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Multiple myeloma is an incurable malignancy of plasma cells resulting from impaired terminal B cell development. Almost all patients with multiple myeloma eventually have a relapse. Many studies have demonstrated the importance of the various genomic mutations that characterize multiple myeloma as a complex heterogeneous disease. In recent years,next-generation sequencing has been used to identify the genomic mutation landscape and clonal heterogeneity of multiple myeloma. This is the first study,a prospective observational study,to identify somatic mutations in plasma cell disorders in the Thai population using targeted next-generation sequencing. Twenty-seven patients with plasma cell disorders were enrolled comprising 17 cases of newly diagnosed multiple myeloma,5 cases of relapsed/refractory multiple myeloma,and 5 cases of other plasma cell disorders. The pathogenic mutations were found in 17 of 27 patients. Seventy percent of those who had a mutation (12/17 patients) habored a single mutation,whereas the others had more than one mutation. Fifteen pathogenic mutation genes were identified: ATM,BRAF,CYLD,DIS3,DNMT3A,FBXW7,FLT3,GNA13,IRF4,KMT2A,NRAS,SAMHD1,TENT5C,TP53,and TRAF3. Most have previously been reported to be involved in the RAS/MAPK pathway,the nuclear factor kappa B pathway,the DNA-repair pathway,the CRBN pathway,tumor suppressor gene mutation,or an epigenetic mutation. However,the current study also identified mutations that had not been reported to be related to myeloma: GNA13 and FBXW7. Therefore,a deep understanding of molecular genomics would inevitably improve the clinical management of plasma cell disorder patients,and the increased knowledge would ultimately result in better outcomes for the patients. View Publication

BACKGROUND Lymphoid neoplasms,including multiple myeloma (MM),non-Hodgkin lymphoma (NHL),and NK/T cell neoplasms,are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies,like daratumumab,have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials. METHODS We generated chimeric antigen receptor (CAR) T cells targeting CD38 and tested its cytotoxicity against multiple CD38high and CD38low lymphoid cancer cells. We evaluated the synergistic effects of all-trans retinoic acid (ATRA) and CAR T cells or daratumumab against cancer cells and xenograft tumors. RESULTS CD38-CAR T cells dramatically inhibited the growth of CD38high MM,mantle cell lymphoma (MCL),Waldenstrom's macroglobulinemia (WM),T-cell acute lymphoblastic leukemia (T-ALL),and NK/T-cell lymphoma (NKTCL) in vitro and in mouse xenografts. ATRA elevated CD38 expression in multiple CD38low cancer cells and enhanced the anti-tumor activity of daratumumab and CD38-CAR T cells in xenograft tumors. CONCLUSIONS These findings may expand anti-CD38 immunotherapy to a broad spectrum of lymphoid malignancies and call for the incorporation of ATRA into daratumumab or other anti-CD38 immunological agents for cancer therapy. View Publication

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay,we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms,we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFN$\gamma$ production. Moreover,TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I,adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings,we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis,acting as a regulator for the induction of different effector functions. View Publication

Tuberculosis is a leading cause of death in mankind due to infectious agents,and Mycobacterium tuberculosis (Mtb) infects and survives in macrophages (MФs). Although MФs are a major niche,myeloid-derived suppressor cells (MDSCs) are an alternative site for pathogen persistence. Both MФs and MDSCs express varying levels of leukocyte immunoglobulin-like receptor B (LILRB),which regulate the myeloid cell suppressive function. Herein,we demonstrate that antagonism of LILRB2 by a monoclonal antibody (mab) induced a switch of human MDSCs towards an M1-macrophage phenotype,increasing the killing of intracellular Mtb. Mab-mediated antagonism of LILRB2 alone and its combination with a pharmacological blockade of SHP1/2 phosphatase increased proinflammatory cytokine responses and phosphorylation of ERK1/2,p38 MAPK,and NF-kB in Mtb-infected MDSCs. LILRB2 antagonism also upregulated anti-mycobacterial iNOS gene expression and an increase in both nitric oxide and reactive oxygen species synthesis. Because genes associated with the anti-mycobacterial function of M1-MФs were enhanced in MDSCs following mab treatment,we propose that LILRB2 antagonism reprograms MDSCs from an immunosuppressive state towards a pro-inflammatory phenotype that kills Mtb. LILRB2 is therefore a novel therapeutic target for eradicating Mtb in MDSCs. View Publication

Na{\{i}}ve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions T cell activation and T cell expansion in vivo. Our results showed that na{\"{i}}ve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient na{\"{i}}ve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse and that these conjugates were less stable than their WT counterparts. Finally we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions which promotes the initiation of stable T cell conjugates during APC scanning. Therefore Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo." View Publication

UNLABELLED Immune-checkpoint blockade (ICB) promotes antitumor immune responses and can result in durable patient benefit. However,response rates in breast cancer patients remain modest,stimulating efforts to discover novel treatment options. Cancer-associated fibroblasts (CAF) represent a major component of the breast tumor microenvironment and have known immunosuppressive functions in addition to their well-established roles in directly promoting tumor growth and metastasis. Here we utilized paired syngeneic mouse mammary carcinoma models to show that CAF abundance is associated with insensitivity to combination $\alpha$CTLA4 and $\alpha$PD-L1 ICB. CAF-rich tumors exhibited an immunologically cold tumor microenvironment,with transcriptomic,flow cytometric,and quantitative histopathologic analyses demonstrating a relationship between CAF density and a CD8+ T-cell-excluded tumor phenotype. The CAF receptor Endo180 (Mrc2) is predominantly expressed on myofibroblastic CAFs,and its genetic deletion depleted a subset of $\alpha$SMA-expressing CAFs and impaired tumor progression in vivo. The addition of wild-type,but not Endo180-deficient,CAFs in coimplantation studies restricted CD8+ T-cell intratumoral infiltration,and tumors in Endo180 knockout mice exhibited increased CD8+ T-cell infiltration and enhanced sensitivity to ICB compared with tumors in wild-type mice. Clinically,in a trial of melanoma patients,high MRC2 mRNA levels in tumors were associated with a poor response to $\alpha$PD-1 therapy,highlighting the potential benefits of therapeutically targeting a specific CAF subpopulation in breast and other CAF-rich cancers to improve clinical responses to immunotherapy. SIGNIFICANCE Paired syngeneic models help unravel the interplay between CAF and tumor immune evasion,highlighting the benefits of targeting fibroblast subpopulations to improve clinical responses to immunotherapy. View Publication

BACKGROUND Obesity has often been associated with severe allergic asthma (AA). Here,we analyzed the frequency of different circulating CD4+T-cell subsets from lean,overweight and obese AA patients. METHODS Mononuclear cells from peripheral blood were obtained from 60 AA patients and the frequency of different CD4+T-cell subsets and type 1 regulatory B cells (Br1) was determined by cytometry. The effect of obese-related leptin dose on cytokine production and Treg cell function in AA-derived CD4+ T cell cultures was evaluated by ELISA and 3H thymidine uptake,respectively. Leptin levels were quantified in the plasma by ELISA. According to the BMI,patients were stratified as lean,overweight and obese. RESULTS AA severity,mainly among obese patients,was associated with an expansion of hybrid Th2/Th17 and Th17-like cells rather than classic Th2-like cells. On the other hand,the frequencies of Th1-like,Br1 cells and regulatory CD4+ T-cell subsets were lower in patients with severe AA. While percentages of the hybrid Th2/Th17 phenotype and Th17-like cells positively correlated with leptin levels,the frequencies of regulatory CD4+ T-cell subsets and Br1 cells negatively correlated with this adipokine. Interestingly,the obesity-related leptin dose not only elevated Th2 and Th17 cytokine levels,but also directly reduced the Treg function in CD4+ T cell cultures from lean AA patients. CONCLUSION In summary,our results indicated that obesity might increase AA severity by favoring the expansion of Th17-like and Th2/Th17 cells and decreasing regulatory CD4+T cell subsets,being adverse effects probably mediated by leptin overproduction. View Publication

BACKGROUND The use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected,and occasionally of distal lesions. However,intralesional-BCG is less effective in patients with visceral metastases and did not significantly improve overall survival. METHODS We generated a novel BCG lysate and developed it into a thermosensitive PLGA-PEG-PLGA hydrogel (BCG hydrogel),which was injected adjacent to the tumor to assess its antitumor effect in syngeneic tumor models (B16F10,MC38). The effect of BCG hydrogel treatment on contralateral tumors,lung metastases,and survival was assessed to evaluate systemic long-term efficacy. Gene expression profiles of tumor-infiltrating immune cells and of tumor-draining lymph nodes from BCG hydrogel-treated mice were analyzed by single-cell RNA sequencing (scRNA-seq) and CD8+ T cell receptor (TCR) repertoire diversity was assessed by TCR-sequencing. To confirm the mechanistic findings,RNA-seq data of biopsies obtained from in-transit cutaneous metastases of patients with melanoma who had received intralesional-BCG therapy were analyzed. RESULTS Here,we show that BCG lysate exhibits enhanced antitumor efficacy compared to live mycobacteria and promotes a proinflammatory tumor microenvironment and M1 macrophage (M$\Phi$) polarization in vivo. The underlying mechanisms of BCG lysate-mediated tumor immunity are dependent on M$\Phi$ and dendritic cells (DCs). BCG hydrogel treatment induced systemic immunity in melanoma-bearing mice with suppression of lung metastases and improved survival. Furthermore,BCG hydrogel promoted cathepsin S (CTSS) activity in M$\Phi$ and DCs,resulting in enhanced antigen processing and presentation of tumor-associated antigens. Finally,BCG hydrogel treatment was associated with increased frequencies of melanoma-reactive CD8+ T cells. In human patients with melanoma,intralesional-BCG treatment was associated with enhanced M1 M$\Phi$,mature DC,antigen processing and presentation,as well as with increased CTSS expression which positively correlated with patient survival. CONCLUSIONS These findings provide mechanistic insights as well as rationale for the clinical translation of BCG hydrogel as cancer immunotherapy to overcome the current limitations of immunotherapies for the treatment of patients with melanoma. View Publication

The high mobility group (HMG) transcription factor TCF-1 is essential for early T cell development. Although in vitro biochemical assays suggest that HMG proteins can serve as architectural elements in the assembly of higher-order nuclear organization,the contribution of TCF-1 on the control of three-dimensional (3D) genome structures during T cell development remains unknown. Here,we investigated the role of TCF-1 in 3D genome reconfiguration. Using gain- and loss-of-function experiments,we discovered that the co-occupancy of TCF-1 and the architectural protein CTCF altered the structure of topologically associating domains in T cell progenitors,leading to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. The TCF-1-dependent gain in long-range interactions was linked to deposition of active enhancer mark H3K27ac and recruitment of the cohesin-loading factor NIPBL at active enhancers. These data indicate that TCF-1 has a role in controlling global genome organization during T cell development. View Publication

Type 1 diabetes is an autoimmune disease with no cure,where clinical translation of promising therapeutics has been hampered by the reproducibility crisis. Here,short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent research centers. Treatment with sRAGE increased regulatory T cells (Tregs) within the islets,pancreatic lymph nodes,and spleen,increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE with use of knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression,migration,and Treg homeostasis (FOXP3,IL7R,TIGIT,JAK1,STAT3,STAT5b,CCR4). Loss of suppressive function was reversed by sRAGE,where Tregs increased proliferation and suppressed conventional T-cell division,confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes,showing efficacy and reproducibility at multiple research centers and in human T cells. View Publication

Allogeneic T cells are key immune therapeutic cells to fight cancer and other clinical indications. High T cell dose per patient and increasing patient numbers result in clinical demand for a large number of allogeneic T cells. This necessitates a manufacturing platform that can be scaled up while retaining cell quality. Here we present a closed and scalable platform for T cell manufacturing to meet clinical demand. Upstream manufacturing steps of T cell activation and expansion are done in-vessel,in a stirred-tank bioreactor. T cell selection,which is necessary for CAR-T-based therapy,is done in the bioreactor itself,thus maintaining optimal culture conditions through the selection step. Platform's attributes of automation and performing the steps of T cell activation,expansion,and selection in-vessel,greatly contribute to enhancing process control,cell quality,and to the reduction of manual labor and contamination risk. In addition,the viability of integrating a closed,automated,downstream process of cell concentration,is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while preserving key factors of cell quality and process control. View Publication

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here,we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state,followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. View Publication