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BACKGROUND & AIMS N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS Mettl3 knockout mice,CD34+ humanized mice,and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay,respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells,and eventually suppressed CRC in ApcMin/+Mettl3+/- mice,CD34+ humanized mice,and syngeneic mice models. Mechanistically,METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing,RNA sequencing,and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner,which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However,the effect was negligible on BHLHE41 depletion,CXCL1 protein or CXCR2 inhibitor SB265610 administration,inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently,depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly,targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC. 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

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

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

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

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

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

The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However,when ART is suspended,the virus rebounds due to the presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats available presently,the Tat/Rev induced limiting dilution assay (TILDA) offers the most robust and technically simple assay strategy. The TILDA formats reported thus far are limited by being selective to one or a few HIV-1 genetic subtypes,thus,restricting them from a broader level application. The novel TILDA,labelled as U-TILDA ('U' for universal),can detect all the major genetic subtypes of HIV-1 unbiasedly,and with comparable sensitivity of detection. U-TILDA is well suited to characterize the latent reservoirs of HIV-1 and aid in the formulation of cure strategies. Graphical abstract. View Publication

The decreased proportion of antigen-inexperienced,na{\{i}}ve T cells is a hallmark of aging in both humans and mice and contributes to reduced immune responses particularly against novel and re-emerging pathogens. Na{\"{i}}ve T cells depend on survival signals received during their circulation among the lymph nodes by direct contacts with stroma in particular fibroblastic reticular cells. Macroscopic changes to the architecture of the lymph nodes have been described but it is unclear how lymph node stroma are altered with age and whether these changes contribute to reduced na{\"{i}}ve T cell maintenance. Here using 2-photon microscopy we determined that the aged lymph node displayed increased fibrosis and correspondingly that na{\"{i}}ve T-cell motility was impaired in the aged lymph node especially in proximity to fibrotic deposition. Functionally adoptively transferred young na{\"{i}}ve T-cells exhibited reduced homeostatic turnover in aged hosts supporting the role of T cell-extrinsic mechanisms that regulate their survival. Further we determined that early development of resident fibroblastic reticular cells was impaired which may correlate to the declining levels of na{\"{i}}ve T-cell homeostatic factors observed in aged lymph nodes. Thus our study addresses the controversy as to whether aging impacts the composition lymph node stroma and supports a model in which impaired differentiation of lymph node fibroblasts and increased fibrosis inhibits the interactions necessary for na{\"{i}}ve T cell homeostasis." View Publication

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens,T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However,the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe,and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC),a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm,which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the ? and ? subunits of a TCR,producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTC + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment,no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably,one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant. View Publication

Immunogenicity following an additional dose of Coronavirus disease 2019 (COVID-19) vaccine was investigated in an extended primary series among kidney transplant (KT) recipients. Eighty-five KT participants were randomized to receive either an mRNA (M group; n =??43) or viral vector (V group; n =??42) vaccine. Among them,62% were male,with a median (IQR) age of 50 (43-59) years and post-transplantation duration of 46 (26-82) months. At 2??weeks post-additional dose,there was no difference in the seroconversion rate between the M and V groups (70% vs. 65%,p =??.63). A median (IQR) of anti-RBD antibody level was not statistically different between the M group compared with the V group (51.8 [5.1-591] vs. 28.5 [2.9-119.3] BAU/ml,p =??.18). Furthermore,the percentage of participants with positive SARS-CoV-2 surrogate virus neutralization test results was not statistically different between groups (20% vs. 15%,p =??.40). S1-specific T cell and RBD-specific B cell responses were also comparable between the M and V groups (230 [41-420] vs. 268 [118-510],p =??.65 and 2 [0-10] vs. 2 [0-13] spot-forming units/106 peripheral blood mononuclear cells,p =??.60). In conclusion,compared with an additional dose of viral vector COVID-19 vaccine,a dose of mRNA COVID-19 vaccine did not elicit significantly different responses in KT recipients,regarding either humoral or cell-mediated immunity. (TCTR20211102003). 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