技术资料

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However,current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel,humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy,left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery,patient received multiple lines of standard of care therapy including sunitinib,axitinib,bevacizumab,everolimus and cabozantinib. While progressing on cabozantinib,he was treated with nivolumab. Seven years after initiation of nivolumab,and 4 years after stopping systemic therapy,he remains in complete remission. To generate autologous PDX model,bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant,the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore,HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells,B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood,our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting. View Publication

Juvenile dermatomyositis (JDM) is a pediatric autoimmune disease associated with characteristic rash and proximal muscle weakness. To gain insight into differential lymphocyte gene expression in JDM,peripheral blood mononuclear cells from 4 new-onset JDM patients and 4 healthy controls were sorted into highly enriched lymphocyte populations for RNAseq analysis. NK cells from JDM patients had substantially greater differentially expressed genes (273) than T (57) and B (33) cells. Upregulated genes were associated with the innate immune response and cell cycle,while downregulated genes were associated with decreased ribosomal RNA. Suppressed ribosomal RNA in JDM NK cells was validated by measuring transcription and phosphorylation levels. We confirmed a population of low ribosome expressing NK cells in healthy adults and children. This population of low ribosome NK cells was substantially expanded in 6 treatment-na{\{i}}ve JDM patients and was associated with decreased NK cell degranulation. The enrichment of this NK low ribosome population was completely abrogated in JDM patients with quiescent disease. Together these data suggest NK cells are highly activated in new-onset JDM patients with an increased population of low ribosome expressing NK cells which correlates with decreased NK cell function and resolved with control of active disease." View Publication

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here,we investigated the role of arginase 1 (ARG1) in V$\kappa$*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in EY-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of V$\kappa$*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However,arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether,these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors. View Publication

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs,focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations,gene expression,and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma,including severe type 2 inflammation,airway fibrosis,and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages,especially M2a and M2c. Furthermore,MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation,that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes,which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts,which were also suppressed by MSC treatment. In conclusion,intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management. View Publication

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore,depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However,identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here,we describe GS-1811,a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs,and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets,demonstrating a window for selective depletion of Tregs in the tumor. Importantly,we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy,which is dependent on Treg depleting activity,and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity. View Publication

BACKGROUND Novel therapies are urgently needed for ovarian cancer (OC),the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA,including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling,recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change,which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors,and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response. METHODS Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1,anti-CD8,or anti-NK1.1 antibodies every 3 days. RESULTS We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-$\beta$ compared with either perturbation alone. Furthermore,DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells. CONCLUSION In summary,we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus,epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC,a disease that does not respond to current immunotherapies. View Publication

BACKGROUND Systemic sclerosis (SSc) is a multiple-organ disease characterized by vascular damage,autoimmunity,and tissue fibrosis. Organ injuries such as interstitial lung diseases (ILD),resulting from inflammatory and fibrosis processes,lead to poor prognosis. Although autoantibodies are detected in the serum of patients with SSc,the mechanisms by which immune cells are involved in tissue inflammation and fibrosis is not fully understood. Recent studies have revealed carcinoembryonic antigen related cell adhesion molecule (CEACAM)-positive monocytes are involved in murine bleomycin-induced lung fibrosis. We investigated CEACAM-positive monocytes in patients with SSc to clarify the role of monocytes in the pathogenesis of SSc. METHODS The proportion of of CEACAM-positive classical monocytes in healthy controls (HCs) and patients with rheumatoid arthritis (RA) and SSc was evaluated using flow cytometry. The correlation between the proportion of CEACAM-positive monocytes and clinical parameters was analyzed in patients with SSc. Gene expression microarrays were performed in CEACAM-positive and negative monocytes in patients with SSc. Infiltration of CEACAM-positive monocytes into scleroderma skin was evaluated by immunohistochemical staining. RESULTS The proportion of CEACAM-positive classical monocytes was increased in patients with early SSc within 2 years after diagnosis,which positively correlated with ESR,serum IgG,and serum KL-6 and negatively correlated with %forced vital capacity. The percentage of CEACAM-positive monocytes decreased after immunosuppressive therapy. CEACAM6-positive cells among classical monocytes were significantly increased in patients with SSc compared with HCs and patients with rheumatoid arthritis. SSc serum induced CEACAM6 expression on monocytes from HCs. Functionally,CEACAM-positive monocytes produced higher levels of TNF-$\alpha$ and IL-1$\beta$ compared to CEACAM-negative cells and showed activation of the NF-$\kappa$B pathway. Furthermore,CEACAM6-positive monocytes infiltrated the dermis of SSc. CONCLUSIONS CEACAM-positive monocytes showed inflammatory phenotypes and may be involved in the tissue inflammation and fibrosis in early SSc. CEACAM-positive monocytes may be one of biomarkers to detect patients with progressive ILD,requiring therapeutic intervention. View Publication

Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and,therefore,often stimulate nonspecific lymphocyte activation,resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse,we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover,tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate,respectively. While Tbc1d10c was not expressed in solid tumor cells,Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically,Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-$\kappa$B pathway activation via regulation of Map3k3-mediated IKK$\beta$ phosphorylation. Strikingly,none of these cellular or molecular perturbations in the NF-$\kappa$B pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-$\kappa$B signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-$\kappa$B activation in tumor cells. View Publication

INTRODUCTION Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently,cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping,which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS The immunomagnetic negative selection was used for the enrichment of B and T cells fractions,and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis,principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as $\beta$-carotene. In addition,the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability,the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system. View Publication

BACKGROUND Ageing is a complex phenomenon that leads to decreased proliferative activity,loss of function of the cells,and cellular senescence. Senescence of the immune system exacerbates individual's immune response,both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+,CD8+,CD19+,CD56+. RESULTS Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively,p??=??0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (na{\{i}}ve and effector memory) CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22 3%) than high-risk patients (9 3%) p??=??0 04. CONCLUSION Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features characteristic for physiological ageing (telomeric shortening immunophenotype) when compared to their respective donors. However a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments." View Publication

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here,we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs,and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL,single-chain-variable-fragment,heavy-chain-only). METHODS Three new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo,for their polyfunctionality,immune synapse formation,memory,exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms,we analyzed BCMA levels and cellular localization and quantified CAR T cell-target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. RESULTS APRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BB$\zeta$ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BB$\zeta$ CAR T cells. After CAR T cell-myeloma cell contact,BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis,and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse,we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell-MM cell interactions activated BCMA pathway,we did not find enhanced tumor growth in vitro or in vivo. CONCLUSION Antitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect,but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies. View Publication

Immunotherapy has revolutionized cancer treatment,but preclinical models are required to understand immunotherapy resistance mechanisms underlying patient relapse. This protocol describes how to generate an acquired resistance humanized in vivo model to immunotherapies in patient-derived xenografts (PDX). We detail steps to inject human CD34+ cells into NSG mice,followed by generation of immunoresistant PDX in humanized mice. This approach recapitulates the human immune system,allowing investigators to generate preclinical resistance models to different immunotherapies for identifying the resistant phenotype. For complete details on the use and execution of this protocol,please refer to Mart{\'{i}}nez-Sabadell et al.,2022 and Arenas et al. (2021). View Publication