Exert Effector Functions Research Articles

Maternal-Fetal Microchimerism: Impacts on Offspring's Immune Development and Transgenerational Immune Memory Transfer.

Maternal-fetal microchimerism is a fascinating phenomenon in which maternal cells migrate to the tissues of the offspring during both pregnancy and breastfeeding. These cells primarily consist of leukocytes and stem cells. Remarkably, these maternal cells possess functional potential in the offspring and play a significant role in shaping their immune system development. T lymphocytes, a cell population mainly found in various tissues of the offspring, have been identified as the major cell type derived from maternal microchimerism. These T lymphocytes not only exert effector functions but also influence the development of the offspring's T lymphocytes in the thymus and the maturation of B lymphocytes in the lymph nodes. Furthermore, the migration of maternal leukocytes also facilitates the transfer of immune memory across generations. Maternal microchimerism has also been observed to address immunodeficiencies in the offspring. This review article focuses on investigating the impact of maternal cells transported within maternal microchimerism on the immune system development of the offspring, as well as elucidating the effector functions of maternal cells that migrate through the placenta and breast milk to reach the offspring.

MORITS: An improved method to predict peptides from heterologous proteins that are recognized by the same T-cell receptor

Antigen-specific priming of T cells results in the activation of T cells that exert effector functions by interaction of their T-cell receptor (TCR) with the corresponding self-MHC molecule presenting a peptide on the surface of a target cell. Such antigen-specific T cells potentially can also interact with peptide-MHC complexes that contain peptides from unrelated antigens, a phenomenon that often is referred to as heterologous immunity. For example, some individuals that were pre-immunized against an allergen, could subsequently mount better anti-viral T-cell responses than non-allergic individuals. So far only few peptide pairs that experimentally have been shown to provoke heterologous immunity were identified, and available prediction tools that can identify potential candidates are imprecise. We developed the MORITS algorithm to rapidly screen large lists of peptides for sequence similarities, while giving enhanced consideration to peptide residues presented by MHC that are particularly relevant for TCR interactions. In combination with established peptide-MHC binding prediction tools, the MORITS algorithm revealed peptide similarities between the SARS-CoV-2 proteome and certain allergens. The method outperformed previously published workflows and may help to identify novel pairs of peptides that mediate heterologous immune responses.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB058.

Clinical and basic science aspects of innate lymphoid cells as novel immunotherapeutic targets in cancer treatment.

Immunotherapy has revolutionised cancer treatment over the past decade, demonstrating remarkable efficacy across a broad range of cancer types. However, not all patients or cancer types respond to contemporary clinically-utilised immunotherapeutic strategies, which largely focus on harnessing adaptive immune T cells for cancer treatment. Accordingly, it is increasingly recognised that upstream innate immune pathways, which govern and orchestrate the downstream adaptive immune response, may prove critical in overcoming cancer immunotherapeutic resistance. Innate lymphoid cells (ILCs) are the most recently discovered major innate immune cell population. They have overarching roles in homeostasis and orchestrating protective immunity against pathogens. As innate immune counterparts of adaptive immune T cells, ILCs exert effector functions through the secretion of cytokines and direct cell-to-cell contact, with broad influence on the overall immune response. Importantly, dysregulation of ILC subsets have been associated with a range of diseases, including immunodeficiency disorders, allergy, autoimmunity, and more recently, cancer. ILCs may either promote or inhibit cancer initiation and progression depending on the cancer type and the specific ILC subsets involved. Critically, therapeutic targeting of ILCs and their associated cytokines shows promise against a wide range of cancer types in both preclinical models and early phase oncology clinical trials. This chapter provides a comprehensive overview of the current understanding of ILC subsets and the associated cytokines they produce in cancer pathogenesis, with specific focus on how these innate pathways are, or can be targeted, therapeutically to overcome therapeutic resistance and ultimately improve patient care.

Predicting a Stable Dimeric Form of the PD1-PDL1 Complex: Implications for Understanding the PD1 Activation Mechanism.

The activation of T cells is typically accompanied by inhibitory mechanisms within which the programmed cell death (PD1) receptor stands out. Upon binding the ligands PDL1 and PDL2, PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling is activated upon the ligands' binding remains an open question. Several experimental facts suggest that the activation of the PD1-PLD1 pathway depends on the interaction with an unknown partner at the cellular membrane. In this work, we investigate the possibility that the target of PD1-PDL1 is the same PD1-PDL1 complex. We combined molecular docking with molecular dynamics and umbrella sampling simulations to explore different binding modes and assess the complexes' stability. We predicted a stable dimeric form of the extracellular domains of the PD1-PDL1 complex. This dimeric complex has an affinity comparable to the PD1-PDL1 interaction and resembles the form of a linear lattice. We proposed a new model for PD1 activation where the PD1-PDL1 dimeric form could facilitate the interaction of the intracellular domains of PD1 and the further binding and activation of the SHP2 phosphatase. This model might explain the inhibitory effect of anti-PD1/PDL1 antibodies through the prevention of the formation of the PD1-PDL1 dimers and, subsequently, the abrogation of the SHP2 phosphatase activation.

Use of CD122+ natural killer cell precursors for superior anti-leukemia response.

Abstract Acute Myeloid Leukemia (AML) is an aggressive blood cancer in adults. Intensive induction therapy successfully induces a complete response in up to 80% of the adult patients but a fraction of them is refractory or relapses. The fraction of non-responsive or relapsing patients is especially high amongst medically unfit and older patients, who cannot undergo aggressive chemotherapy treatment. Therefore, offering patients failing on first line intensive chemotherapy and medically unfit and older patients an effective treatment option for long-term control or even cure of disease represents a significant yet unmet clinical need. As a component of the innate immunity, NK cells have recently shown great promise for the treatment of patients with different malignancies. Here, we show that injection of human interleukin-2 (IL-2) complexes (IL-2cx) induce the de novo generation and massive expansion of NK cell precursors in vivo. Furthermore, IL-2cx-expanded NK cells exert effector functions with the capacity to control the growth of non-self MHC class I-deficient RMA-S lymphoma cells in vivo, while remaining tolerant towards MHC class-expressing self RMA cells. In an experimental setup mimicking the clinical case of refractory patients after intensive AML induction therapy, IL-2cx treatment mediated strong anti-AML responses following haploidentical bone marrow transplantation without the need for adoptive transfer of donor-derived or in vitro autologous expanded NK cells. Thus, this study demonstrates that IL-2cx immunotherapy allows the in vivo generation and expansion of functionally mature NK cells to achieve long-term response in AML paving the way to an effective treatment option. We are sponsored partly by the Cell Evaluation & Therapy Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313), and an ACS-IRG to CK.

IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata

IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata

IgE glycans promote anti-IgE IgG autoantibodies that facilitate IgE serum clearance via Fc Receptors.

Recent studies have shown that IgE glycosylation significantly impacts the ability of IgE to bind to its high-affinity receptor FcεRI and exert effector functions. We have recently demonstrated that immunizing mice with IgE in a complex with an allergen leads to a protective, glycan-dependent anti-IgE response. However, to what extent the glycans on IgE determine the induction of those antibodies and how they facilitate serum clearance is unclear.Therefore, we investigated the role of glycan-specific anti-IgE IgG autoantibodies in regulating serum IgE levels and preventing systemic anaphylaxis by passive immunization. Mice were immunized using glycosylated or deglycosylated IgE-allergen-immune complexes (ICs) to induce anti-IgE IgG antibodies. The anti-IgE IgG antibodies were purified and used for passive immunization. Glycosylated IgE-ICs induced a significantly higher anti-IgE IgG response and more IgG-secreting plasma cells than deglycosylated IgE-ICs. Passive immunization of IgE-sensitized mice with purified anti-IgE IgG increased the clearance of IgE and prevented systemic anaphylaxis upon allergen challenge. Anti-IgE IgG purified from the serum of mice immunized with deglycosylated IgE-ICs, led to a significantly reduced elimination and protection, confirming that the IgE glycans themselves are the primary drivers of the protectivity induced by the IgE-immune complexes. IgE glycosylation is essential for a robust anti-IgE IgG response and might be an important regulator of serum IgE levels.

Unraveling a Conserved Conformation of the FG Loop upon the Binding of Natural Ligands to the Human and Murine PD1.

The activation of T cells is normally accompanied by inhibitory mechanisms within which the PD1 receptor stands out. PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions upon binding the ligands PDL1 and PDL2. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling pathway is activated upon the ligands' binding remains an open question. In this work, we used molecular dynamics simulations to assess the differences of the PD1 motion in the free state and in complex with the ligands. We found that, in both human and murine systems, the binding of PDL1 and PDL2 stabilizes the conformation of the FG loop similarly. This result, combined with the conservation of the FG loop residues across species, suggests that the conformation of the FG loop is somehow related to the signaling process. We also found a high similarity between the PD1-PDL1 structures with the variable region of an antibody structure, where the FG loop occupies a similar position to the CDR3 light chain.

Regulation of activated T cell survival in rheumatic autoimmune diseases.

Adaptive immune responses rely on the proliferation of T lymphocytes able to recognize and eliminate pathogens. The magnitude and duration of the expansion of activated T cell clones are finely regulated to minimize immunopathology and avoid autoimmunity. In patients with rheumatic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, activated lymphocytes survive and exert effector functions for prolonged periods, defying the mechanisms that normally curb their capacities during acute and chronic infections. Here, we review the molecular mechanisms that limit the duration of immune responses in health and discuss the factors that alter such regulation in the setting of systemic lupus erythematosus and rheumatoid arthritis. We highlight defects that could contribute to the development and progression of autoimmune disease and describe how chronic inflammation can alter the regulation of activated lymphocyte survival, promoting its perpetuation. These concepts might contribute to the understanding of the mechanisms that underlie the chronicity of inflammation in the context of autoimmunity.

Dynamic Microtubule Arrays in Leukocytes and Their Role in Cell Migration and Immune Synapse Formation.

The organization of microtubule arrays in immune cells is critically important for a properly operating immune system. Leukocytes are white blood cells of hematopoietic origin, which exert effector functions of innate and adaptive immune responses. During these processes the microtubule cytoskeleton plays a crucial role for establishing cell polarization and directed migration, targeted secretion of vesicles for T cell activation and cellular cytotoxicity as well as the maintenance of cell integrity. Considering this large spectrum of distinct effector functions, leukocytes require flexible microtubule arrays, which timely and spatially reorganize allowing the cells to accommodate their specific tasks. In contrast to other specialized cell types, which typically nucleate microtubule filaments from non-centrosomal microtubule organizing centers (MTOCs), leukocytes mainly utilize centrosomes for sites of microtubule nucleation. Yet, MTOC localization as well as microtubule organization and dynamics are highly plastic in leukocytes thus allowing the cells to adapt to different environmental constraints. Here we summarize our current knowledge on microtubule organization and dynamics during immune processes and how these microtubule arrays affect immune cell effector functions. We particularly highlight emerging concepts of microtubule involvement during maintenance of cell shape and physical coherence.

Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches

Induction of memory CD8 T cells residing in peripheral tissues is of interest for T cell-based vaccines as these cells are located at mucosal and barrier sites and can immediately exert effector functions, thus providing protection in case of local pathogen encounter. Different memory CD8 T cell subsets patrol peripheral tissues, but it is unclear which subset is superior in providing protection upon secondary infections. We used influenza virus to induce predominantly tissue resident memory T cells or cytomegalovirus to elicit a large pool of effector-like memory cells in the lungs and determined their early protective capacity and mechanism of reactivation. Both memory CD8 T cell pools have unique characteristics with respect to their phenotype, localization, and maintenance. However, these distinct features do not translate into different capacities to control a respiratory vaccinia virus challenge in an antigen-specific manner, although differential activation mechanisms are utilized. While influenza-induced memory CD8 T cells respond to antigen by local proliferation, MCMV-induced memory CD8 T cells relocate from the vasculature into the tissue in an antigen-independent and partially chemokine-driven manner. Together these results bear relevance for the development of vaccines aimed at eliciting a protective memory CD8 T cell pool at mucosal sites.

Interplay between γδT-Cell Metabolism and Tumour Microenvironment Offers Opportunities for Therapeutic Intervention

Solid tumour targeting using adoptive cell therapy has failed to reproduce the spectacular clinical successes seen with chimeric antigen receptor T cell therapies and B cell malignancies. Low in glucose, oxygen, pH and populated with suppressive cells, the solid tumour microenvironment (TME) remains a formidable obstacle to successful immune targeting. The use of atypical, tissue-tropic lymphocytes, such as γδT cells, may offer enhanced tumour trafficking over canonical αβT cells. Nonetheless, γδT cells too interact with the TME. The consequences of this interaction are poorly understood and of high translational relevance. Lopes and colleagues show that, in a murine context, low glucose environments preferentially retained pro-tumorigenic IL-17-producing γδT cells. Anti-tumorigenic IFN-γ-producing γδT cells, meanwhile, required high ambient glucose to survive and exert effector function. Unexpectedly, this metabolic imprinting was evident in the murine thymus, suggesting that the ontological separation of these functional subsets occurs early in their development. Elucidation of this relationship between TME glucose levels and γδT cell functionality in a human context is likely to carry significant implications for the development of γδT cell-based oncoimmunotherapeutics.

The downregulation of IL-18R defines bona fide kidney-resident CD8+ Tcells.

SummaryStepwise induction of CD69 and CD103 marks distinct differentiation stages of mucosal Trms. But the majority of non-mucosal Trm lacks CD103 expression. The expression of CD69 alone cannot faithfully define Trm cells in heavily vascularized non-mucosal tissues, such as the kidney. Here, we found that a subset of kidney Trms downregulated IL-18 receptor during differentiation. Via global transcriptional analysis and parabiosis experiments, we have discovered that the downregulation of interleukin-18 receptor (IL-18R) is associated with the establishment of tissue residency. Together with the expression of CD69, IL-18Rlo exclusively identify tissue-resident cells whereas IL-18Rhi population contains both tissue-resident and migratory ones. Local cytokines including transforming growth factor β (TGF-β) and interferon α (IFN-α)/β as well as TGF-β-dependent suppression of transcription factor Tcf-1 are essential for IL-18R downregulation during kidney Trm differentiation. Together, we identified a convenient surface marker to distinguish bona fide kidney-resident CD8+ T cells as well as underlying molecular mechanisms controlling this differentiation process.

Cross-reactivity of mouse IgG subclasses to human Fc gamma receptors: Antibody deglycosylation only eliminates IgG2b binding

Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs.

Exploring the conformational dynamics of PD1 in complex with different ligands: What we can learn for designing novel PD1 signaling blockers?

Activation of T cells triggers the expression of regulatory molecules like the programmed cell death 1 (PD1) protein. The association of PD1 with the natural ligands PDL1 and PDL2 induces an inhibitory signal that prevents T cells from proliferating and exerting effector functions. However, little is known about how the binding of the ligands induce the PD1 inhibitory signal over T cells effector functions. Here, we explore the dynamics of PD1 free, and in complex with different PDL1 variants as well as the therapeutic antibodies nivolumab and pembrolizumab in order to assess the conformational changes in PD1 related to the signaling process. Our simulations suggest a pre-conformational selection mechanism for the binding of the different PDL1 variants, while an induced-fit model fits better for the molecular recognition process of the therapeutic antibodies. A deep analysis of the changes on PD1 movement upon the binding to different ligands revealed that as larger is the difference in the conformation adopted by loop C'D with respect to the complex with PDL1 is higher the ligand ability to reduce the PD1 inhibitory signaling. This behavior suggests that targeting specific conformations of this loop can be useful for designing therapies able to recover T cells effector functions.

Chromatin accessibility of CD8 T cell differentiation and metabolic regulation.

CD8+T cells play an important role in controlling infections and tumorigenesis in vivo. naïve CD8+T cells exponentially expand and exert effector functions in response to TCR ligation. After antigen clearance, most effector CD8+T cells (Teff) experience activation-induced cell death, only a small portion becomes long-lived memory T cells (Tmem). The cell-intrinsic mechanisms driving the differentiation need further understanding. Here we used combined transposase-accessible chromatin (ATAC-seq) technology and RNA-seq analysis to explore chromatin accessibility in CD8+T cell subsets (naïve T cells, Teff, and Tmem). The data demonstrates different chromatin openness of CD8+T cell states is associated with metabolic regulation and the high accessibility of upstream binding site SP1 emerged as critical transcription factor for both Teff and Tmem in fatty acid oxidation (FAO) and glycolysis. The different presence of accessible regions in CD8+T cell subsets provides a novel perspective for understanding epigenetic mechanisms underlying T cell differentiation and related immune response.

NK and NKT Cell-Mediated Immune Surveillance against Hematological Malignancies.

Recent cancer treatment modalities have been intensively focused on immunotherapy. The success of chimeric antigen receptor T cell therapy for treatment of refractory B cell acute lymphoblastic leukemia has pushed forward research on hematological malignancies. Among the effector types of innate lymphocytes, natural killer (NK) cells show great importance in immune surveillance against infectious and tumor diseases. Particularly, the role of NK cells has been argued in either elimination of target tumor cells or escape of tumor cells from immune surveillance. Therefore, an NK cell activation approach has been explored. Recent findings demonstrate that invariant natural killer T (iNKT) cells capable of producing IFN-γ when optimally activated can promptly trigger NK cells. Here, we review the role of NKT and/or NK cells and their interaction in anti-tumor responses by highlighting how innate immune cells recognize tumors, exert effector functions, and amplify adaptive immune responses. In addition, we discuss these innate lymphocytes in hematological disorders, particularly multiple myeloma and acute myeloid leukemia. The immune balance at different stages of both diseases is explored in light of disease progression. Various types of innate immunity-mediated therapeutic approaches, recent advances in clinical immunotherapies, and iNKT-mediated cancer immunotherapy as next-generation immunotherapy are then discussed.

When liposomes met antibodies: Drug delivery and beyond.

Drug encapsulated liposomes and monoclonal antibodies (Mabs) are two distinctively different classes of therapeutics, but both aim to become the ultimate "magic bullet". While PEGylated liposomes rely on the enhanced permeability and retention (EPR) effect for accumulation in solid tumor tissues, Mabs are designed to bind tightly to specific surface antigens on target cells to exert effector functions. Immunoliposome (IL) refers to the structural combination of liposomes and antibodies, whereas the antibodies are usually decorated on the liposome surface. ILs can therefore take advantage of interactions between antibodies and cancer cells for more efficient endocytosis and intracellular drug delivery. The antibody structure, affinity, density, as well as the liposome surface properties and drug to lipid ratios all contribute to the IL pharmacokinetic(PK) and pharmacodynamic(PD) behaviors. The optimal formulation parameters may vary for different target cells and tissues. Furthermore, besides the delivery of cytotoxic drugs to cancer cells, new ILs are being developed to interact with multiple target receptors, multiple target cells and trigger multiple therapeutic effects. We envision that the IL format can be a great platform for the molecular engineering of multi-valent, multi-specific interactions to achieve complex biological functions for therapeutic benefits, especially in the area of cancer immunotherapy.

Sorafenib attenuated the function of natural killer cells infiltrated in HCC through inhibiting ERK1/2

Sorafenib has been systemically utilized to therapy the advanced hepatocellular carcinoma (HCC). Natural killer cells (NKs) are important cytotoxic innate lymphocytes, which can exert effector functions especially in liver and tumor. However, how Sorafenib affects the function of NKs remains to be elucidated. Here, we utilized the subcutaneous and in situ tumor bearing mice with mouse hepatoma cell line hepa 1–6. At the endpoint, the number and function of NKs in blood, liver, TDLN, and tumor were explored using FACS, ELISA, WB, etc. To confirm the direct effects of Sorafenib on NKs, the NKs were sorted using FACS, which were then stimulated with Sorafenib to detect the functions and the relevant mechanisms using qPCR, western blot, and FACS in vitro. Finally, we found that Sorafenib led to a significant block of tumor progression, but reduced the number of NK cells through suppressing the proliferation of NK cells. This phenotype made us study the terminal function of NK cells, revealing that Sorafenib could decrease the production of effector molecules and cytokines, such as perforin, granzyme B, TNF-α, IFN-γ, etc. Besides, p-ERK1/2 in NK cells was inhibited after treatment with Sorafenib, and a similar tendency of NK cells could be achieved using ERK1/2 inhibitor. Collectively, our data suggested that Sorafenib functioned as a critical inhibitor that controlled the number and function of NK cells through inhibiting ERK1/2.