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Among different cancer immunotherapy approaches,bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However,most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study,we describe a bispecific antibody,BiHC (bispecific Her2-CD3 antibody),constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format. In contrast to most IgG-like BsAbs,the two arms in BiHC have different molecular weights,making it easier to separate hetero- or homodimers. BiHC can be expressed in Escherichia coli and purified via Protein A affinity chromatography. The purified BiHC can recruit T cells and induce specific cytotoxicity of Her2-expressing tumor cells in vitro. The BiHC can also efficiently inhibit the tumor growth in vivo. Thus,BiHC is a promising candidate for the treatment of Her2-positive cancers. View Publication

Lung macrophage subpopulations have been identified based on size. We investigated characteristics of small and large macrophages in the alveolar spaces and lung interstitium of COPD patients and controls. Alveolar and interstitial cells were isolated from lung resection tissue from 88 patients. Macrophage subpopulation cell-surface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Inflammatory related gene expression was measured. Alveolar and interstitial macrophages had subpopulations of small and large macrophages based on size and granularity. Alveolar macrophages had similar numbers of small and large cells; interstitial macrophages were mainly small. Small macrophages expressed significantly higher cell surface HLA-DR,CD14,CD38 and CD36 and lower CD206 compared to large macrophages. Large alveolar macrophages showed lower marker expression in COPD current compared to ex-smokers. Small interstitial macrophages had the highest pro-inflammatory gene expression levels,while large alveolar macrophages had the lowest. Small alveolar macrophages had the highest phagocytic ability. Small alveolar macrophage CD206 expression was lower in COPD patients compared to smokers. COPD lung macrophages include distinct subpopulations; Small interstitial and small alveolar macrophages with more pro-inflammatory and phagocytic function respectively,and large alveolar macrophages with low pro-inflammatory and phagocytic ability. View Publication

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However,whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study,we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5,a principal Tfh transcription factor Bcl6,and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle,express B cell costimulatory proteins,and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease,in part,through CD4 follicular-like differentiation and functionality. View Publication

Cellular senescence,which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2,can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing,including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7-9; however,a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis,hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition,and degeneration of cortical and hippocampal neurons,thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively,these results show that senescent cells have a role in the initiation and progression of tau-mediated disease,and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies. View Publication

Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks,such as PD-1 expression,human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast,melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality,despite PD-1 expression,and suggest that they may be harnessed for effective immunotherapies. View Publication

Zika virus is a mosquito-borne flavivirus closely related to dengue virus that can cause severe disease in humans,including microcephaly in newborns and Guillain-Barr{\'{e}} syndrome in adults. Specific treatments and vaccines for Zika virus are not currently available. Here,we isolate and characterize four monoclonal antibodies (mAbs) from an infected patient that target the non-structural protein NS1. We show that while these antibodies are non-neutralizing,NS1-specific mAbs can engage Fc$\gamma$R without inducing antibody dependent enhancement (ADE) of infection in vitro. Moreover,we demonstrate that mAb AA12 has protective efficacy against lethal challenges of African and Asian lineage strains of Zika virus in Stat2-/- mice. Protection is Fc-dependent,as a mutated antibody unable to activate known Fc effector functions or complement is not protective in vivo. This study highlights the importance of the ZIKV NS1 protein as a potential vaccine antigen. View Publication

Pathogen immune responses are profoundly attenuated in fetuses and premature infants,yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic,metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-$\gamma$-regulated metabolic pathways,limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast,they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants,and suggest that the fetal immune system is metabolically programmed to avoid energetically costly,dispensable and potentially harmful immune responses during ontogeny. View Publication

Vascular-deposited IgG immune complexes promote neutrophil recruitment,but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1,in its bent state,interacts with the IgG receptor Fc$\gamma$RIIA in cis to reduce the affinity of Fc$\gamma$RIIA for IgG and inhibit Fc$\gamma$RIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of Fc$\gamma$RIIA,as does a Mac-1 ligand and a mutation in Mac-1's ligand binding $\alpha$I-domain. Sialylated complex glycans on Fc$\gamma$RIIA interact with the $\alpha$I-domain via divalent cations,and this interaction is required for Fc$\gamma$RIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented Fc$\gamma$RIIA-dependent recruitment to IgG-coated endothelium. In mice,CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary,cis interaction between sialylated Fc$\gamma$RIIA and the $\alpha$I-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases. View Publication

Rheumatoid arthritis is characterized by progressive joint inflammation and affects {\~{}}1{\%} of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1,DOCK2,and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment,we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly,Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints,without general inhibition of inflammatory responses. Further,neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity,whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis. View Publication

Precise control of Wnt signaling is necessary for immune system development. In this study,we detected severely impaired development of all lymphoid lineages in mice,resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l),which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/beta-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation acting as a negative regulator of the Wnt/beta-catenin pathway." View Publication

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here,we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model,HMBA did not reactivate HIV-1,yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However,HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells,hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside,HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall,our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells,providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug. View Publication

In the inner ear,endolymph fluid surrounds the organ of Corti,which is important for auditory function; notably,even slight environmental changes mediated by trauma or infection can have significant consequences. However,it is unclear how the immune response is modulated in these tissues. Here,we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C,Cochlin,and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection,the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly,hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss. View Publication