Natural killer cells in tumor immunotherapy

Cancer is the second most common cause of death worldwide and remains one of the critical public health problems of our time1.Recently,immunotherapy has considerably improved the outcomes of patients with advanced cancers.Immune checkpoint blockade and chimeric antigen receptor(CAR)-T cell-based therapies have achieved remarkable success in recent decades,thus placing the host immune response in the spotlight as a potential new approach in antitumor therapy.

Trispecific killer engager 161519 enhances natural killer cell function and provides anti-tumor activity against CD19-positive cancers

Objective:Natural killer(NK)cells have gained considerable attention due to their potential in treating"cold tumors,"and are therefore considered as one of the new strategies for curing cancer,by using worldwide development of their new possibilities and interventions with NK cell-related therapeutic products.Methods:We constructed a trispecific killer engager(TriKE)consisting of anti-CD16,IL-15,and anti-CD19.This TriKE was designed to attract CD19^(+)tumor cells to CD16^(+)NK cells,whereas IL-15 sustained the proliferation,development,and survival of NK cells.Results:Treatment with 161519 TriKE in the presence of CD19^(+)targets upregulated expression of CD69,CD107 a,TRAIL,IFN-γ,and TNF-α in NK cells,and significantly improved the proliferation and cytotoxicity of NK cells.NK cells"armed"with 161519 TriKE showed stronger cytolysis against CD19+targets compared with that of"unarmed"NK cells.A preclinical model of B-cell lymphoma in human peripheral blood mononuclear cell-reconstituted xenograft mice showed significant inhibition of tumor growth and prolonged overall survival after treatment with 161519 TriKE,when compared with that in control mice or mice treated with 1619 BiKE.Combined use of IL-2 was a more effective treatment with 1619 BiKE,when compared with that using 161519 TriKE.Conclusions:The newly generated 161519 TriKE enhanced the proliferation,activation,cytokine secretion,and cytotoxicity of NK cells in the presence of CD19+tumor cells.The 161519 TriKE aided inhibition of tumor growth and prolonged the overall survival of murine xenografts,and could be used to treat CD19-positive cancers.

Fetuin-A is an immunomodulator and a potential therapeutic option in BMP4-dependent heterotopic ossification and associated bone mass loss

Heterotopic ossification(HO)is the abnormal formation of bone in extraskeletal sites.However,the mechanisms linking HO pathogenesis with bone mass dysfunction remain unclear.Here,we showed that mice harboring injury-induced and BMP4-dependent HO exhibit bone mass loss similar to that presented by patients with HO.Moreover,we found that injury-induced hyperinflammatory responses at the injury site triggered HO initiation but did not result in bone mass loss at 1 day post-injury(dpi).In contrast,a suppressive immune response promoted HO propagation and bone mass loss by 7 dpi.Correcting immune dysregulation by PD1/PDL1 blockade dramatically alleviated HO propagation and bone mass loss.We further demonstrated that fetuin-A(FetA),which has been frequently detected in HO lesions but rarely observed in HO-adjacent normal bone,acts as an immunomodulator to promote PD1 expression and M2 macrophage polarization,leading to immunosuppression.Intervention with recombinant FetA inhibited hyperinflammation and prevented HO and associated bone mass loss.Collectively,our findings provide new insights into the osteoimmunological interactions that occur during HO formation and suggest that FetA is an immunosuppressor and a potential therapeutic option for the treatment of HO.

B-cell-derived IL-10 promotes allergic sensitization in asthma regulated by Bcl-3

Aeroallergen sensitization,mainly mediated by lung epithelium and dendritic cells(DCs),is integral to allergic asthma pathogenesis and progression.IL-10 has a dual role in immune responses,as it inhibits myeloid cell activation but promotes B-cell responses and epithelial cell proliferation.Here,we report a proinflammatory function of B-cell-derived IL-10 modulated by Bcl-3 in allergic asthma.Specifically,Bcl-3^(−/−)mice showed elevated IL-10 levels and were found to be highly vulnerable to allergic asthma induced by house dust mites(HDMs).IL-10 had a positive correlation with the levels of the DC chemoattractant CCL-20 in HDM-sensitized mice and in patients with asthma and induced a selective increase in CCL-20 production by mouse lung epithelial cells.Blockade of IL-10 or IL-10 receptors during sensitization dampened both HDM-induced sensitization and asthma development.IL-10 levels peaked 4 h post sensitization with HDM and IL-10 was primarily produced by B cells under Bcl-3–Blimp-1–Bcl-6 regulation.Mice lacking B-cell-derived IL-10 displayed decreased lung epithelial CCL-20 production and diminished DC recruitment to the lungs upon HDM sensitization,thereby demonstrating resistance to HDM-induced asthma.Moreover,responses to HDM stimulation in Bcl-3^(−/−)mice lacking B-cell-derived IL-10 were comparable to those in Bcl-3^(+/+)mice.The results revealed an unexpected role of B-cell-derived IL-10 in promoting allergic sensitization and demonstrated that Bcl-3 prevents HDM-induced asthma by inhibiting B-cell-derived IL-10 production.Thus,targeting the Bcl-3/IL-10 axis to inhibit allergic sensitization is a promising approach for treating allergic asthma.

Cytokine storm and translating IL-6 biology into effective treatments for CoVID-19

As of May 3,2023,the Coronavirus disease 2019(COVID-19)pandemic has resulted in more than 760 million confirmed cases and over 6.9 million deaths.Several patients have developed pneumonia,which can deteriorate into acute respiratory distress syndrome.The primary etiology may be attributed to cytokine storm,which is triggered by the excessive release of proinflammatory cytokines and subsequently leads to immune dysregulation.Considering that high levels of interleukin-6(IL-6)have been detected in several highly pathogenic coronavirus-infected diseases,such as severe acute respiratory syndrome in 2002,the Middle East respiratory syndrome in 2012,and COVID-19,the IL-6 pathway has emerged as a key in the pathogenesis of this hyperinflammatory state.Thus,we review the history of cytokine storm and the process of targeting IL-6 signaling to elucidate the pivotal role played by tocilizumab in combating COVID-19.

Decidual natural killer cells and the immune microenvironment at the maternal-fetal interface

During early pregnancy,an orchestrated evolutionary maternal adaption toward tolerance of the semiallogeneic fetus is required to ensure decidualization and early embryo development.Remodeling of the immune system involves natural killer cells(NKs),macrophages,T cells and dendritic cells(DCs) altering the microenvironment in the deciduas.In particular,a unique population of NK cells with a CD56^(bright)CD16^- phenotype in the decidua has been proposed to play a key role in the maternal adaptation to pregnancy.However,there is a tendency for pregnancy immunology to reflect transplantation immunology regarding the assumption that the maternal immune system should be suppressed.This tendency is misleading.We discuss how the immune system is formed in early deciduas and the interactions between maternal NK cells and fetal growth.We propose that the maternal immune response must not be fully suppressed and is even necessary for the local response of uterine NK cells.

Isolation of Lymphocytes and Their Innate Immune Characterizations from Liver,Intestine,Lung and Uterus

In steady-state conditions, the number and distribution of lymphocyte populations are under homeostatic control. New lymphocytes are continuously produced in primary and secondary lymphoid organs and then achieve immune-competence within different tissues, and they must challenge with resident cells for survival. The first step in the study of tissue lymphoid cells is their isolation in intact and viable form appropriate for establishment of in vitro culture systems. For reasons of simplicity, cell purity, cell yields and various purposes, lymphocytes obtained from different tissues in different labs were subjected to diverse protocols. To fully elucidate the nature of the local immune system as well as to adequately study the innate role of lymphocytes in liver, intestine, lung and uterus, we briefly reviewed the characterization of resident lymphocytes, and additional information on those cells from non-lymphoid tissues by using the recommended operation procedure was also presented.

Immunotherapeutical Potential of Mycobacterium Vaccae on M. Tuberculosis Infection in Mice

Tuberculosis remains the worldwide infectious disease. To identify the therapeutic potential of M. vaccae in treating tuberculosis, M. vaccae was injected into Mycobacterium tuberculosis （M. tuberculosis） infected mice. The optimal dose of M. vaccae （22.5 μg/mouse） treated mice showed lower pathological change index, spleen weight index, lung weight index and vital M. tuberculosis count than those of the untreated group. Treatment with M. vaccae enhanced the percentages of CD3^＋ and CD4^＋ T cells, IFN-γ^＋CD4^＋ T cells, innate immune cells including NK cells, NK1.1^＋ T cells and γδ T cells, and reduced the percentage of IL-4^＋CD4^＋ T cells. Therefore, M. vaccae could protect the mice from M. tuberculosis infection and improved mouse innate and adaptive cell-mediated immunity, suggesting that M. vaccae is a potential immunotherapeutic agent in pulmonary tuberculosis. Cellular ＆ Molecular Immunology.

Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors

The spatiotemporal distribution of cytokines orchestrates immune responses in vivo,yet the underlying mechanisms remain to be explored.We showed here that the spatial distribution of interleukin-4(IL4)in invariant natural killer T(iNKT)cells regulated crosstalk between iNKT cells and dendritic cells(DCs)and controlled iNKT cell-mediated T-helper type 1(Th1)responses.The persistent polarization of IL4 induced by strong lipid antigens,that is,α-galactosylceramide(αGC),caused IL4 accumulation at the immunological synapse(IS),which promoted the activation of the IL4R-STAT6(signal transducer and activator of transcription 6)pathway and production of IL12 in DCs,which enhanced interferon-γ(IFNγ)production in iNKT cells.Conversely,the nonpolarized secretion of IL4 induced by Th2 lipid antigens with a short or unsaturated chain was incapable of enhancing this iNKT cell-DC crosstalk and thus shifted the immune response to a Th2-type response.The nonpolarized secretion of IL4 in response to Th2 lipid antigens was caused by the degradation of Cdc42 in iNKT cells.Moreover,reduced Cdc42 expression was observed in tumorinfiltrating iNKT cells,which impaired IL4 polarization and disturbed iNKT cell-DC crosstalk in tumors.

Application of Optical Tweezers in the Research of Molecular Interaction between Lymphocyte Function Associated Antigen-1 and Its Monoclonal Antibody

Lymphocyte function associated antigen-1 （CD11a/CD18, LFA-1） plays an important role in the structure of the immunological synapse and is required for efficient lysis of cytotoxic T lymphocytes （CTLs） and natural killer （NK） cells. To study the activation mode of LFA-1 on the NK cell surface, optical tweezers were used in the work. As an emerging technology, optical tweezers are widely used to manipulate microscopic objects and measure the forces of molecular interactions in the field of biological research. In our study, a new platform was constructed to study the single molecular behavior of receptor on cell surface using optical tweezers. Based on the platform, the interaction between an NK cell and a polystyrene microsphere coated with anti-LFA-1 antibody was observed. The result confirmed that the adhesion forces between an NK cell and a polystyrene bead were time-dependent. According to our findings, we propose that anti-LFA-1 antibody may cause the clustering of LFA-1 on NK cell surface. Cellular ＆ Molecular Immunology.

Analysis of uterine CD49a^(+) NK cell subsets in menstrual blood reflects endometrial status and association with recurrent spontaneous abortion

Maternal uterine immune cells,especially natural killer(NK)cells,are important for successful pregnancy,and an abnormal number or function of uterine NK cells is closely correlated with an impaired endometrial environment and miscarriage.However,there are currently no noninvasive testing methods using reliable indicators that accurately predict uterine changes leading to miscarriage.Here,we confirmed that before implantation,menstrual blood(MB)from healthy donors had 70%CD49a^(+)tissue-resident NK(trNK)cells,the majority of which were CD49a^(+)Eomes+NK cells.Importantly,MB from patients with recurrent spontaneous abortion(RSA)had many fewer CD49a^(+)trNK cells than MB from controls.Analysis of uNK cell populations in MB may better predict an abnormal endometrial environment associated with miscarriage than peripheral blood(PB)analysis.Therefore,we suggest using MB-and CD49a-related markers as a promising noninvasive way to evaluate endometrial status.

CD49a^(+)CD49b^(+) NK cells induced by viral infection reflect an activated state of conventional NK cells

Natural killer(NK) cells are important innate effectors that play a pivotal role in the defense against tumors and infections and participate in regulating adaptive immunity. Recent studies have revealed phenotypic and functional heterogeneity of NK cells.Here, using murine models of acute and chronic lymphocytic choriomeningitis virus infection, we observed that a CD49a^(+)CD49b^(+) NK cell subset emerged in the liver and other tissues, and underwent vigorous expansion following viral infection,before progressively decreasing in cell number. These viral infection-induced CD49a^(+)CD49b^(+) NK cells displayed an activated and mature phenotype. Moreover, compared with liver-resident NK cells and conventional NK(cNK) cells, CD49a^(+)CD49b^(+) NK cells showed increased functional competence, as evidenced by higher amounts of IFN-γ production and stronger cytotoxic capabilities during viral infection. Generation of these CD49a^(+)CD49b^(+) NK cells was shown to be independent of the T-bet transcription factor. Adoptive transfer experiments revealed that c NK cells could convert into CD49a^(+)CD49b^(+) NK cells following viral infection. Collectively, these results suggest that viral infection-induced CD49a^(+)CD49b^(+) NK cells represent a transiently activated state of cNK cells.

Restoration of HBV-specific CD8+T-cell responses by sequential low-dose IL-2 treatment in non-responder patients after IFN-αtherapy

Patients with chronic hepatitis B(CHB)undergoing interferon(IFN)-α-based therapies often exhibit a poor HBeAg serological response.Thus,there is an unmet need for new therapies aimed at CHB.This study comprised two clinical trials,including 130 CHB patients,who were treatment-naïve;in the first,92 patients were systematically analyzed ex vivo for interleukin-2 receptor(IL-2R)expression and inhibitory molecules expression after receiving Peg-IFN-α-2b therapy.In our second clinical trial,38 non-responder patients,in whom IFN-αtherapy had failed,were treated with or without low-dose IL-2 for 24 weeks.We then examined the hepatitis B virus(HBV)-specific CD8+T-cell response and the clinical outcome,in these patients.Although the majority of the participants undergoing Peg-IFN-α-2b therapy were non-responders,we observed a decrease in CD25 expression on their CD4+T cells,suggesting that IFN-αtherapy may provide a rationale for sequential IL-2 treatment without increasing regulatory T cells(Tregs).Following sequential therapy with IL-2,we demonstrated that the non-responders experienced a decrease in the numbers of Tregs and programmed cell death protein 1(PD-1)expression.In addition,sequential IL-2 administration rescued effective immune function,involving signal transducer and activator of transcription 1(STAT1)activation.Importantly,IL-2 therapy significantly increased the frequency and function of HBV-specific CD8+T cells,which translated into improved clinical outcomes,including HBeAg seroconversion,among the non-responder CHB patients.Our findings suggest that sequential IL-2 therapy shows efficacy in rescuing immune function in non-responder patients with refractory CHB.

HBsAg-specific CD8^(+)T cells as an indispensable trigger to induce murine hepatocellular carcinoma

Hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC)is mediated by an inappropriate attack by HBV-specific T cells in patients.However,this immunopathogenic process has not been clarified because of the lack of a suitable animal model.Here,we used immunocompetent Fah^(-/-)mice as the recipients in the adoptive transfer of HBsAg^(+)hepatocytes from HBs-Tg mice to replace the recipient hepatocytes(HBs-HepR).HBs-HepR mice exhitited persistent HBsAg expression with chronic hepatitis and eventually developed HCC with a prevalence of 100%.HBsAg-specific CD8^(+)T cells were generated and specifically and continuously induced hepatocyte apoptosis with progressive chronic inflammation,and the depletion of CD8^(+)T cells or their deficiency prevented HCC,which could then be reproduced by the transfer of HBsAg-specific CD8^(+)T cells.In summary,our results demonstrated that CD8^(+)T cells plays a critical role in HBsAg-driven inflammtion and HCC tumorigenesis.

Tocilizumab in patients with moderate or severe COVID-19: a randomized, controlled, open-label, multicenter trial

Tocilizumab has been reported to attenuate the“cytokine storm”in COVID-19 patients.We attempted to verify the effectiveness and safety of tocilizumab therapy in COVID-19 and identify patients most likely to benefit from this treatment.We conducted a randomized,controlled,open-label multicenter trial among COVID-19 patients.The patients were randomly assigned in a 1:1 ratio to receive either tocilizumab in addition to standard care or standard care alone.The cure rate,changes of oxygen saturation and interference,and inflammation biomarkers were observed.Thirty-three patients were randomized to the tocilizumab group,and 32 patients to the control group.The cure rate in the tocilizumab group was higher than that in the control group,but the difference was not statistically significant(94.12%vs.87.10%,rate difference 95%CI−7.19%–21.23%,P=0.4133).The improvement in hypoxia for the tocilizumab group was higher from day 4 onward and statistically significant from day 12(P=0.0359).In moderate disease patients with bilateral pulmonary lesions,the hypoxia ameliorated earlier after tocilizumab treatment,and less patients(1/12,8.33%)needed an increase of inhaled oxygen concentration compared with the controls(4/6,66.67%;rate difference 95%CI−99.17%to−17.50%,P=0.0217).No severe adverse events occurred.More mild temporary adverse events were recorded in tocilizumab recipients(20/34,58.82%)than the controls(4/31,12.90%).Tocilizumab can improve hypoxia without unacceptable side effect profile and significant influences on the time virus load becomes negative.For patients with bilateral pulmonary lesions and elevated IL-6 levels,tocilizumab could be recommended to improve outcome.

All-trans retinoic acid induces leukemia resistance to NK cell cytotoxicity by down-regulating B7-H6 expression via c-Myc signaling

Background:The interaction between activating receptor NKp30 and its major tumor ligand B7-H6 is important for NK cell-mediated tumor rejection.However,the regulation of B7-H6 by tumor therapeutics remains largely unknown.In this study,we investigated the regulation of B7-H6 by all-trans retinoic acid(atRA),a terminal differentiation inducer of tumor cells that is extensively used for clinical leukemia therapy.Methods:We investigated the role of NKp30:B7-H6 axis in NK cell-mediated tumor lysis against leukemia cells and the influence of atRA treatment on the cytotoxicity of NK cells using NK cell lines(NK92 and NKG)and leukemia cell lines(U-937 and THP-1).We evaluated the effect of atRA treatment on the expression of B7-H6 using real-time PCR,flow cytometry and western blotting.We used CRISPR/Cas9 to knockdown B7-H6 expression and siRNA to knockdown c-Myc in U-937 cells to evaluate the role of B7-H6 and c-Myc in atRA-induced tumor resistance against NK cells.Results:NK cell-mediated U-937 cell lysis was mainly dependent on NKp30/B7-H6 interaction.Blockade of B7-H6 by monoclonal antibody significantly impaired NK cytotoxicity.atRA treatment induced U-937 resistance to NK cell cytotoxicity by reducing B7-H6 expression,and showed no effect on NK cytotoxicity against B7-H6 knockdown U-937 cells.Epigenetic modifications,such as DNA methylation and histone deacetylase(HDAC),were not responsible for atRA-mediated B7-H6 down-regulation as inhibitors of these pathways could not restore B7-H6 mRNA expression.On the other hand,atRA treatment reduced c-Myc expression,which in turn inhibited the transcription of B7-H6 on leukemia cells.Conclusion:atRA treatment promotes tumor cell resistance against NK cellmediated lysis by down-regulating B7-H6 expression via the c-Myc signaling pathway,suggesting that more attention needs to be paid to the immunological adverse effects in the clinical use of atRA treatment.

Landscape and Dynamics of the Transcriptional Regulatory Network During Natural Killer Cell Differentiation

Natural killer(NK)cells are essential in controlling cancer and infection.However,little is known about the dynamics of the transcriptional regulatory machinery during NK cell differentiation.In this study,we applied the assay of transposase accessible chromatin with sequencing(ATAC-seq)technique in a home-developed in vitro NK cell differentiation system.Analysis of ATAC-seq data illustrated two distinct transcription factor(TF)clusters that dynamically regulate NK cell differentiation.Moreover,two TFs from the second cluster,FOS-like 2(FOSL2)and early growth response 2(EGR2),were identified as novel essential TFs that control NK cell maturation and function.Knocking down either of these two TFs significantly impacted NK cell differentiation.Finally,we constructed a genome-wide transcriptional regulatory network that provides a better understanding of the regulatory dynamics during NK cell differentiation.

Pyroptotic macrophages stimulate the SARS-CoV-2-associated cytokine storm

Coronavirus disease 2019(COVID-19)is an unprecedented pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).As of 22 February 2021,the worldwide pandemic has resulted in more than 110 million cases and 2.4 million deaths.1 Clinical investigation of COVID-19 patients has shown that a systemic cytokine storm can occur,especially in severe cases.2 Treatment of the SARS-CoV-2-associated cytokine storm with tocilizumab3 or anakinra4 has been shown to immediately improve the clinical outcome in most severe and critical COVID-19 patients.These data highlight the systemic cytokine storm as an important exacerbating event in severe COVID-19;however,our understanding of the molecular mechanisms involved in the initiation of the SARS-CoV-2-associated cytokine storm is limited.In the present study,we uncovered a reasonable explanation for cytokine storm initiation through the analysis of 13 autopsy samples from severe COVID-19 patients.

Transcriptomic characteristics and impaired immune function of patients who retest positive for SARS-CoV-2 RNA

Coronavirus disease 2019(COVID-19),caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection,has become a global public health crisis.Some patients who have recovered from COVID-19 subsequently test positive again for SARS-CoV-2 RNA after discharge from hospital.How such retest-positive(RTP)patients become infected again is not known.In this study,30 RTP patients,20 convalescent patients,and 20 healthy controls were enrolled for the analysis of immunological characteristics of their peripheral blood mononuclear cells.We found that absolute numbers of CD4^(+)T cells,CD8^(+)T cells,and natural killer cells were not substantially decreased in RTP patients,but the expression of activation markers on these cells was significantly reduced.The percentage of granzyme B-producing T cells was also lower in RTP patients than in convalescent patients.Through transcriptome sequencing,we demonstrated that high expression of inhibitor of differentiation 1(ID1)and low expression of interferon-induced transmembrane protein 10(IFITM10)were associated with insufficient activation of immune cells and the occurrence of RTP.These findings provide insight into the impaired immune function associated with COVID-19 and the pathogenesis of RTP,which may contribute to a better understanding of the mechanisms underlying RTP.