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T cell therapies,such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cells,are a growing class of anti-cancer treatments. However,expansion to novel indications and beyond last-line treatment requires engineering cells' dynamic population behaviors. Here we develop the tools for cellular behavior analysis of T cells from live-cell imaging,a common and inexpensive experimental setup used to evaluate engineered T cells. We first develop a state-of-the-art segmentation and tracking pipeline,Caliban,based on human-in-the-loop deep learning. We then build the Occident pipeline to collect a catalog of phenotypes that characterize cell populations,morphology,movement,and interactions in co-cultures of modified T cells and antigen-presenting tumor cells. We use Caliban and Occident to interrogate how interactions between T cells and cancer cells differ when beneficial knock-outs of RASA2 and CUL5 are introduced into TCR T cells. We apply spatiotemporal models to quantify T cell recruitment and proliferation after interactions with cancer cells. We discover that,compared to a safe harbor knockout control,RASA2 knockout T cells have longer interaction times with cancer cells leading to greater T cell activation and killing efficacy,while CUL5 knockout T cells have increased proliferation rates leading to greater numbers of T cells for hunting. Together,segmentation and tracking from Caliban and phenotype quantification from Occident enable cellular behavior analysis to better engineer T cell therapies for improved cancer treatment. View Publication

Galectin-1 is implicated in several pro-tumourigenic mechanisms and is considered immune-suppressive. The pharmacological inhibition of galectin-1 may be beneficial in cancers in which galectin-1 is overexpressed and driving cancer progression. This study aimed to further characterise the immunosuppressive cytokines influenced by galectin-1 in in vitro immune cell cultures and an in vivo inflammatory model using a recently discovered selective inhibitor of galectin-1,GB1908. To enable a translational approach and link mouse and human pharmacology,anti-CD3/anti-CD28 stimulated T cells cultured from human whole blood and mouse spleens were compared. For in vivo studies of T cell-mediated inflammation,the concanavalin-A (Con-A) mouse model was used to induce a T lymphocyte-driven acute liver injury phenotype. The inhibition of galectin-1 with GB1908 reduced IL-17A,IFNγ and TNFα in a concentration-dependent manner in both mouse and human T cells in vitro. The immunosuppressive cytokines measured in Con-A-treated mice were all upregulated compared to naïve mice. Subsequently,mice treated with GB1908 demonstrated a significant reduction in IL-17A,IFNγ,IL-6 and TNFα compared to vehicle-treated mice. In conclusion,galectin-1 induced the production of several important immune-suppressive cytokines from T cells in vitro and in vivo. This result suggests that,in the context of cancer therapy,a selective galectin-1 could be a viable approach as a monotherapy,or in combination with chemotherapeutic agents and/or checkpoint inhibitors,to enhance the numbers and activity of cytotoxic T cells in the tumour microenvironment of high galectin-1 expressing cancers. View Publication

BackgroundThe characteristics of the tumor immunosuppressive microenvironment represent a major challenge that limits the efficacy of immunotherapy. Our previous results suggested that cryo-thermal therapy,a tumor ablation system developed in our laboratory,promotes macrophage M1-type polarization and the complete maturation of DCs to remodel the immunosuppressive environment. However,the cells that respond promptly to CTT have not yet been identified. CTT can cause extensive cell death and the release of danger-associated molecular patterns and antigens. Neutrophils are the first white blood cells recruited to sites of damage and acute inflammation. Therefore,we hypothesized that neutrophils are the initial cells that respond to CTT and are involved in the subsequent establishment of antitumor immunity.MethodsIn this study,we examined the kinetics of neutrophil recruitment after CTT via flow cytometry and immunofluorescence staining and explored the effect of neutrophils on the establishment of systemic antitumor immunity by in vivo neutrophil depletion and in vitro co-culture assays.ResultsWe found that CTT led to a rapid and strong proinflammatory neutrophil response,which was essential for the long-term survival of mice. CTT-induced neutrophils promoted the activation of monocytes via reactive oxygen species and further upregulated the expression of IFN-γ and cytotoxic molecules in T and NK cells. Adoptive neutrophil transfer further enhanced the antitumor efficacy of CTT in tumor models of spontaneous and experimental metastasis.ConclusionThese results reveal the important role of neutrophil‒monocyte interactions in the development of anti-tumor immunity and highlight that CTT could be used as an immunotherapy for targeting neutrophils and monocytes to enhance antitumor immunity. View Publication

AbstractMyeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells due to the acquisition of mutations such as JAK2V617F. TGF‐β,whose secretion is increased in MPN patients,is known to negatively regulate haematopoietic stem cell (HSC) proliferation. Using an isogenic JAK2V617F or JAK2 wild‐type UT‐7 cell line we observed that JAK2V617F cells resist to TGF‐β antiproliferative activity. Although TGF‐β receptors and SMAD2/3 expressions are similar in both cell types,TGF‐β‐induced phosphorylation of SMAD2/3 is reduced in UT‐7 JAK2V617F cells compared with JAK2 WT cells. We confirmed that JAK2V617F mutated cells are resistant to the antiproliferative effect of TGF‐β in a competitive assay as we observed a positive selection of JAK2V617F cells when exposed to TGF‐β. Using cell lines,CD34‐positive cells from MPN patients and bone marrow cells from JAK2V617F knock‐in mice we identified a down regulation of the SHP‐1 phosphatase,which is required for the regulation of HSC quiescence by TGF‐β. The transduction of SHP‐1 cDNA (but not a phosphatase inactive cDNA) restores the antiproliferative effect of TGF‐β in JAK2V617F mutated cells. Finally,SC‐1,a known agonist of SHP‐1,antagonized the selection of JAK2V617F mutated cells in the presence of TGF‐β. In conclusion,we show a JAK2‐dependent down regulation of SHP‐1 in MPN patients' cells which is related to their resistance to the antiproliferative effect of TGF‐β. This may participate in the clonal selection of cancer cells in MPNs. View Publication

While adoptive cell therapies (ACT) have been successful as therapies for blood cancers,they have limited efficacy in treating solid tumours,where the tumour microenvironment excludes and suppresses adoptively transferred tumour-specific immune cells. A major obstacle to improving cell therapies for solid tumours is a lack of accessible and quantitative imaging modalities capable of tracking the migration and immune functional activity of ACT products for an extended duration in vivo.Methods: A high-efficiency magnetophoretic method was developed for facile magnetic labelling of hard-to-label immune cells,which were then injected into tumour-bearing mice and imaged over two weeks with a compact benchtop Magnetic Particle Imager (MPI) design.Results: Labelling efficiency was improved more than 10-fold over prior studies enabling longer-term tracking for at least two weeks in vivo of the labelled immune cells and their biodistribution relative to the tumour. The new imager showed 5-fold improved throughput enabling much larger density of data (up to 20 mice per experiment).Conclusions: Taken together,our innovations enable the convenient and practical use of MPI to visualise the localisation of ACT products in in vivo preclinical models for longitudinal,non-invasive functional evaluation of therapeutic efficacy. View Publication

Sensory neurons sense pathogenic infiltration to drive innate immune responses,but their role in humoral immunity is unclear. Here,using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma,we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae,sensory neuron depletion increases bacterial burden and reduces B cell numbers,IgG release,and neutrophil stimulation. Meanwhile,during A. alternata-induced airway inflammation,sensory neuron depletion decreases B cell population sizes,IgE levels,and asthmatic characteristics. Mechanistically,during bacterial infection,sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma,sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden,while VIPR1 deficiency increases infection. Similarly,exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice,while substance P deficiency ameliorates asthma. Our data,thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen. Sensory neurons may regulate innate immune cells,but their roles in humoral immunity is still unclear. Here the authors show that bacterial infection and asthma induction induce sensory neuron production of distinct neurotransmitters to dampen B cell responses but differentially target IgG and IgE,respectively,to specifically modulate the symptoms. View Publication

Physical frailty as a sign of accelerated aging is not well characterized in breast cancer (BC) and hematopoietic cell transplant (HCT) survivors and its correlation with outcomes and quality of life (QOL) is not defined. We conducted a prospective study to determine the prevalence of frailty in adult BC and HCT survivors,examine its impact on QOL,and determine its association with p16INK4a,a molecular biomarker for biological aging. The study included 59 BC and 65 HCT survivors. Median age was 60 years (range 27-81),68.5% were female and 49.2% were 18-59 vs. 51.8% ≥60 years old. A total of 71 (57.3%) were “fit” (frailty score 0) vs. 53 (42.7%) were pre-frailty/frail (frailty scores ≥1),and of the latter 17 (32.1%) were BC and 36 (67.9%) HCT patients. On multivariate analysis,patients >60 years were twice as likely to be frail (OR 2.04,95% CI,0.96-4.33; p=0.07),HCT were more likely to be frail compared to BC patients,and female HCT had 2.43 (95% CI,0.92-6.40) and male HCT patients had 3.25 (95% CI,1.37-7.72) times higher risk of frail; p=0.02. Frailty was associated with significant decline in QOL,measured by Medical Outcomes Study (MOS) Short Form 36 (SF-36) Physical Component Summary (PCS) and Mental Component Summary (MCS),and FACT (Functional Assessment of Cancer Therapy) scores. p16INK4a expression was higher in those who were frail,older than 60,and with higher expression in frail vs. fit patients who are 18-59 years. Our study highlights the high prevalence of frailty in survivors with detrimental effects on physical and overall wellbeing,and supports an association between frailty and the senescence marker p16INK4a. View Publication

BackgroundFANCA mutations have been detected in a variety of cancers and found to be pro-carcinogenic. However,no functional studies have been identified regarding the involvement of FANCA in the occurrence and the immune response of LUAD.MethodsThe mRNA expression and overall survival rates of FANCA were evaluated by the TIMER,PrognoScan and TCGA database in LUAD tissues,and FANCA expression was further validated by clinical serum samples using ELISA. The correlation between FANCA and immune infiltration level was investigated via TISIDB database and CIBERSORT algorithm. The Kaplan–Meier plotter was used to further evaluate the prognostic value based on the expression levels of FANCA in related immune cells. Then,the influence of FANCA knockout on the proliferation,migration,and invasion of A549 and H1299 cells was validated using CCK8,cloning formation,and Transwell assays. Subsequently,HLA-A2-restricted FANCA antigenic peptides were predicted and synthesized by NetMHC4.0 and SYFPEITHI,and DCs were induced and cultured in vitro. Finally,DCs loaded with HLA-A2-restricted FANCA antigenic peptides were co-cultured with autologous peripheral blood lymphocyte to generate specific CTLs. The killing effects of different CTLs on LUAD cells were studied.ResultsThe results showed that high levels of FANCA in patients with LUAD were significantly correlated with worse OS survival,which was correlated with age,clinical stage,pathological T stage,M stage,and N stage in LUAD. Knockdown of FANCA in A549 and H1299 cells significantly inhibited proliferation,metastasis,and invasion in vitro. In addition,FANCA was significantly related to immune infiltrate,genomic alterations and TMB. FANCA expression infuenced the prognosis of LUAD patients by directly affecting immune cell infltration. Finally,HLA-A2-restricted FANCA antigenic peptides were synthesized. And FANCA 146–154 (SLLEFAQYL) antigenic peptide exhibit a stronger affinity for DCs,and induce CTLs to produce stronger targeted killing ability for LUAD cells at an effector-to-target ratio of 40:1.ConclusionThese results demonstrated that the elevation of FANCA promotes malignant phenotype of LUAD,and the potential peptide P2 (SLLEFAQYL) derived from FANCA may be used as an epitope vaccine for the treatment of LUAD. View Publication

IntroductionCytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However,the mechanism of CRS induced by SARS-CoV-2 is vague.MethodsUsing spike protein combined with IL-2,IFN-γ,and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines,the content of cytokines in the supernatant was detected,and the effects of NK,T,and monocytes were analyzed.ResultsThis study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1β,IL-6,and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ),as well as T cells to release IFN-γ Mechanistically,IFN-γ and TNF-α enhance the transcription of CD40,and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result,there is a constant interaction between spike protein and TLR4,leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore,TNF-α also activates NF-κB signaling in monocytes,which further cooperates with IFN-γ and spike protein to modulate NF-κB–dependent transcription of CRS-related inflammatory cytokines.DiscussionTargeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19. View Publication

Mature erythrocytes are known to lack major histocompatibility complex (MHC) proteins. However,the presence of MHC molecules on erythrocytes has been occasionally reported,though without a defined function. In this study,we designed erythrocyte conjugated solely with a fusion protein consisting of an antigenic peptide linked to MHC class I (MHC-I) protein,termed MHC-I‒Ery. The modified erythrocyte,decorated with the peptide derived from human papillomavirus (HPV) 16 oncoprotein E6/E7,effectively activated antigen-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from HPV16+ cervical cancer patients. Additionally,MHC-I‒Ery monotherapy was shown to inhibit antigen-positive tumor growth in mice. This treatment immediately activated CD8+ T cells and reduced suppressive myeloid cells in the spleen,leading to systemic anti-tumor activity. Safety and tolerability evaluations of MHC-I‒Ery in non-human primates further supported its clinical potential. Our results first demonstrated that erythrocytes equipped solely with antigen peptide‒MHC-I complexes can robustly stimulate the immune system,suggesting a novel and promising approach for advancing cancer immunotherapy. View Publication

Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells,but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited,especially from widely-used 3′-barcoded libraries. Here,we report a method that can recover paired,full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate,full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys,indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination. A method that recovers full-length,paired heavy- and light-chain variable regions of B cell receptor transcripts from 3’barcoded scRNA-seq libraries reveals a convergent response to pneumococcus vaccination in rhesus macaques. View Publication

A well-balanced maternal immune system is crucial to maintain fetal tolerance in case of infections during pregnancy. Immune adaptations include an increased secretion of soluble mediators to protect the semi-allogeneic fetus from excessive pro-inflammatory response. B lymphocytes acquire a higher capacity to express CD83 and secrete soluble CD83 (sCD83) upon exposure to bacteria-derived components such as LPS. CD83 possesses immune modulatory functions and shows a promising therapeutic potential against inflammatory conditions. The administration of sCD83 to pregnant mice reduces LPS-induced abortion rates. The increased CD83 expression by endometrial B cells as compared to peripheral blood B cells suggests its modulatory role in the fetal tolerance,especially in the context of infection. We postulate that in pregnancy,CD83 expression and release is controlled by pregnancy-related hormones. The intra- and extracellular expression of CD83 in leukocytes from peripheral blood or decidua basalis and parietalis at term were analyzed by flow cytometry. After treatment with pregnancy-related hormones and LPS,ELISA and qPCR were performed to study sCD83 release and CD83 gene expression,respectively. Cleavage prediction analysis was used to find potential proteases targeting CD83. Expression of selected proteases was analyzed by ELISA. Higher levels of CD83 were found in CD11c+ dendritic cells,CD3+ T cells and CD19+ B cells from decidua basalis and decidua parietalis after LPS-stimulation in vitro. An increase of intracellular expression of CD83 was also detected in CD19+ B cells from both compartments. Stimulated B cells displayed significantly higher percentages of CD83+ cells than dendritic cells and T cells from decidua basalis and peripheral blood. Treatment of B lymphocytes with pregnancy-related molecules (E2,P4,TGF-β1 and hCG) enhanced the LPS-mediated increase of CD83 expression,while dexamethasone led to a reduction. Similarly,the release of sCD83 was increased under TGF-β1 treatment but decreased upon dexamethasone stimulation. Finally,we found that the hormonal regulation of CD83 expression is likely a result from a balance between gene transcription from CD83 and the modulation of the metalloproteinase MMP-7. Thus,data supports and complements our previous murine studies on hormonal regulation of CD83 expression,reinforcing its immunomodulatory relevance in anti-bacterial responses during pregnancy. View Publication