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Detection and characterization of rare circulating tumor cells (CTCs) in patients' blood is important for the diagnosis and monitoring of cancer. The traditional way of counting CTCs via fluorescent images requires a series of tedious experimental procedures and often impacts the viability of cells. Here we present a method for label-free detection of CTCs from patient blood samples, by taking advantage of data analysis of bright field microscopy images. The approach uses the convolutional neural network, a powerful image classification and machine learning algorithm to perform label-free classification of cells detected in microscopic images of patient blood samples containing white blood cells and CTCs. It requires minimal data pre-processing and has an easy experimental setup. Through our experiments, we show that our method can achieve high accuracy on the identification of rare CTCs without the need for advanced devices or expert users, thus providing a faster and simpler way for counting and identifying CTCs. With more data becoming available in the future, the machine learning model can be further improved and can serve as an accurate and easy-to-use tool for CTC analysis.

Lymphatic endothelial cells (LECs) chemoattract na{\{i}}ve T cells and promote their survival in the lymph nodes and can cross-present antigens to na{\"{i}}ve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However the functional consequence of LEC priming of CD8+ T cells is unknown. Here we show that while many proliferating LEC-educated T cells enter early apoptosis the remainders comprise a long-lived memory subset with transcriptional metabolic and phenotypic features of central memory and stem cell-like memory T cells. In vivo these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge."""

Type III interferon (interferon lambda [IFN-$\lambda$]) is known to be a potential immune modulator, but the mechanisms behind its immune-modulatory functions and its impact on plasmablast differentiation in humans remain unknown. Human B cells and their subtypes directly respond to IFN-$\lambda$. Using B cell transcriptome profiling, we investigate the immune-modulatory role of IFN-$\lambda$ in B cells. We find that IFN-$\lambda$-induced gene expression in B cells is steady, prolonged, and importantly, cell type specific. Furthermore, IFN-$\lambda$ enhances the mTORC1 (mammalian/mechanistic target of rapamycin complex 1) pathway in B cells activated by the B cell receptor (BCR/anti-IgM). Engagement of mTORC1 by BCR and IFN-$\lambda$ induces cell-cycle progress in B cells. Subsequently, IFN-$\lambda$ boosts the differentiation of naive B cells into plasmablasts upon activation, and the cells gain effector functions such as cytokine release (IL-6 and IL-10) and antibody production. Our study shows how IFN-$\lambda$ systematically boosts the differentiation of naive B cells into plasmablasts by enhancing the mTORC1 pathway and cell-cycle progression in activated B cells.

Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2, such as TEPP-46 and DASA-58, limit tumorigenesis and inflammation. To understand how these compounds alter T cell function, we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology, in part, through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition, we found that activation of PKM2 interfered with TGF-$\beta$1 signaling, which is necessary for the development of TH17 and regulatory T cells. Collectively, our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function.

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by defects in the NADPH oxidase complex. Mutations in NCF2 encoding the cytosolic factor p67phox result in autosomal recessive CGD. We describe three patients with a novel c.855G{\textgreater}C NCF2 mutation presenting with diverse clinical phenotype. Two siblings were heterozygous for the novel mutation and for a previously described exon 8-9 duplication, while a third unrelated patient was homozygous for the novel mutation. Mutation pathogenicity was confirmed by abnormal DHR123 assay and absent p67phox production and by sequencing of cDNA which showed abnormal RNA splicing. Clinically, the homozygous patient presented with suspected early onset interstitial lung disease and NCF2 mutation was found on genetic testing performed in search for surfactant-related defects. The two siblings also had variable presentation with one having history of severe pneumonia, lymphadenitis, and recurrent skin abscesses and the other presenting in his 30s with discoid lupus erythematosus and without significant infectious history. We therefore identified a novel pathogenic NCF2 mutation causing diverse and unusual clinical phenotype.

Immune checkpoint blockade therapies have shown clinical promise in a variety of cancers, but how tumor-infiltrating T cells are activated remains unclear. In this study, we explore the functions of PD-L1 on dendritic cells (DCs), which highly express PD-L1. We observe that PD-L1 on DC plays a critical role in limiting T cell responses. Type 1 conventional DCs are essential for PD-L1 blockade and they upregulate PD-L1 upon antigen uptake. Upregulation of PD-L1 on DC is mediated by type II interferon. While DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, subsequent PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Blocking PD-L1 in established tumors promotes re-activation of tumor-infiltrating T cells for tumor control. Our study identifies a critical and dynamic role of PD-L1 on DC, which needs to be harnessed for better invigoration of antitumor immune responses.

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P {\textless} .0001) and death (P {\textless} .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

B cell acute lymphoblastic leukemia (B-ALL) blasts hijack the bone marrow (BM) microenvironment to form chemoprotective leukemic BM niches facilitating chemoresistance and, ultimately, disease relapse. However, the ability to dissect these evolving, heterogeneous interactions among distinct B-ALL subtypes and their varying BM niches is limited with current in vivo methods. Here, we demonstrated an in vitro organotypic leukemia-on-a-chip" model to emulate the in vivo B-ALL BM pathology and comparatively studied the spatial and genetic heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. We revealed the heterogeneous chemoresistance mechanisms across various B-ALL cell lines and patient-derived samples. We showed that the leukemic perivascular endosteal and hematopoietic niche-derived factors maintain B-ALL survival and quiescence (e.g. CXCL12 cytokine signal VCAM-1/OPN adhesive signals and enhanced downstream leukemia-intrinsic NF-$\kappa$B pathway). Furthermore we demonstrated the preclinical use of our model to test niche-cotargeting regimens which may translate to patient-specific therapy screening and response prediction."

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ T cells, and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.