cytokine Release from Bone Marrow-Derived Eosinophils Independently of the ITIM and ITIM-like Motif Phosphorylation.

Eosinophils are potent innate effector cells associated mainly with type 2 immune responses elicited by helminths and allergens. Their activity needs to be tightly controlled to prevent severe inflammation and tissue damage. Eosinophil degranulation and secretion of inflammatory effector molecules, including cytokines, chemokines, and lipid mediators, can be regulated by activating and inhibitory receptors on the cell surface. In this study, we investigated the modulation of proliferation, apoptosis, gene expression, and cytokine/chemokine secretion from IL-33-activated Mus musculus eosinophils on cross-linking of the transmembrane receptor Sialic acid-binding Ig-like lectin F (Siglec-F). Siglec-F contains an ITIM plus an ITIM-like motif in its intracellular tail and is mainly regarded as an inhibitory and apoptosis-inducing receptor. In vitro costimulation of bone marrow-derived eosinophils with anti-Siglec-F and IL-33 compared with treatment with either alone led to enhanced STAT6 phosphorylation, stronger induction of hypoxia/glycolysis-related proinflammatory genes, and elevated secretion of type 2 cytokines (IL-4, IL-13) and chemokines (CCL3, CCL4) with only minor effects on proliferation and apoptosis. Using a competitive mixed bone marrow chimera approach with wild-type and Siglec-F-deficient eosinophils, we observed no evidence for Siglec-F-regulated inhibition of Aspergillus fumigatus-elicited lung eosinophilia. Truncation of the Siglec-F cytoplasmic tail, but not mutation of the ITIM and ITIM-like motifs, ablated the effect of enhanced cytokine/chemokine secretion. This provides evidence for an ITIM phosphorylation-independent signaling pathway from the cytoplasmic tail of the Siglec-F receptor that enhances effector molecule release from activated eosinophils.

The Route of Vaccine Administration Determines Whether Blood Neutrophils Undergo Long-Term Phenotypic Modifications.

Innate immunity modulates adaptive immunity and defines the magnitude, quality, and longevity of antigen-specific T- and B- cell immune memory. Various vaccine and administration factors influence the immune response to vaccination, including the route of vaccine delivery. We studied the dynamics of innate cell responses in blood using a preclinical model of non-human primates immunized with a live attenuated vaccinia virus, a recombinant Modified vaccinia virus Ankara (MVA) expressing a gag-pol-nef fusion of HIV-1, and mass cytometry. We previously showed that it induces a strong, early, and transient innate response, but also late phenotypic modifications of blood myeloid cells after two months when injected subcutaneously. Here, we show that the early innate effector cell responses and plasma inflammatory cytokine profiles differ between subcutaneous and intradermal vaccine injection. Additionally, we show that the intradermal administration fails to induce more highly activated/mature neutrophils long after immunization, in contrast to subcutaneous administration. Different batches of antibodies, staining protocols and generations of mass cytometers were used to generate the two datasets. Mass cytometry data were analyzed in parallel using the same analytical pipeline based on three successive clustering steps, including SPADE, and categorical heatmaps were compared using the Manhattan distance to measure the similarity between cell cluster phenotypes. Overall, we show that the vaccine per se is not sufficient for the late phenotypic modifications of innate myeloid cells, which are evocative of innate immune training. Its route of administration is also crucial, likely by influencing the early innate response, and systemic inflammation, and vaccine biodistribution.

Natural Killer (NK) Cells: An Innate Defense Shield Against Respiratory Viral Infections

In general, B and T lymphocytes, which are involved in adaptive immunity, are in charge of cell-mediated response and antibody-mediated immunity, respectively. Another subset of lymphocytes, known as natural killer (NK) cells, are innate effector cells. They serve as the body’s initial line of defence against viral infections. They perform the task of eliminating stressed cells and are crucial for tumour immunity. These cells are capable of performing their killing function without clonal expansion and differentiation following activation. The NK cells will immediately eliminate infected host cells but other lymphocytes need lymphocyte proliferative response which takes several days and further differentiate into effector cells, so that they eliminate host cells infected by the viral pathogen. The NK cells also form a bridge between the adaptive and innate immunity and play significant roles during respiratory infection. Number and the role of NK cells correlate with the severity of severe acute respiratory syndrome (SARS); the number and the percentage of CD158b+ NK cells in severe SARS infection were significantly less in number than those with mild cases. Innate defence mechanisms, particularly NK cells, are able to control SARS infection even in the absence of T cells and antibodies, according to cellular immunological responses to SARS infection in mice. As a result, NK cells are crucial in the fight against viral infections of the respiratory system. As an innate immune system, they serve as the initial line of virus protection. It is possible to do additional research to take advantage of this NK cell trait and develop a cutting-edge therapeutic approach to fight developing respiratory viral diseases.

Interaction Between Innate Lymphoid Cells and the Nervous System.

The interaction between the immune system and the nervous system remains an intriguing enigma. Recent studies indicate that innate lymphoid cells (ILCs), a unique family of innate effector cells, participate in intense cross talk with the nervous system. In the mucosal barrier sites, ILCs have been found to co-localize with neurons, nerves, glial cell projectors, and neuroendocrine cells. The cross talk between ILCs and peripheral nervous system orchestrates mucosal homeostasis and immunity. In addition, the barrier tissues of the central nervous system (CNS) also provide conductive microenvironment for ILC development and maintenance. Activities of CNS-associated ILCs impact the outcome of various CNS disorders. In this chapter, we review and discuss the intricate and bidirectional interaction between ILCs and nervous system.

Glycoprotein Targeted CAR-NK Cells for the Treatment of SARS-CoV-2 Infection

H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.

The Sumoylation Inhibitor TAK-981 in Combination with the CD19-Targeting Antibody Tafasitamab Shows Enhanced Anti-Tumor Activity in Preclinical B-Cell Lymphoma Models

IntroductionTAK-981 is a first-in-class small molecule inhibitor of the SUMO activating enzyme currently in Phase I/II clinical trials. TAK-981 has been shown to increase NK cell activation and M1 macrophage polarization via upregulation of Type I interferon (IFN) signaling, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in combination with rituximab (Nakamura 2019, AACR).Tafasitamab (MOR208) is a CD19-targeting antibody with enhanced Fc effector function mediating ADCC, ADCP and direct cytotoxic activities against B-lymphoma cells. Based on the Phase II clinical study L-MIND (Salles et al., 2020 and Duell et al., 2021), tafasitamab in combination with lenalidomide received accelerated approval by the Food and Drug Administration for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL).Due to the potential for TAK-981 to enhance the activity of tafasitamab via activation of innate effector cells, we aimed to investigate the effects of this drug combination on ADCC, ADCP and tumor cell viability in vitro. Additionally, combinatorial activity of TAK-981 plus tafasitamab was evaluated in lymphoma xenograft models.MethodsA panel of 9 aggressive lymphoma cell lines was analyzed (7 DLBCL and 2 Burkitt lymphoma). For ADCC, PBMC effector cells from healthy human donors were pre-treated with 0.1 or 1 µM TAK-981 or dimethyl sulfoxide (DMSO) control for 24 hours. Tumor cells were incubated with/without 1 nM tafasitamab in the presence of TAK-981 pretreated PBMCs at effector-to-target (E:T) ratios of 5:1 to 10:1 for 2 hours. Degranulation of NK cells was determined via CD107a surface expression after co-incubation of TAK-981 pre-treated PBMCs with tumor cells and 0.1 or 10 nM tafasitamab for 3 hours. Cytokine levels in the supernatant were investigated upon incubation of PBMCs with lymphoma cells, 1 µM TAK-981 and/or 10 nM tafasitamab for 24 hours. For the ADCP assays, in vitro differentiated macrophages were treated with 1 µM TAK-981 for 24 hours. Next, macrophages were incubated with lymphoma cells and 1 or 10 nM tafasitamab at an E:T ratio of 2:1 for 3 hours. For cell viability assays, tumor cells were treated with 1-1000 nM TAK-981 and/or 5 nM tafasitamab for 24 hours in the absence of effector cells. Cytotoxicity, phagocytosis, degranulation and cytokine release were analyzed by flow cytometry. Cell viability was assessed by determination of ATP levels. For in vivo analysis, effects of TAK-981 (7.5 mg/kg IV twice weekly) in combination with tafasitamab (3, 10 or 20 mg/kg IP twice weekly) on tumor growth were evaluated in Daudi and WSU-DLCL2 xenograft models of Burkitt lymphoma and DLBCL grown in SCID mice.ResultsIn ADCC experiments, increased cytotoxicity was observed upon combination treatment with TAK-981 and tafasitamab compared to the respective mono treatments in 5/8 tested lymphoma cell lines (Daudi, SU-DHL-2, SU-DHL-6, TMD8, OCI-LY10). Moreover, TAK-981 plus tafasitamab enhanced degranulation of NK cells and cytokine release compared to mono treatments. In ADCP assays, combination of TAK-981 and tafasitamab resulted in increased phagocytosis rates in comparison to mono treatments in 2/2 tested cell lines (Daudi, Ramos). Cell viability analysis revealed a combination benefit by increased direct cytotoxic effects against SU-DHL-6 cells. Finally, TAK-981 and tafasitamab were investigated in Daudi and WSU-DLCL2 xenograft models with 3 weeks of dosing. In the Daudi model, the combination treatments of TAK-981 with 10 or 20 mg/kg tafasitamab performed better than either treatment alone, and in the WSU-DLCL2 model, the combination treatments of TAK-981 with 3, 10 or 20 mg/kg tafasitamab performed better than the single agent treatments.ConclusionsThe combination of TAK-981 with tafasitamab significantly enhanced anti-tumor effects compared to the respective monotherapies in vitro and in vivo. These preclinical data support a clinical evaluation of this drug combination in patients with lymphoma including aggressive subtypes such as Burkitt lymphoma and DLBCL.The study was funded by MorphoSys AG and Takeda Development Center Americas, Inc. DisclosuresPatra-Kneuer: MorphoSys AG: Current Employment. Nakamura: Takeda Development Center Americas, Inc.: Current Employment. Song: Takeda Pharmaceuticals International Co.: Current Employment. Grossman: Takeda Development Center, Cambridge MA: Current Employment. Polzer: MorphoSys: Current Employment. Ginzel: MorphoSys: Current Employment. Steidl: MorphoSys AG: Current Employment. Berger: Takeda Development Center Americas, Inc.: Current Employment. Proscurshim: Takeda Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company. Heitmüller: MorphoSys AG: Current Employment.

IgE cross-linking induces activation of human and mouse mast cell progenitors

The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation invivo, we hypothesized that they are activated by IgE cross-linking. Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.

Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss-of-function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of “redeemed” Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease, and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice.

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

Abstract 2712: Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers

Abstract The success of early cancer immunotherapies has led to the development of novel therapeutic approaches including multi-specific antibodies engaging innate and adaptive effector cells. To evaluate the antitumor potency of these multi-specific cell engagers, we have established a protocol to select human PBMCs from donors of a commercial frozen source by their potency to produce inflammatory cytokines and the capacity to engraft into NOD-Scid IL2Rgammanull (NSG)mice. The selected human PBMCs were used to reconstitute NSG mice, or as donors of effector cells that upon in vitro expansion were adoptively transferred to NSG mice for humanization. We have employed these humanization strategies to evaluate T cell engagers, which are typically multi-specific antibodies directed against the T cell and one or multiple tumor-associated antigen (TAA), whose therapeutic strategy is to 1) engage T cells, 2) activate the T cells and 3) engage tumor cells, and thus induce tumor cell killing. We will present representative data from several humanization approaches using liquid and solid orthotopic tumor models to assess the effectiveness of multi-specific T cell engagers in mediating antitumor responses. Thus, pre-clinical evaluations rely on the development of optimized tumor bearing humanized models that mirror key properties of a human setting, to assess the therapeutic potential of anti-tumor multi-specific biologics. Citation Format: Liqing Cheng, Jane Chen, Qunyan Yu, Natalia Malkova, Virna F. Cortez-Retamozo, Fangxian Sun, Sukhvinder Sidhu. Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2712.

Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells.

SUMMARYHIV monoclonal antibodies for viral reservoir eradication strategies will likely need to recognize reactivated infected cells and potently drive Fc-mediated innate effector cell activity. We systematically characterize a library of 185 HIV-envelope-specific antibodies derived from 15 spontaneous HIV controllers (HCs) that selectively exhibit robust serum Fc functionality and compared them to broadly neutralizing antibodies (bNAbs) in clinical development. Within the 10 antibodies with the broadest cell-recognition capability, seven originated from HCs and three were bNAbs. V3-loop-targeting antibodies are enriched among the top cell binders, suggesting the V3-loop may be selectively exposed and accessible on the cell surface. Fc functionality is more variable across antibodies, which is likely influenced by distinct binding topology and corresponding Fc accessibility, highlighting not only the importance of target-cell recognition but also the need to optimize for Fc-mediated elimination. Ultimately, our results demonstrate that this comprehensive selection process can identify monoclonal antibodies poised to eliminate infected cells.

Recent Advances to Augment NK Cell Cancer Immunotherapy Using Nanoparticles.

Among various immunotherapies, natural killer (NK) cell cancer immunotherapy using adoptive transfer of NK cells takes a unique position by targeting tumor cells that evade the host immune surveillance. As the first-line innate effector cell, it has been revealed that NK cells have distinct mechanisms to both eliminate cancer cells directly and amplify the anticancer immune system. Over the last 40 years, NK cell cancer immunotherapy has shown encouraging reports in pre-clinic and clinic settings. In total, 288 clinical trials are investigating various NK cell immunotherapies to treat hematologic and solid malignancies in 2021. However, the clinical outcomes are unsatisfying, with remained challenges. The major limitation is attributed to the immune-suppressive tumor microenvironment (TME), low activity of NK cells, inadequate homing of NK cells, and limited contact frequency of NK cells with tumor cells. Innovative strategies to promote the cytolytic activity, durable persistence, activation, and tumor-infiltration of NK cells are required to advance NK cell cancer immunotherapy. As maturing nanotechnology and nanomedicine for clinical applications, there is a greater opportunity to augment NK cell therapeutic efficacy for the treatment of cancers. Active molecules/cytokine delivery, imaging, and physicochemical properties of nanoparticles are well equipped to overcome the challenges of NK cell cancer immunotherapy. Here, we discuss recent clinical trials of NK cell cancer immunotherapy, NK cell cancer immunotherapy challenges, and advances of nanoparticle-mediated NK cell therapeutic efficacy augmentation.

Antibodies for Human Immunodeficiency Virus-1 Cure Strategies.

Human immunodeficiency virus (HIV) infection leads to the establishment of a long-lived latent cellular reservoir. One strategy to eliminate quiescent reservoir cells is to reactivate virus replication to induce HIV envelope glycoprotein (Env) expression on the cell surface exposing them to subsequent antibody targeting. Via the interactions between the antibody Fc domain and Fc-γ receptors (FcγRs) that are expressed on innate effector cells, such as natural killer cells, monocytes, and neutrophils, antibodies can mediate the elimination of infected cells. Over the last decade, a multitude of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes have been identified and are currently being explored for HIV eradication strategies. Antibody development also includes novel Fc engineering approaches to increase engagement of effector cells and optimize antireservoir efficacy. In this review, we discuss the usefulness of antibodies for HIV eradication approaches specifically focusing on antibody-mediated strategies to target latently infected cells and options to increase antibody efficacy.

Expansion of dysfunctional CD56-CD16+ NK cells in chronic hepatitis B patients.

Natural killer (NK) cells are primary innate effector cells that play an important role in the control of human viral infections. During chronic viral infection, NK cells undergo significant changes in phenotype, function and subset distribution, including the appearance of CD56-CD16+ (CD56-) NK cells, previously identified in chronic human immunodeficiency virus (HIV) and hepatitis C virus infection. However, the presence of CD56- NK cells in the pathogenesis of chronic hepatitis B (CHB) remains unknown. Phenotype and function of CD56- NK cells from patients with CHB (n=28) were assessed using flow cytometry and in vitro stimulation with HBV antigen. CHB patients had a higher frequency of CD56- NK cells compared to healthy controls in peripheral blood (6.2% vs 1.4%, P<.0001). Compared to CD56+ NK cells, CD56- NK cells had increased expression of inhibitory receptors, and reduced expression of activating receptors, as measured by MFI and qPCR. CD56- NK cells were less responsive to target cell and cytokine stimulation compared to their CD56+ counterparts. In addition, CD56- NK cells demonstrated defective dendritic cells (DCs) interactions resulting in reduced DCs maturation, lower expression of NK CD69 and impaired capacity of NK cells to eliminate immature DCs in co-culture studies. Finally, frequency of CD56- NK cells was positively correlated with serum HBV DNA levels. Chronic HBV infection induces the expansion of highly dysfunctional of CD56- NK cells that likely contribute to inefficient innate and adaptive antiviral immune response in chronic HBV infection.