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Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations1, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality2,3. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by CCL4 (MIP-1β) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1β and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.

Human behavior as they engaged in financial activities is intimately connected to the observed market dynamics. Despite many existing theories and studies on the fundamental motivations of the behavior of humans in financial systems, there is still limited empirical deduction of the behavioral compositions of the financial agents from a detailed market analysis. Blockchain technology has provided an avenue for the latter investigation with its voluminous data and its transparency of financial transactions. It has enabled us to perform empirical inference on the behavioral patterns of users in the market, which we explore in the bitcoin and ethereum cryptocurrency markets. In our study, we first determine various properties of the bitcoin and ethereum users by a temporal complex network analysis. After which, we develop methodology by combining k-means clustering and Support Vector Machines to derive behavioral types of users in the two cryptocurrency markets. Interestingly, we found four distinct strategies that are common in both markets: optimists, pessimists, positive traders and negative traders. The composition of user behavior is remarkably different between the bitcoin and ethereum market during periods of local price fluctuations and large systemic events. We observe that bitcoin (ethereum) users tend to take a short-term (long-term) view of the market during the local events. For the large systemic events, ethereum (bitcoin) users are found to consistently display a greater sense of pessimism (optimism) towards the future of the market.

Current immunotherapy paradigms aim to reinvigorate CD8+ T cells, but the contribution of humoral immunity to antitumor immunity remains understudied. Here, we demonstrate that in head and neck squamous cell carcinoma (HNSCC) caused by human papillomavirus infection (HPV+), patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with tertiary lymphoid structures (TLS) with GCs, both of which correlate with favorable outcome. GC TIL-Bs in HPV+ HNSCC are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. Semaphorin 4a expression is enhanced on GC TIL-Bs present in TLS of HPV+ HNSCC and during the differentiation of TIL-Bs. Our study suggests that therapeutics to enhance TIL-B responses in HNSCC should be prioritized in future studies to determine if they can complement current T cell mediated immunotherapies., Recent studies have highlighted the importance of B cells and tertiary lymphoid structures (TLS) in the modulation of anti-tumor immune responses. Here, the authors characterize how HPV status influences the phenotype of tumor infiltrating B cells in patients with head and neck squamous cell carcinoma and demonstrate that TLS with germinal centres are associated with better survival.

Background Current FDA-approved immunotherapies aim to reinvigorate CD8+ T cells, but the contribution of the humoral arm of the immune response in human cancer remains poorly understood. B cells within tissues can mediate anti-tumor immunity and regulate immune responses by presenting antigen and producing tumor-specific antibodies and immunomodulatory cytokines. Head and neck squamous cell carcinoma (HNSCC) can be induced by human papillomavirus (HPV+) and carcinogens such as tobacco and alcohol (HPV-), and the immune infiltrate is quite distinct in the two etiologies, in particular, increased B cells in HPV+ HNSCC patients. Further, increased B cells in HNSCC patients correlate with improved patient survival. Our study seeks to differentiate B cell phenotype, function and location in HPV+ and HPV- HNSCC to identify putative B cell-centric immunotherapeutic targets. Methods We utilized a multi-level approach to clearly categorize B cells in HNSCC patients. Single cell RNA sequencing (scRNAseq) was performed on CD45+ tumor infiltrating lymphocytes (TIL) from HPV+ and HPV- HNSCC patients. HNSCC TIL and PBL were stained via spectral cytometry (Cytek Aurora,25 parameters) for unbiased analysis of B cell subsets via computational spectral unmixing. Paraffin embedded slides from HNSCC primary tumors were utilized for multispectral immunofluorescence (mIF) to identify tertiary lymphoid structures (TLS) and identify differences in HPV+ and HPV- disease. Results We demonstrated distinct trajectories for B cells in HPV+ and HPV- disease. HPV- HNSCC tumors mainly contained memory B cells and plasma cells, while the B cells in HPV+ HNSCC were naive and germinal center (GC). Further, we quantified B cells and CD4+ T cells in TLS, and germinal center-like TLS were associated with improved outcome in HPV+ disease. We also observed that transcriptional and protein expression of Semaphorin A (SEMA4a) was restricted to GC B cells and increased on GC B cells in HNSCC patients compared to healthy tonsils. Additionally, we identified distinct waves of gene expression in GC B cells in HNSCC tumors, ultimately revealing a novel transitional state for GC B cells in the tumor microenvironment (TME). Conclusions Understanding B cell function in human cancers and how different TMEs influence B cells and TLS are important for devising novel therapeutic options for cancer patients. Ultimately, development of therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.

While T cell-based cancer immunotherapies have shown great promise, there remains a need to understand how individual metastatic tumor environments impart local T cell dysfunction. At advanced stages, cancers that metastasize to the pleural space can result in a malignant pleural effusion (MPE) that harbors abundant tumor and immune cells, often exceeding 108 leukocytes per liter. Unlike other metastatic sites, MPEs are readily and repeatedly accessible via indwelling catheters, providing an opportunity to study the interface between tumor dynamics and immunity. In the current study, we examined CD8+ T cells within MPEs collected from patients with heterogeneous primary tumors and at various stages in treatment to determine (1) if these cells possess anti-tumor activity following removal from the MPE, (2) factors in the MPE that may contribute to their dysfunction, and (3) the phenotypic changes in T cell populations that occur following ex vivo expansion. Co-cultures of CD8+ T cells with autologous CD45― tumor containing cells demonstrated cytotoxicity (p = 0.030) and IFN&gamma, production (p = 0.003) that inversely correlated with percent of myeloid derived suppressor cells, lactate, and lactate dehydrogenase (LDH) within the MPE. Ex vivo expansion of CD8+ T cells resulted in progressive differentiation marked by distinct populations expressing decreased CD45RA, CCR7, CD127, and increased inhibitory receptors. These findings suggest that MPEs may be a source of tumor-reactive T cells and that the cellular and acellular components suppress optimal function.

Current immunotherapy paradigms aim to reinvigorate CD8+T cells, but the contribution of humoral immunity to antitumor immunity remains understudied1,2. Head and neck squamous cell carcinoma (HNSCC) is caused by either human papillomavirus (HPV+) or environmental carcinogens (i.e. tobacco and alcohol; HPV–)3,4. Here, we demonstrate that HPV+ HNSCC patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with GC-like tertiary lymphoid structures (TLS), both of which correlate with favorable outcomes in HNSCC patients. Further, our single-cell RNAseq data also indicate that GC TIL-Bs are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. High-dimensional spectral flow cytometry permitted in depth characterization of activated, memory and GC TIL-Bs. Further, single cell RNAseq analysis and subsequent protein validation identified a role for semaphorin 4a (Sema4a) in the differentiation of GC TIL-Bs and indicated that expression of Sema4a was enhanced on GC TIL-Bs and within GC-like TLS in the TME. Thus, in contrast to some reports on the detrimental role of TIL-Bs in human tumors, our findings suggest that TIL-Bs play an instrumental role in antitumor immunity5,6. Novel therapeutics to enhance TIL-B responses in HNSCC should be prioritized as a compliment to current T-cell mediated immunotherapies.

3D In painting is a method for changing a 2D picture over to a3D photograph and saving the harmed, debilitating, or missing regions of the photograph by filling it into a perfect image. The principal issue in this cycle happens when making the profundity map, recognizing the missing segments, filling the missing bits utilizing fitting filling techniques. These issues are fundamental and regular in 3D photograph in painting procedures. So the fundamental point of this review is recognizing each progression in 3D in painting procedures and distinguishing their disappointment cases. Numerous strategies utilize different pictures as information and numerous techniques utilize a single picture as input sources.

Bitcoin is the earliest cryptocurrency and among the most successful ones to date. Recently, its dynamical evolution has attracted the attention of the research community due to its completeness and richness in historical records. In this paper, we focus on the detailed evolution of bitcoin trading with the aim of elucidating the mechanism that drives the formation of the bitcoin transaction network. Our empirical investigation reveals that although the temporal properties of the transaction network possesses scale-free degree distribution like many other networks, its formation mechanism is different from the commonly assumed models of degree preferential attachment or wealth preferential attachment. By defining the fitness value of each node as the ability of the node to attract new connections, we have instead uncovered that the observed scale-free degree distribution results from the intrinsic fitness of each node following a power-law distribution. Our finding thus suggests that the “good-get-richer” rather than the “rich-get-richer” paradigm operates within the bitcoin ecosystem. Based on these findings, we propose a model that captures the temporal generative process by means of a fitness preferential attachment and data-driven birth/death mechanism. Our proposed model is able to produce structural properties in good agreement with those obtained from the empirical bitcoin network. Published version

Blockade of the inhibitory PD1 pathway on CD8+ and CD4+ tumor infiltrating lymphocytes (TILs) has revolutionized standard of care for cancer patients. However, these approaches only benefit approximately 20% of cancer patients, thus a more comprehensive understanding of the immune response is paramount for the development of new therapeutic approaches and enhancement of the anti-tumor immune response. The presence of tertiary lymphoid structures (TLS) correlates with enhanced anti-tumor immunity and improved prognosis in several solid tumors. These ectopic lymphoid aggregates can exhibit features like secondary lymphoid organs (SLOs), including high endothelial venules, a T cell zone with mature dendritic cells (DCs), and a germinal center (GC) with follicular DCs and B cells, that facilitate the induction of immune responses in situ. Furthermore, the presence of TLS correlate with superior responses to immunotherapy and mature, GC-containing TLS correlate with increased T cell function in human tumors. However, current immunotherapies do not target TLS despite their predominance in the tumor microenvironment (TME) and key role in the adaptive immune response. Developing therapies that adequately induce TLS is limited by a lack of objective, bioinformatic analyses to elucidate the cellular and locational heterogeneity of TLS. TLS differences in composition are thought to define maturation levels i.e. early TLS have B and T cell aggregates but lack follicular DC and GC appear as the TLS mature. However, cellular composition is just one aspect of TLS heterogeneity. To successfully increase mature TLS in cancer patients for maximal humoral immunity, we must first understand the complete transcriptomic and proteomic profile of TLS in cancer patients. We have utilized the innovative Nanostring GeoMx platform to spatially interrogate the transcriptomics and proteomics associated with TLS. By pairing this technology with high level multispectral immunofluorescence via the Vectra Polaris system, we can link TLS heterogeneity to TLS position within the tumor. Further, we can analyze the complete activation profile of B and T cells to understand how TLS heterogeneity affects lymphocyte function. Our data have indicated that TLS can be linked to increased CD8+ T cell infiltrate and function. In fact, when mature TLS are not present, CD8+ T cell and Treg interactions increase, ultimately creating a more immunosuppressive TME. Lastly, utilizing a novel, robust bioinformatic pipeline, we have identified new genes that will aid in an objective definition of TLS. Specifically, CD27 is increased within TLS that are proximal to the tumor compared to those in the normal adjacent tissue. These studies will revolutionize the way in which TLS are defined within the TME, ultimately offering comprehensive gene and protein analysis of TLS that includes spatial geography of the structures and an unbiased, objective definition of TLS for future clinical utility and immunotherapeutic targeting. Citation Format: Dongyan Liu, Xiang Li, Rajesh Acharya, Ernest M. Meyer, Shelley Reynolds, Ayana Ruffin, Robert L. Ferris, Dario A.A. Vignali, Riyue Bao, Tullia C. Bruno. Utilizing spatial transcriptomics to elucidate tertiary lymphoid structure heterogeneity in human cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-083.

Immunotherapy, specifically anti-PD1, has improved patient survival in a range of tumor types including head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC). Despite the success of anti-PD1 therapy, only 20% of patients produce a durable response to this treatment. Thus, a need exists to develop additional therapeutic strategies to treat these patients, which includes evaluation of other tumor-infiltrating immune cells that could further augment the CD8+ and CD4+ T-cell response. Tumor-infiltrating B cells (TIL-B) represent a possible target for immunotherapy due to their predominance in the tumor microenvironment (TME) and crucial role in the immune response. However, TIL-B function in cancer and in the context of immunotherapy has been understudied. In fact, conclusions on an anti- or protumor role for TIL-Bs in the TME is dependent on the study. However, in HNSCC and NSCLC patients, current evidence suggests an antitumor role for TIL-Bs. Specifically, detection of TIL-Bs within tertiary lymphoid structures (TLS) correlates with better prognosis. While TIL-Bs have been identified in HNSCC and NSCLC patients, their complete phenotypic signature and function in the TME has been understudied with no focus on their role as antigen presenting cells (APCs) and their influence on CD8+ and CD4+ tumor infiltrating lymphocytes (TILs). We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs within TLS.Using unmanipulated, primary human B cells from fresh tumor, we quantified and further characterized TIL-Bs in HNSCC and NSCLC utilizing single-cell RNAseq and multiparameter flow cytometry. We observed increased numbers of activated TIL-Bs in these primary tumors compared to other immune subsets, specifically CD27+ TIL-Bs. We further assessed the TIL-Bs by correlating phenotype of the TIL-B with its location in the TME, predominantly separating out differences between TIL-Bs within TLS and outside TLS. In addition, we generated a specific antigen presentation assay in vitro, and we observed three types of CD4+ TIL responses when TIL-Bs presented autologous tumor antigens. There were activated responder CD4+ TILs that proliferated when combined with TIL-Bs alone, which indicates stimulation with endogenous tumor antigens. There were antigen-associated responders that required exogenous autologous tumor lysate to elicit a CD4+ TIL response, and there were patient CD4 TILs that did not respond to antigen presentation by TIL-Bs. Within the activated and antigen-associated responders, the TIL-B phenotype influenced the CD4+ TIL phenotype; if the TIL-Bs were activated (CD27+), the CD4+ TILs were T helper (antitumor) CD4+ T-cells and if the TIL-Bs were non-activated (CD27-), the CD4+ TILs were T regulatory cells (protumor). These data suggest that TIL-Bs influence the phenotype and function of CD4+ TILs in patient tumors. In conclusion, activated TIL-Bs are increased in human primary tumors, they can present antigen to CD4+ TILs and influence their overall phenotype. Determining the complete activation signature of TIL-Bs in HNSCC and NSCLC patients will determine the extent of their antitumor function in these cancers. Comparison of TIL-Bs in HNSCC and NSCLC is important as there are not many unified studies on TIL-B function across tumor types. Further, because HNSCC has two etiologies (viral vs. carcinogen induced), we are able to better study the differential function of activated and nonactivated TIL-Bs in solid tumors. Ultimately, results from this study will help predict how to target TIL-B functions in future TIL-B-specific immunotherapies or in combination with current immunotherapies for HNSCC and NSCLC patients like blockade of the inhibitory receptor, PD-1. Citation Format: Tullia C. Bruno, Ayana T. Ruffin, Anthony R. Cillo, Robert L. Ferris, Dario A.A. Vignali. Activated B cells in human primary tumors present antigen and increase antitumor function of CD4 T-cells in tertiary lymphoid structures [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A053.