参考文献

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity,we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity,non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition,we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells,termed iDuo NK cells,displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models,iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore,iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19,features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies. View Publication

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2$\alpha$ and/or CK2$\alpha$') and two regulatory subunits (CK2$\beta$). CK2 promotes cancer progression by activating the NF-$\kappa$B,PI3K/AKT/mTOR,and JAK/STAT pathways,and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2$\alpha$ deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore,CK2$\alpha$ is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly,using a mouse Listeria monocytogenes infection model,we demonstrate that CK2$\alpha$ is required for CD8+ T cell expansion,maintenance,and effector function in both primary and memory immune responses. Collectively,our study implicates CK2$\alpha$ as an important regulator of mouse CD8+ T cell activation,metabolic reprogramming,and differentiation both in vitro and in vivo. View Publication

BACKGROUND Adoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However,even for the best-studied and clinically validated CD19-targeted chimeric antigen receptor (CAR) T-cell therapy,many patients face the challenge of lack of response or occurrence of relapse. There is increasing need to improve the efficacy of ACT so that durable,curative outcomes can be achieved in a broad patient population. METHODS Here,we investigated the impact of indomethacin (indo),a non-steroidal anti-inflammatory drug (NSAID),on the efficacy of ACT in multiple preclinical models. Mice with established B-cell lymphoma received various combinations of preconditioning chemotherapy,infusion of suboptimal dose of tumor-reactive T cells,and indo administration. Donor T cells used in the ACT models included CD4+ T cells expressing a tumor-specific T cell receptor (TCR) and T cells engineered to express CD19CAR. Mice were monitored for tumor growth and survival. The effects of indo on donor T cell phenotype and function were evaluated. The molecular mechanisms by which indo may influence the outcome of ACT were investigated. RESULTS ACT coupled with indo administration led to improved tumor growth control and prolonged mouse survival. Indo did not affect the activation status and tumor infiltration of the donor T cells. Moreover,the beneficial effect of indo in ACT did not rely on its inhibitory effect on the immunosuppressive cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis. Instead,indo-induced oxidative stress boosted the expression of death receptor 5 (DR5) in tumor cells,rendering them susceptible to donor T cells expressing TNF-related apoptosis-inducing ligand (TRAIL). Furthermore,the ACT-potentiating effect of indo was diminished against DR5-deficient tumors,but was amplified by donor T cells engineered to overexpress TRAIL. CONCLUSION Our results demonstrate that the pro-oxidative property of indo can be exploited to enhance death receptor signaling in cancer cells,providing rationale for combining indo with genetically modified T cells to intensify tumor cell killing through the TRAIL-DR5 axis. These findings implicate indo administration,and potentially similar use of other NSAIDs,as a readily applicable and cost-effective approach to augment the efficacy of ACT. View Publication

T cell antigen-receptor (TCR) signaling controls the development,activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing,translation and stability of transcripts. In the present study,we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes,control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut ROR?t+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells. View Publication

Plasmacytoid dendritic cells (pDCs) have been implicated as having a role in antifungal immunity,but mechanisms of their interaction with fungi and the resulting cellular responses are not well understood. In this study,we identify the direct and indirect biological response of human pDCs to the fungal pathogen Aspergillus fumigatus and characterize the expression and regulation of antifungal receptors on the pDC surface. Results indicate pDCs do not phagocytose Aspergillus conidia,but instead bind hyphal surfaces and undergo activation and maturation via the upregulation of costimulatory and maturation markers. Measuring the expression of C-type lectin receptors dectin-1,dectin-2,dectin-3,and mannose receptor on human pDCs revealed intermediate expression of each receptor compared with monocytes. The specific dectin-1 agonist curdlan induced pDC activation and maturation in a cell-intrinsic and cell-extrinsic manner. The indirect activation of pDCs by curdlan was much stronger than direct stimulation and was mediated through cytokine production by other PBMCs. Overall,our data indicate pDCs express various C-type lectin receptors,recognize and respond to Aspergillus hyphal Ag,and serve as immune enhancers or modulators in the overarching fungal immune response. View Publication

Myeloid-derived suppressor cells (MDSCs) are known to promote tumor growth in part by their immunosuppressive activities and their angiogenesis support. It has been shown that Bv8 blockade inhibits the recruitment of MDSCs to tumors,thereby delaying tumor relapse associated with resistance to antiangiogenic therapy. However,the impact of Bv8 blockade on tumors resistant to the new immunotherapy drugs based on the blockade of immune checkpoints has not been investigated. Here,we demonstrate that granulocytic-MDSCs (G-MDSCs) are enriched in anti-PD1 resistant tumors. Importantly,resistance to anti-PD1 monotherapy is reversed upon switching to a combined regimen comprised of anti-Bv8 and anti-PD1 antibodies. This effect is associated with a decreased level of G-MDSCs and enrichment of active cytotoxic T cells in tumors. The blockade of anti-Bv8 has shown efficacy also in hyperprogressive phenotype of anti-PD1-treated tumors. In vitro,anti-Bv8 antibodies directly inhibit MDSC-mediated immunosuppression,as evidenced by enhanced tumor cell killing activity of cytotoxic T cells. Lastly,we show that anti-Bv8-treated MDSCs secrete proteins associated with effector immune cell function and T cell activity. Overall,we demonstrate that Bv8 blockade inhibits the immunosuppressive function of MDSCs,thereby enhancing anti-tumor activity of cytotoxic T cells and sensitizing anti-PD1 resistant tumors. Our findings suggest that combining Bv8 blockade with anti-PD1 therapy can be used as a strategy for overcoming therapy resistance. View Publication

Recent studies suggest that highly activated,polyfunctional CD4+ T cells are incredibly effective in strengthening and sustaining overall host antitumor immunity,promoting tumor-specific CD4+ T-cell responses and effectively enhancing antitumor immunity by immunotherapy. Previously,we developed a novel cryo-thermal therapy for local tumor ablation and achieved long-term survival rates in several tumor models. It was discovered that cryo-thermal therapy remodeled the tumor microenvironment and induced an antigen-specific CD4+ T-cell response,which mediated stronger antitumor immunity in vivo. In this study,the phenotype of bulk T cells in spleen was analyzed by flow cytometry after cryo-thermal therapy and both CD4+ Th1 and CD8+ CTL were activated. In addition,by using T-cell depletion,isolation,and adoptive T-cell therapy,it was found that cryo-thermal therapy induced Th1-dominant CD4+ T cells that directly inhibited the growth of tumor cells,promoted the maturation of MDSCs via CD4+ T-cell-derived IFN-? and enhanced the cytotoxic effector function of NK cells and CD8+ T cells,and promoted the maturation of APCs via cell-cell contact and CD4+ T-cell-derived IFN-?. Considering the multiple roles of cryo-thermal-induced Th1-dominant CD4+ T cells in augmenting antitumor immune memory,we suggest that local cryo-thermal therapy is an attractive thermo-immunotherapy strategy to harness host antitumor immunity and has great potential for clinical application. View Publication

Innate lymphoid cells 2 (ILC2) play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). An important aspect of ILC2-mediated tumorigenesis is the expansion of the resident ILC2 and simultaneous recruitment of the peripheral ILC2. Here,we describe a protocol for isolation,enrichment,and DiD labeling of ILC2 for in vivo tracking of ILC2s in the mouse. Further,we describe steps for the adoptive transfer of ILC2 to a recipient mouse model of PDAC. For complete details on the use and execution of this protocol,please refer to Alam et al. (2022). View Publication

OBJECTIVES Rheumatoid arthritis (RA) is a chronic autoimmune disease,that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS Plasma levels of soluble (s) Tim-3 in early RA (n=98),were followed,to evaluate association with treatment and disease activity,acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies,either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS Long-time stimulated CD4 T-cells expressed high levels of Tim-3,but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3,CD4,CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFN$\gamma$ and MCP-1,in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally,we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION Taken together,our findings indicate disease-suppressive functions of Tim-3 in RA. View Publication

Cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) inhibitors such as pentoxifylline (PTX) suppress cAMP degradation and promote cAMP-dependent signal transduction. PDE inhibitors increase bone formation and bone mass in preclinical models and are used clinically to treat psoriatic arthritis by targeting inflammatory mediators including activated T cells. T cell activation requires two signals: antigen-dependent CD3-activation,which stimulates cAMP production; and CD28 co-stimulation,which downregulates cAMP-signaling,through PDE activation. PDE-inhibitors consequently suppress T cell activation by disrupting CD28 co-stimulation. Interestingly,we have reported that when CD8+ T cells are activated in the absence of CD28 co-stimulation,they secrete Wnt-10b,a bone anabolic Wnt ligand that promotes bone formation. In the present study,we investigated whether the bone anabolic activity of the PDE-inhibitor PTX,has an immunocentric basis,involving Wnt-10b production by CD8+ T cells. When wild-type (WT) mice were administered PTX,biochemical markers of both bone resorption and formation were significantly increased,with net bone gain in the axial skeleton,as quantified by micro-computed tomography ($\mu$CT). By contrast,PTX increased only bone resorption in T cell knockout (KO) mice,causing net bone loss. Reconstituting T cell-deficient mice with WT,but not Wnt-10b knockout (KO) CD8+ T cells,rescued bone formation and prevented bone loss. To study the role of cAMP signaling in Wnt-10b expression,reverse-transcription polymerase chain reaction (RT-PCR) and luciferase-reporter assays were performed using primary T cells. PDE inhibitors intensified Wnt-10b promoter activity and messenger RNA (mRNA) accumulation in CD3 and CD28 activated CD8+ T cells. In contrast,inhibiting the cAMP pathway mediators protein kinase A (PKA) and cAMP response element-binding protein (CREB),suppressed Wnt-10b expression by T cells activated in the absence of CD28 co-stimulation. In conclusion,the data demonstrate a key role for Wnt-10b production by CD8+ T cells in the bone anabolic response to PDE-inhibitors and reveal competing T cell-independent pro-resorptive properties of PTX,which dominate under T cell-deficient conditions. Selective targeting of CD8+ T cells by PDE inhibitors may be a beneficial approach for promoting bone regeneration in osteoporotic conditions. {\textcopyright} 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. View Publication