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Background: Immunotherapy provided significant survival benefits for recurrent and metastatic patients with head and neck cancer. These improvements could not be reproduced in patients treated with curative-intent chemoradiotherapy (CRT) and the optimal radio-immunotherapy (RIT) concepts have yet to be designed. Exploration and analysis of the pre-therapeutic immune status of these patients and the changes occurring during the treatment course could be crucial in rationally designing future combined treatments. Methods: Blood samples were collected from a cohort of 25 head and neck cancer patients treated with curative-intended (C)-RT prior to therapy, after the first week of treatment, and three months after treatment completion. Peripheral blood mononuclear cells (PBMCs) or all nucleated blood cells were isolated and analyzed via flow cytometry. Results: At baseline, patients showed reduced monocyte and lymphocyte counts compared to healthy individuals. Although overall CD8+ T-cell frequencies were reduced, the proportion of memory subsets were increased in patients. Radiotherapy (RT) treatment led to a further increase in CD8+ effector memory T-cells. Among myeloid populations, tumor-promoting subsets became less abundant after RT, in favor of pro-inflammatory cells. Conclusion: The present study prospectively demonstrated a complex interplay and distinct longitudinal changes in the composition of lymphocytic and myeloid populations during curative (C)-RT of head and neck cancer. Further validation of this method in a larger cohort could allow for better treatment guidance and tailored incorporation of immunotherapies (IT) in the future. [ABSTRACT FROM AUTHOR]

Phelan–McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by a developmental delay and autism spectrum disorder (ASD)-like behaviors. Emerging research suggests a link between gut microbiota and neuropsychiatric conditions, including PMS. This study aimed to investigate the fecal microbiota and immune profiles of children with PMS compared to healthy controls. Fecal samples were collected from children diagnosed with PMS and age-matched healthy controls. The bacterial composition was analyzed using 16S rRNA gene sequencing, while short-chain fatty acids (SCFAs) were quantified through gas chromatography. Immunological profiling was conducted using a multiplex cytokine assay. Significant differences were observed in the gut microbiota composition between PMS patients and controls, including a lower abundance of key bacterial genera such as Faecalibacterium and Agathobacter in PMS patients. SCFA levels were also reduced in PMS patients. Immunological analysis revealed higher levels of several pro-inflammatory cytokines in the PMS group, although these differences were not statistically significant. The findings indicate that children with PMS have distinct gut microbiota and SCFA profiles, which may contribute to the gastrointestinal and neurodevelopmental symptoms observed in this syndrome. These results suggest potential avenues for microbiota-targeted therapies in PMS. [ABSTRACT FROM AUTHOR]

Simple Summary: Salivary duct carcinomas (SDC) can present with distinct immunologic profiles similar to breast cancer, such as androgen receptor (AR) and HER-2/Neu-positivity, raising the hypothesis that these tumors may respond to hormonal signaling. No consensus exists on how to best manage this entity. The data evaluating the use of targeted therapies, such as androgen deprivation therapy and HER-2 receptor inhibitors, in the front-line setting when treating curatively is limited. We studied patients with AR+ SDC, demonstrating high rates of control and survival using an aggressive approach to treatment. Immunoprofiles were highly variable, highlighting the potential for future treatment individualization. We hope that this may allow for personalization of treatment in the future, using molecular profiling to determine whether the addition of biological agents in the definitive setting against specific targets, such as AR and HER-2/Neu, will further improve outcomes for these patients. Background: Salivary duct carcinomas (SDC) are a rare and aggressive subtype of salivary gland neoplasm. They can present with distinct immunoprofiles, such as androgen receptor (AR) and HER-2/Neu-positivity. To date, no consensus exists on how to best manage this entity. Methods: All patients diagnosed with nonmetastatic AR+ SDC of the parotid from 2013 to 2019 treated with curative intent were included. Immunologic tumor profiling was conducted using 24 distinct markers. Kaplan–Meier analyses were used to estimate locoregional recurrence (LRR), distant control, and overall survival (OS). Results: Fifteen patients were included. Nine (60%) patients presented with T4 disease and eight (53%) had positive ipsilateral cervical lymphadenopathy. Ten (67%) patients underwent trimodality therapy, including surgery followed by adjuvant radiation and concurrent systemic therapy. The median follow-up was 5.5 years (interquartile range, 4.8–6.1). The estimated 5-year rates of LRR, distant progression, and OS were 6%, 13%, and 87%, respectively. Conclusion: Despite only including AR+ SDC of the parotid, immunoprofiles, such as expression of HER-2, were highly variable, highlighting the potential to tailor systemic regimens based on individual histologic profiles in the future. Studies with larger patient numbers using tumor-specific molecular profiling and tumor heterogeneity analyses are justified to better understand the biology of these tumors. Molecularly informed treatment approaches, including the potential use of AR- and HER-2/Neu-directed therapies upfront in the definitive setting, may hold future promise to further improve outcomes for these patients. [ABSTRACT FROM AUTHOR]

Phelan–McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by a developmental delay and autism spectrum disorder (ASD)-like behaviors. Emerging research suggests a link between gut microbiota and neuropsychiatric conditions, including PMS. This study aimed to investigate the fecal microbiota and immune profiles of children with PMS compared to healthy controls. Fecal samples were collected from children diagnosed with PMS and age-matched healthy controls. The bacterial composition was analyzed using 16S rRNA gene sequencing, while short-chain fatty acids (SCFAs) were quantified through gas chromatography. Immunological profiling was conducted using a multiplex cytokine assay. Significant differences were observed in the gut microbiota composition between PMS patients and controls, including a lower abundance of key bacterial genera such as Faecalibacterium and Agathobacter in PMS patients. SCFA levels were also reduced in PMS patients. Immunological analysis revealed higher levels of several pro-inflammatory cytokines in the PMS group, although these differences were not statistically significant. The findings indicate that children with PMS have distinct gut microbiota and SCFA profiles, which may contribute to the gastrointestinal and neurodevelopmental symptoms observed in this syndrome. These results suggest potential avenues for microbiota-targeted therapies in PMS.

Brucellosis remains a worldwide zoonotic disease with a serious impact on public health and livestock productivity. Controlling brucellosis in livestock is crucial for limiting human infections in the absence of effective human vaccines. Brucellosis control measures are majorly dependent on rigorous monitoring of disease outbreaks and mass vaccination of livestock. Live attenuated vaccines are available for livestock vaccination that play a vital role in brucellosis control programs in many countries. Even though the existing animal vaccines confer protection against brucellosis, they carry some drawbacks, including their infectivity to humans and interference with sero-monitoring. The available serodiagnostic assays for brucellosis depend on detecting anti-LPS antibodies in the serum. Since diagnosis plays a vital role in controlling brucellosis, developing improved serodiagnostic assays with enhanced specificity, sensitivity and DIVA capability is required. Therefore, it is essential to identify novel antigens for developing improved vaccines and serodiagnostic assays for brucellosis. In the present study, we performed a high throughput immunoprofiling of B. melitensis protein microarray using brucellosis-positive human and animal serum samples. The screening identified several serodominant proteins of Brucella that exhibited common or differential reactivity with sera from animals and humans. Subsequently, we cloned, expressed, and purified ten serodominant proteins, followed by analyzing their potential to develop next-generation vaccines and improved serodiagnostic assays for brucellosis. Further, we demonstrated the protective efficacy of one of the serodominant proteins against the B. melitensis challenge in mice. We found that the seroreactive protein, Dps (BMEI1980), strongly reacted with brucellosis-positive serum samples, but it did not react with sera from B. abortus S19-vaccinated cattle, indicating DIVA capability. A prototype lateral flow assay and indirect ELISA based on Dps protein exhibited high sensitivity, specificity, and DIVA capability. Thus, the present study identified promising candidates for developing improved vaccines and affordable, DIVAcapable serodiagnostic assays for animal and human brucellosis. [ABSTRACT FROM AUTHOR]

Abstract Background Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by development of autoantibodies and multiorgan involvement. Kidney involvement, termed lupus nephritis, has major impact on life expectancy. It is increasingly recognized that SLE is likely a common clinical manifestation of pathophysiologically diverse processes, and lupus nephritis has similarly been associated with several distinct immunological processes. We compared the immune cell phenotypes of individuals with SLE in the presence or absence of nephritis. Methods Cryopreserved peripheral blood mononuclear cells from SLE patients with and without kidney involvement underwent flow cytometric analysis to identify major populations in T cells, B cells and myeloid lineages. Results We compared the frequencies of lymphocyte populations in 69 SLE patients without nephritis, 20 SLE patients with nephritis, and 92 healthy blood donors. Patients with SLE and lupus nephritis (LN) had reduced marginal zone B cells (P

Brucellosis remains a worldwide zoonotic disease with a serious impact on public health and livestock productivity. Controlling brucellosis in livestock is crucial for limiting human infections in the absence of effective human vaccines. Brucellosis control measures are majorly dependent on rigorous monitoring of disease outbreaks and mass vaccination of livestock. Live attenuated vaccines are available for livestock vaccination that play a vital role in brucellosis control programs in many countries. Even though the existing animal vaccines confer protection against brucellosis, they carry some drawbacks, including their infectivity to humans and interference with sero-monitoring. The available serodiagnostic assays for brucellosis depend on detecting anti-LPS antibodies in the serum. Since diagnosis plays a vital role in controlling brucellosis, developing improved serodiagnostic assays with enhanced specificity, sensitivity and DIVA capability is required. Therefore, it is essential to identify novel antigens for developing improved vaccines and serodiagnostic assays for brucellosis. In the present study, we performed a high throughput immunoprofiling of B. melitensis protein microarray using brucellosis-positive human and animal serum samples. The screening identified several serodominant proteins of Brucella that exhibited common or differential reactivity with sera from animals and humans. Subsequently, we cloned, expressed, and purified ten serodominant proteins, followed by analyzing their potential to develop next-generation vaccines and improved serodiagnostic assays for brucellosis. Further, we demonstrated the protective efficacy of one of the serodominant proteins against the B. melitensis challenge in mice. We found that the seroreactive protein, Dps (BMEI1980), strongly reacted with brucellosis-positive serum samples, but it did not react with sera from B. abortus S19-vaccinated cattle, indicating DIVA capability. A prototype lateral flow assay and indirect ELISA based on Dps protein exhibited high sensitivity, specificity, and DIVA capability. Thus, the present study identified promising candidates for developing improved vaccines and affordable, DIVA-capable serodiagnostic assays for animal and human brucellosis.

Abstract Background Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease with a heterogenous clinical presentation and unpredictable response to available therapies. This personalized transcriptomics study sought proof-of-concept for single-cell RNA sequencing to characterize patient-specific immune profiles. Methods Whole blood samples from six untreated children, newly diagnosed with JIA, and two healthy controls were cultured for 24 h with or without ex vivo TNF stimulation and subjected to scRNAseq to examine cellular populations and transcript expression in PBMCs. A novel analytical pipeline, scPool, was developed wherein cells are first pooled into pseudocells prior to expression analysis, facilitating variance partitioning of the effects of TNF stimulus, JIA disease status, and individual donor. Results Seventeen robust immune cell-types were identified, the abundance of which was significantly affected by TNF stimulus, which resulted in notable elevation of memory CD8 + T-cells and NK56 cells, but down-regulation of naïve B-cell proportions. Memory CD8 + and CD4 + T-cells were also both reduced in the JIA cases relative to two controls. Significant differential expression responses to TNF stimulus were also characterized, with monocytes showing more transcriptional shifts than T-lymphocyte subsets, while the B-cell response was more limited. We also show that donor variability exceeds the small degree of possible intrinsic differentiation between JIA and control profiles. An incidental finding of interest was association of HLA-DQA2 and HLA-DRB5 expression with JIA status. Conclusions These results support the development of personalized immune-profiling combined with ex-vivo immune stimulation for evaluation of patient-specific modes of immune cell activity in autoimmune rheumatic disease.

Simple Summary: Currently, multiple myeloma is mostly an incurable disease. Given the poor survival and frequent disease relapses, there is an urgent need for new treatment approaches and patient stratification. In an attempt to predict response to treatment, we comprehensively analyzed the immune profiles of longitudinal bone marrow samples collected from 18 multiple myeloma patients, separating them into good and poor responders to therapy. We identified several distinctive features that might have an influence on the therapy response, such as the abundance of tumor cells and the functional state of T and NK cells. These results suggest that deep immune profiling could be used for the guidance of treatment. The bone marrow microenvironment interacts with malignant cells and regulates cancer survival and immune evasion in multiple myeloma (MM). We investigated the immune profiles of longitudinal bone marrow samples from patients with newly diagnosed MM (n = 18) using cytometry by time-of-flight. The results before and during treatment were compared between patients with good (GR, n = 11) and bad (BR, n = 7) responses to lenalidomide/bortezomib/dexamethasone-based treatment. Before treatment, the GR group had a lower tumor cell burden and a higher number of T cells with a phenotype shifted toward CD8+ T cells expressing markers attributed to cytotoxicity (CD45RA and CD57), a higher abundance of CD8+ terminal effector cells, and a lower abundance of CD8+ naïve T cells. On natural killer (NK) cells, increased expression of CD56 (NCAM), CD57, and CD16 was seen at baseline in the GR group, indicating their maturation and cytotoxic potential. During lenalidomide-based treatment, the GR patients showed an increase in effector memory CD4+ and CD8+ T-cell subsets. These findings support distinct immune patterns in different clinical contexts, suggesting that deep immune profiling could be used for treatment guidance and warrants further exploration. [ABSTRACT FROM AUTHOR]

Adjuvants are critical components of vaccines that enhance the host immune response to the vaccine antigen, however, only a small number of adjuvants are used in vaccines approved for human use. This is in part due to the slow process of novel adjuvants advancing from preclinical models to human studies, and modest mechanistic insights obtained using standard immunological methods to justify selection of a particular adjuvant for clinical evaluation. Here, we discuss several aspects of current adjuvant research and strategies to better assess the complex pathways triggered by adjuvant candidates that can increase adjuvanticity and vaccine efficacy while minimizing reactogenicity. We propose a more systematic use of broad immunoprofiling, coupled with data integration using computational and mathematical modeling. This comprehensive evaluation of the host immune response will facilitate the selection of the most appropriate adjuvant for a vaccine, ultimately leading to the expeditious evaluation of novel adjuvants for vaccines against emerging infectious diseases, which will prove especially valuable during a pandemic where speed is of the essence when developing vaccines.

Immunoprofiling has become a crucial tool for understanding the complex interactions between the immune system and diseases or interventions, such as therapies and vaccinations. Immune response biomarkers are critical for understanding those relationships and potentially developing personalized intervention strategies. Single-cell data have emerged as a promising source for identifying immune response biomarkers. In this review, we discuss the current state-of-the-art methods for immunoprofiling, including those for reducing the dimensionality of high-dimensional single-cell data and methods for clustering, classification, and prediction. We also draw attention to recent developments in data integration.

Background: Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease with a heterogenous clinical presentation and unpredictable response to available therapies. This personalized transcriptomics study sought proof-of-concept for single-cell RNA sequencing to characterize patient-specific immune profiles. Methods: Whole blood samples from six untreated children, newly diagnosed with JIA, and two healthy controls were cultured for 24 h with or without ex vivo TNF stimulation and subjected to scRNAseq to examine cellular populations and transcript expression in PBMCs. A novel analytical pipeline, scPool, was developed wherein cells are first pooled into pseudocells prior to expression analysis, facilitating variance partitioning of the effects of TNF stimulus, JIA disease status, and individual donor. Results: Seventeen robust immune cell-types were identified, the abundance of which was significantly affected by TNF stimulus, which resulted in notable elevation of memory CD8 + T-cells and NK56 cells, but down-regulation of naïve B-cell proportions. Memory CD8 + and CD4 + T-cells were also both reduced in the JIA cases relative to two controls. Significant differential expression responses to TNF stimulus were also characterized, with monocytes showing more transcriptional shifts than T-lymphocyte subsets, while the B-cell response was more limited. We also show that donor variability exceeds the small degree of possible intrinsic differentiation between JIA and control profiles. An incidental finding of interest was association of HLA-DQA2 and HLA-DRB5 expression with JIA status. Conclusions: These results support the development of personalized immune-profiling combined with ex-vivo immune stimulation for evaluation of patient-specific modes of immune cell activity in autoimmune rheumatic disease. [ABSTRACT FROM AUTHOR]

Introduction: SARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants. Methods: We combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19. Results: We observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions. Discussion: We propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NKlike CD8+ T cells in COVID-19 severity. [ABSTRACT FROM AUTHOR]

IntroductionSARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants.MethodsWe combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19.ResultsWe observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions.DiscussionWe propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity.

While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. Single-cell analyses identified CXCR3+CD8+ effector memory T (T EM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ T EM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. NCT03695952. Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events. [Display omitted] • Distinct immune subsets are linked to response and toxicity in immunotherapy. • Antigen-presenting cells and CD8 T EM cell function act as an interface between response and toxicity. • Diverging immune trajectories of immune subsets contribute to either response or toxicity. • TNFR2 inhibition uncouples response and toxicity in anti-PD-1 immunotherapy. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICI) have been applied in treating advanced hepatocellular carcinoma (aHCC) patients, but few patients exhibit stable and lasting responses. Moreover, identifying aHCC patients suitable for ICI treatment is still challenged. This study aimed to evaluate whether dissecting peripheral immune cell subsets by Mann-Whitney U test and artificial intelligence (AI) algorithms could serve as predictive biomarkers of nivolumab treatment for aHCC. Disease control group carried significantly increased percentages of PD-L1+ monocytes, PD-L1+ CD8 T cells, PD-L1+ CD8 NKT cells, and decreased percentages of PD-L1+ CD8 NKT cells via Mann-Whitney U test. By recursive feature elimination method, five featured subsets (CD4 NKTreg, PD-1+ CD8 T cells, PD-1+ CD8 NKT cells, PD-L1+ CD8 T cells and PD-L1+ monocytes) were selected for AI training. The featured subsets were highly overlapping with ones identified via Mann-Whitney U test. Trained AI algorithms committed valuable AUC from 0.8417 to 0.875 to significantly separate disease control group from disease progression group, and SHAP value ranking also revealed PD-L1+ monocytes and PD-L1+ CD8 T cells exclusively and significantly contributed to this discrimination. In summary, the current study demonstrated that integrally analyzing immune cell profiling with AI algorithms could serve as predictive biomarkers of ICI treatment.

The impact of pre-existing immunity on the efficacy of artemisinin combination therapy is largely unknown. We performed in-depth profiling of serological responses in a therapeutic efficacy study [comparing artesunate-mefloquine (ASMQ) and artemether-lumefantrine (AL)] using a proteomic microarray. Responses to over 200 Plasmodium antigens were significantly associated with ASMQ treatment outcome but not AL. We used machine learning to develop predictive models of treatment outcome based on the immunoprofile data. The models predict treatment outcome for ASMQ with high (72–85%) accuracy, but could not predict treatment outcome for AL. This divergent treatment outcome suggests that humoral immunity may synergize with the longer mefloquine half-life to provide a prophylactic effect at 28–42 days post-treatment, which was further supported by simulated pharmacokinetic profiling. Our computational approach and modeling revealed the synergistic effect of pre-existing immunity in patients with drug combination that has an extended efficacy on providing long term treatment efficacy of ASMQ.

The top cause of mortality in patients with nonalcoholic fatty liver disease (NAFLD) is cardiovascular complications. However, mechanisms of NAFLD‐associated vasculopathy remain understudied. Here, we show that blood outgrowth endothelial cells (BOECs) from NAFLD subjects exhibit global transcriptional upregulation of chemokines and human leukocyte antigens. In mouse models of diet‐induced NAFLD, we confirm heightened endothelial expressions of CXCL12 in the aortas and the liver vasculatures, and increased retention of infiltrated leukocytes within the vessel walls. To elucidate endothelial‐immune crosstalk, we performed immunoprofiling by single‐cell analysis, uncovering T cell intensification in NAFLD patients. Functionally, treatment with a CXCL12‐neutralizing antibody is effective at moderating the enhanced chemotactic effect of NAFLD BOECs in recruiting CD8+ T lymphocytes. Interference with the CXCL12‐CXCR4 axis using a CXCR4 antagonist also averts the impact of immune cell transendothelial migration and restores endothelial barrier integrity. Clinically, we detect threefold more circulating damaged endothelial cells in NAFLD patients than in healthy controls. Our work provides insight into the modulation of interactions with effector immune cells to mitigate endothelial injury in NAFLD. SYNOPSIS: Endothelial chemokine activation is evident in non‐alcoholic fatty liver disease patients and mouse diet‐induced disease models. The CXCL12‐CXCR4 axis mediates the interaction of patient endothelial cells with effector immune cells, potentially leading to vascular injury. NAFLD patient‐derived endothelial cells exhibit increased chemokine hallmarks and human leukocyte antigens.Aortas and liver vasculatures of diet‐induced NAFLD mice show increased endothelial CXCL12 and leukocyte infiltration.Inhibition of the CXCL12‐CXCR4 axis diminishes chemotaxis of CD8+ T cells and endothelial barrier impairment.NAFLD patients manifest elevated levels of circulating damaged endothelial cells. [ABSTRACT FROM AUTHOR]

PURPOSE: Primary malignant solitary fibrous tumor (SFT) of the orbit is a rare spindle cell neoplasm that requires excisional biopsy for histopathological diagnosis. We present a clinical case using contemporary immunohistochemical stains, report on the latest World Health Organization classification, and provide a review of the literature. OBSERVATIONS: Report of a single case of a 65 year old male who presented with right-sided proptosis, limited adduction, ptosis, lateral globe displacement, and cheek festooning. Neuroimaging revealed a 2.2 cm, extraconal heterogeneous mass that diffusely enhanced.En-bloc tumor resection confirmed SFT malignancy based upon nuclear atypia, hypercellularity, and increased mitotic activity (13 mitotic figures/10 high powered fields). Ki-67 showed 2% nuclear staining in the benign tumor and 10-15% staining in the malignant counterpart. Immunohistochemical analysis revealed diffuse Stat6 positivity, CD 34 positivity with partial lack of staining within the malignant portion, S-100 positivity in the malignant portion, and overall negativity for CAM 5.2, desmin, actin, CD 31, and CD 117. CONCLUSIONS AND IMPORTANCE: Immunoprofiling is helpful to making the diagnosis of malignant solitary fibrous tumor of the orbit. Complete tumor resection continues to be the preferred treatment. The behavior of extrathoracic SFT is unpredictable, and patients with SFT in all locations require careful, long-term follow-up.

Simple Summary: Immunotherapy has shown clinical benefits in several solid cancers; still, glioblastoma remains very challenging to treat. Glioblastoma is the most frequent brain cancer and displays great heterogeneity. The standard of care has remained the same for over fifteen years, and to overcome the therapeutic limitations, emerging immune correlates of therapy responses and improved prognosis should be further developed for a more personalized therapy approach and increased clinical responses. Immunotherapy has shown clinical benefits in several solid malignancies—in particular, melanoma and non-small cell lung cancer. However, in other solid tumours such as glioblastoma (GBM), the response to immunotherapy has been more variable, and except for anti-PD-1 for patients with microsatellite instable (MSI)+ cancers, no immunotherapy is currently approved for GBM patients. GBM is the most common and most aggressive brain cancer with a very poor prognosis and a median overall survival of 15 months. A few prognostic biomarkers have been identified and are used to some extent, but apart from MSI, no biomarkers are used for patient stratification for treatments other than the standard of care, which was established 15 years ago. Around 25% of new treatments investigated in GBM are immunotherapies. Recent studies indicate that the use of integrated and validated immune correlates predicting the response and guiding treatments could improve the efficacy of immunotherapy in GBM. In this review, we will give an overview of the current status of immunotherapy and biomarkers in use in GBM with the main challenges of treatment in this disease. We will also discuss emerging biomarkers that could be used in future immunotherapy strategies for patient stratification and potentially improved treatment efficacy. [ABSTRACT FROM AUTHOR]

Abstract Acquisition of recurrent/metastatic potential by a tumor cell defines a critical step in malignant progression. However, understanding of metastatic progression at the molecular level is scarce for cervical carcinomas (CES). In this study, we performed genomic, transcriptomic, and viral profiling of five pairs of primary (CES‐P) and matched recurrent/metastatic tumors (CES‐R/M) with high risk human papillomavirus. Whole exome sequencing revealed mutation features of CES‐R/M including elevated mutation burdens and prevalent copy number alterations compared to their matched CES‐P. A relative deficit of APOBEC‐related mutation signatures accompanying the transcriptional downregulation of APOBEC3A was observed for CES‐R/M. Mutations in genes encoding epigenetic regulators were commonly observed as CES‐R/M‐specific alterations. Immunoprofiling and gene set analysis revealed CES‐Ps were enriched with transcripts representing activated anticancer immunity such as interferon‐gamma pathway, while CES‐R/M exhibited upregulation of genes involved in epithelial‐mesenchymal transition and angiogenesis. Viral capture sequencing revealed that integration sites remained enriched in viral E1 protein domain during malignant progression. Moreover, we found transcriptional upregulation of POSTN and downregulation of APOBEC3A were associated with unfavorable clinical outcomes in CES. Comprehensive genomic and transcriptomic profiling of a rare cohort including CES‐R/M identified metastases‐specific features to advance the molecular understanding into CES metastatic progression with potential clinical implications.

Abstract Background The tumor immune microenvironment is a heterogeneous entity. Gene expression analysis allows us to perform comprehensive immunoprofiling and may assist in dissecting the different components of the immune infiltrate. As gene expression analysis also provides information regarding tumor cells, differences in interactions between the immune system and specific tumor characteristics can also be explored. This study aims to gain further insights in the composition of the tumor immune infiltrate and to correlate these components to histology and overall survival in non-small cell lung cancer (NSCLC). Methods Archival tissues from 530 early stage, resected NSCLC patients with annotated tumor and patient characteristics were analyzed using the NanoString nCounter Analysis system. Results Unsupervised clustering of the samples was mainly driven by the overall level of inflammation, which was not correlated with survival in this patient set. Adenocarcinoma (AD) showed a significantly higher degree of immune infiltration compared to squamous cell carcinoma (SCC). A 34-gene signature, which did not correlate with the overall level of immune infiltration, was identified and showed an OS benefit in SCC. Strikingly, this benefit was not observed in AD. This difference in OS in SCC specifically was confirmed in two independent NSCLC cohorts. The highest correlation between expression of the 34-gene signature and specific immune cell populations was observed for NK cells, but although a plausible mechanism for NK cell intervention in tumor growth could be established in SCC over AD, this could not be translated back to immunohistochemistry, which showed that NK cell infiltration is scarce irrespective of histology. Conclusions These findings suggest that the ability of immune cell infiltration and the interaction between tumor and immune cells may be different between AD and SCC histology and that a subgroup of SCC tumors seems more susceptible to Natural Killer cell recognition and killing, whereas this may not occur in AD tumors. A highly sensitive technique like NanoString was able to detect this subgroup based on a 34-gene signature, but further research will be needed to assist in explaining the biological rationale of such low-level expression signatures.

Snakebite envenoming is a public health issue linked to high mortality and morbidity rates worldwide. Although antivenom has been the mainstay treatment for envenomed victims receiving medical care, the diverse therapeutic efficacy of the produced antivenom is a major limitation. Deinagkistrodon acutus is a venomous snake that poses significant concern of risks to human life in Taiwan, and successful production of antivenom against D. acutus envenoming remains a considerable challenge. Among groups of horses subjected to immunization schedules, few or none subsequently meet the quality required for further scale-up harvesting. The determinants underlying the variable immune responses of horses to D. acutus venom are currently unknown. In this study, we assessed the immunoprofiles of high-potency and low-potency horse plasma against D. acutus venom and explored the conspicuous differences between these two groups. Based on the results of liquid chromatography with tandem mass spectrometry (LC-MS/MS), acutolysin A was identified as the major component of venom proteins that immunoreacted differentially with the two plasma samples. Our findings indicate underlying differences in antivenoms with variable neutralization efficacies, and may provide valuable insights for improvement of antivenom production in the future.

The lack of comprehensive diagnostics and consensus analytical models for evaluating the status of a patient's immune system has hindered a wider adoption of immunoprofiling for treatment monitoring and response prediction in cancer patients. To address this unmet need, we developed an immunoprofiling platform that uses multiparameter flow cytometry to characterize immune cell heterogeneity in the peripheral blood of healthy donors and patients with advanced cancers. Using unsupervised clustering, we identified five immunotypes with unique distributions of different cell types and gene expression profiles. An independent analysis of 17,800 open-source transcriptomes with the same approach corroborated these findings. Continuous immunotype-based signature scores were developed to correlate systemic immunity with patient responses to different cancer treatments, including immunotherapy, prognostically and predictively. Our approach and findings illustrate the potential utility of a simple blood test as a flexible tool for stratifying cancer patients into therapy response groups based on systemic immunoprofiling. [Display omitted] • Development of a machine learning-based clinical immunoprofiling platform • Depiction of immune states by multiparameter flow cytometry and bulk RNA-seq using peripheral blood • Identification and validation of five immunotypes conserved across diverse diagnoses • Potential clinical utility in stratifying treatment responses via a simple blood test Dyikanov et al. developed a machine learning platform that uses a simple blood test to reveal cellular compositions reflective of a person's immune system. These compositions, delineated into five conserved immunotypes, can reflect a person's disease status or how someone responds to specific treatments, thus underscoring their potential clinical utility for cancer patients. [ABSTRACT FROM AUTHOR]

Immunotherapy is a cornerstone in the treatment of melanoma, and is intended to modulate the host immunity against the tumor. Immunotherapy can be used in an adjuvant setting, after complete surgical excision in patients with a high risk of disease relapse and as a treatment in advanced (unresectable or metastatic) stages. Development of novel therapeutic approaches and the optimization of existing therapies hold a great promise in the field of melanoma therapy research. Different clinical trials are ongoing, and immunotherapy is showing the ability to confirm durable clinical benefits in selected groups of melanoma patients. The aim of this review is to summarize different types of immunotherapy agents, as well as to discuss different strategies, complementary regimens, and possible biomarkers of response to the treatment.

Background: Alemtuzumab is a highly effective drug for the treatment of multiple sclerosis (MS), characterized by specific patterns of depletion and repopulation. As an induction-like treatment concept, two mandatory infusion courses can inhibit long-term disease activity in the majority of patients, and additional courses can successfully manage subsequent re-emergence of disease activity. Currently, there are no biomarkers to identify patients with re-emergent disease activity requiring retreatment.Methods: In this study, we systematically characterized 16 MS patients commencing alemtuzumab. Clinical parameters, MRI and detailed immunoprofiling were conducted every 3 months for up to 84 months.Results: Alemtuzumab led to significant decrease in clinical disease activity in all evaluated patients. Nine out of 16 patients presented with no evidence of disease activity (NEDA)-3 up to 84 months (“complete-responder”), while 7 patients demonstrated clinical or/and subclinical MRI disease activity and received alemutzumab retreatment (“partial-responder”). In both response categories, all T- and B-cell subsets were markedly depleted after alemtuzumab therapy. In particular, absolute numbers of Th1 and Th17 cells were markedly decreased and remained stable below baseline levels—this effect was particularly pronounced in complete-responders. While mean cell numbers did not differ significantly between groups, analysis of event-driven immunoprofiling demonstrated that absolute numbers of Th1 and Th17 cells showed a reproducible increase starting 6 months before relapse activity. This change appears to predict emergent disease activity when compared with stable disease.Conclusion: Studies with larger patient populations are needed to confirm that frequent immunoprofiling may assist in evaluating clinical decision-making of alemtuzumab retreatment.

Acquisition of recurrent/metastatic potential by a tumor cell defines a critical step in malignant progression. However, understanding of metastatic progression at the molecular level is scarce for cervical carcinomas (CES). In this study, we performed genomic, transcriptomic, and viral profiling of five pairs of primary (CES‐P) and matched recurrent/metastatic tumors (CES‐R/M) with high risk human papillomavirus. Whole exome sequencing revealed mutation features of CES‐R/M including elevated mutation burdens and prevalent copy number alterations compared to their matched CES‐P. A relative deficit of APOBEC‐related mutation signatures accompanying the transcriptional downregulation of APOBEC3A was observed for CES‐R/M. Mutations in genes encoding epigenetic regulators were commonly observed as CES‐R/M‐specific alterations. Immunoprofiling and gene set analysis revealed CES‐Ps were enriched with transcripts representing activated anticancer immunity such as interferon‐gamma pathway, while CES‐R/M exhibited upregulation of genes involved in epithelial‐mesenchymal transition and angiogenesis. Viral capture sequencing revealed that integration sites remained enriched in viral E1 protein domain during malignant progression. Moreover, we found transcriptional upregulation of POSTN and downregulation of APOBEC3A were associated with unfavorable clinical outcomes in CES. Comprehensive genomic and transcriptomic profiling of a rare cohort including CES‐R/M identified metastases‐specific features to advance the molecular understanding into CES metastatic progression with potential clinical implications. [ABSTRACT FROM AUTHOR]

Background: The tumor immune microenvironment is a heterogeneous entity. Gene expression analysis allows us to perform comprehensive immunoprofiling and may assist in dissecting the different components of the immune infiltrate. As gene expression analysis also provides information regarding tumor cells, differences in interactions between the immune system and specific tumor characteristics can also be explored. This study aims to gain further insights in the composition of the tumor immune infiltrate and to correlate these components to histology and overall survival in non-small cell lung cancer (NSCLC).Methods: Archival tissues from 530 early stage, resected NSCLC patients with annotated tumor and patient characteristics were analyzed using the NanoString nCounter Analysis system.Results: Unsupervised clustering of the samples was mainly driven by the overall level of inflammation, which was not correlated with survival in this patient set. Adenocarcinoma (AD) showed a significantly higher degree of immune infiltration compared to squamous cell carcinoma (SCC). A 34-gene signature, which did not correlate with the overall level of immune infiltration, was identified and showed an OS benefit in SCC. Strikingly, this benefit was not observed in AD. This difference in OS in SCC specifically was confirmed in two independent NSCLC cohorts. The highest correlation between expression of the 34-gene signature and specific immune cell populations was observed for NK cells, but although a plausible mechanism for NK cell intervention in tumor growth could be established in SCC over AD, this could not be translated back to immunohistochemistry, which showed that NK cell infiltration is scarce irrespective of histology.Conclusions: These findings suggest that the ability of immune cell infiltration and the interaction between tumor and immune cells may be different between AD and SCC histology and that a subgroup of SCC tumors seems more susceptible to Natural Killer cell recognition and killing, whereas this may not occur in AD tumors. A highly sensitive technique like NanoString was able to detect this subgroup based on a 34-gene signature, but further research will be needed to assist in explaining the biological rationale of such low-level expression signatures. [ABSTRACT FROM AUTHOR]

Background: Alemtuzumab is a highly effective drug for the treatment of multiple sclerosis (MS), characterized by specific patterns of depletion and repopulation. As an induction-like treatment concept, two mandatory infusion courses can inhibit long-term disease activity in the majority of patients, and additional courses can successfully manage subsequent re-emergence of disease activity. Currently, there are no biomarkers to identify patients with re-emergent disease activity requiring retreatment. Methods: In this study, we systematically characterized 16 MS patients commencing alemtuzumab. Clinical parameters, MRI and detailed immunoprofiling were conducted every 3 months for up to 84 months. Results: Alemtuzumab led to significant decrease in clinical disease activity in all evaluated patients. Nine out of 16 patients presented with no evidence of disease activity (NEDA)-3 up to 84 months ("complete-responder"), while 7 patients demonstrated clinical or/and subclinical MRI disease activity and received alemutzumab retreatment ("partial-responder"). In both response categories, all T- and B-cell subsets were markedly depleted after alemtuzumab therapy. In particular, absolute numbers of Th1 and Th17 cells were markedly decreased and remained stable below baseline levels—this effect was particularly pronounced in complete-responders. While mean cell numbers did not differ significantly between groups, analysis of event-driven immunoprofiling demonstrated that absolute numbers of Th1 and Th17 cells showed a reproducible increase starting 6 months before relapse activity. This change appears to predict emergent disease activity when compared with stable disease. Conclusion: Studies with larger patient populations are needed to confirm that frequent immunoprofiling may assist in evaluating clinical decision-making of alemtuzumab retreatment. [ABSTRACT FROM AUTHOR]

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420–0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.

Purpose: Primary malignant solitary fibrous tumor (SFT) of the orbit is a rare spindle cell neoplasm that requires excisional biopsy for histopathological diagnosis. We present a clinical case using contemporary immunohistochemical stains, report on the latest World Health Organization classification, and provide a review of the literature. Observations: Report of a single case of a 65 year old male who presented with right-sided proptosis, limited adduction, ptosis, lateral globe displacement, and cheek festooning. Neuroimaging revealed a 2.2 cm, extraconal heterogeneous mass that diffusely enhanced. En-bloc tumor resection confirmed SFT malignancy based upon nuclear atypia, hypercellularity, and increased mitotic activity (13 mitotic figures/10 high powered fields). Ki-67 showed 2% nuclear staining in the benign tumor and 10–15% staining in the malignant counterpart. Immunohistochemical analysis revealed diffuse Stat6 positivity, CD 34 positivity with partial lack of staining within the malignant portion, S-100 positivity in the malignant portion, and overall negativity for CAM 5.2, desmin, actin, CD 31, and CD 117. Conclusions and importance: Immunoprofiling is helpful to making the diagnosis of malignant solitary fibrous tumor of the orbit. Complete tumor resection continues to be the preferred treatment. The behavior of extrathoracic SFT is unpredictable, and patients with SFT in all locations require careful, long-term follow-up.

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK‐cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti‐CTLA‐4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL‐15 stimulation improved NK cell‐mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56bright and CD56dim NK subsets and increased serum concentrations of the cytokines IL‐10, IL‐8 and TNF‐α. After tremelimumab treatment, the ratio between the CD56bright and CD56dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM‐3+CD8+ T‐cell frequency, high DNAM‐1+CD56dim NK‐cell frequency and high expression levels of NKp46 on the CD56dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab. What's new? Treatment options for malignant mesothelioma (MM) have not significantly improved over the past four decades. A better understanding of the immune response in MM might guide new treatment strategies. In this study, the authors found evidence that natural killer (NK) cells can infiltrate, recognize, and kill MM cells. However, MM patients have distinct alterations in NK, CD8+ T‐cell, and cytokine profiles. Some of these alterations correlated with prognosis, or were affected by treatment with tremelimumab. [ABSTRACT FROM AUTHOR]

The increasing global incidence and prevalence of non-tuberculous mycobacteria (NTM) infection is of growing concern. New evidence of person-to-person transmission of multidrug-resistant NTM adds to the global concern. The reason why certain individuals are at risk of NTM infections is unknown. Using high definition flow cytometry, we studied the immune profiles of two groups that are at risk of Mycobacterium abscessus complex infection and matched controls. The first group was cystic fibrosis (CF) patients and the second group was elderly individuals. CF individuals with active M. abscessus complex infection or a history of M. abscessus complex infection exhibited a unique surface T cell phenotype with a marked global deficiency in TNFα production during mitogen stimulation. Importantly, immune-based signatures were identified that appeared to predict at baseline the subset of CF individuals who were at risk of M. abscessus complex infection. In contrast, elderly individuals with M. abscessus complex infection exhibited a separate T cell phenotype underlined by the presence of exhaustion markers and dysregulation in type 1 cytokine release during mitogen stimulation. Collectively, these data suggest an association between T cell signatures and individuals at risk of M. abscessus complex infection, however, validation of these immune anomalies as robust biomarkers will require analysis on larger patient cohorts.

The immune response is a dynamic system that maintains the integrity of the body, and more specifically fight against infections. However, an unbalanced host immune response is highlighted in many diseases. Exacerbated responses lead to autoimmune and allergic diseases, whereas, low or inefficient responses favor opportunistic infections and viral reactivations. Conflicting situations may also occur, such as in sepsis where inflammation and compensatory immunosuppression make it difficult to deploy the appropriate drug treatment. Until the current day, assessing the immune profile of patients remains a challenge. This is especially due to the inter-individual variability—a key feature of the immune system—which hinders precise diagnosis, prognosis, and therapeutic stratification. Our incapacity to practically interpret the host response may contribute to a high morbidity and mortality, such as the annual 6 million worldwide deaths in sepsis alone. Therefore, there is a high and increasing demand to assess patient immune function in routine clinical practice, currently met by Immune Functional Assays. Immune Functional Assays (IFA) hold a plethora of potentials that include the precise diagnosis of infections, as well as prediction of secondary and latent infections. Current available products are devoted to indirect pathogen detection such as Mycobacteria tuberculosis interferon gamma release assays (IGRA). In addition, identifying the status and the underlying factors of immune dysfunction (e.g., in septic patients) may guide immune targeted therapies. Tools to monitor and stratify the immune status are currently being studied but they still have many limitations such as technical standardization, biomarkers relevance, systematic interpretation and need to be simplified, in order to set the boundaries of "healthy," "ill," and "critically ill" responses. Thus, the design of new tools that give a comprehensive insight into the immune functionality, at the bedside, and in a timely manner represents a leap toward immunoprofiling of patients. [ABSTRACT FROM AUTHOR]

The increasing global incidence and prevalence of non-tuberculous mycobacteria (NTM) infection is of growing concern. New evidence of person-to-person transmission of multidrug-resistant NTM adds to the global concern. The reason why certain individuals are at risk of NTM infections is unknown. Using high definition flow cytometry, we studied the immune profiles of two groups that are at risk of Mycobacterium abscessus complex infection and matched controls. The first group was cystic fibrosis (CF) patients and the second group was elderly individuals. CF individuals with active M. abscessus complex infection or a history of M. abscessus complex infection exhibited a unique surface T cell phenotype with a marked global deficiency in TNFα production during mitogen stimulation. Importantly, immune-based signatures were identified that appeared to predict at baseline the subset of CF individuals who were at risk of M. abscessus complex infection. In contrast, elderly individuals with M. abscessus complex infection exhibited a separate T cell phenotype underlined by the presence of exhaustion markers and dysregulation in type 1 cytokine release during mitogen stimulation. Collectively, these data suggest an association between T cell signatures and individuals at risk of M. abscessus complex infection, however, validation of these immune anomalies as robust biomarkers will require analysis on larger patient cohorts. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy with antibodies targeting immune checkpoints, such as programmed cell death protein 1 (PD-1), shows encouraging results, but reliable biomarkers predicting response to this costly and potentially toxic treatment approach are still lacking. To explore an immune signature predictive for response, we performed liquid biopsy immunoprofiling in 18 cancer patients undergoing PD-1 inhibition before and shortly after initiation of treatment by multicolor flow cytometry and next-generation T- and B-cell immunosequencing ( TCRß/ IGH). Findings were correlated with clinical outcomes. We found almost complete saturation of surface PD-1 on all T-cell subsets after the first dose of the antibody. Both T- and B-cell compartments quantitatively expanded during treatment. These expansions were mainly driven by an increase in the activated T-cell compartments, as well as of naïve B- and plasma cells. Deep immunosequencing revealed a clear diversification pattern of the clonal T-cell space indicative of antigenic selection in 47% of patients, while the remaining patients showed stable repertoires. 43% of the patients with a diversification pattern showed disease control in response to the PD-1 inhibitor. No disease stabilizations were observed without clonal T-cell space diversification. Our data show for the first time a clear impact of PD-1 targeting not only on circulating T-cells, but also on B-lineage cells, shedding light on the complexity of the anti-tumor immune response. Liquid biopsy T-cell next-generation immunosequencing should be prospectively evaluated as part of a composite response prediction biomarker panel in the context of clinical studies. [ABSTRACT FROM AUTHOR]

Immunoprofiling has become a crucial tool for understanding the complex interactions between the immune system and diseases or interventions, such as therapies and vaccinations. Immune response biomarkers are critical for understanding those relationships and potentially developing personalized intervention strategies. Single-cell data have emerged as a promising source for identifying immune response biomarkers. In this review, we discuss the current state-of-the-art methods for immunoprofiling, including those for reducing the dimensionality of high-dimensional single-cell data and methods for clustering, classification, and prediction. We also draw attention to recent developments in data integration.

Adjuvants are critical components of vaccines that enhance the host immune response to the vaccine antigen, however, only a small number of adjuvants are used in vaccines approved for human use. This is in part due to the slow process of novel adjuvants advancing from preclinical models to human studies, and modest mechanistic insights obtained using standard immunological methods to justify selection of a particular adjuvant for clinical evaluation. Here, we discuss several aspects of current adjuvant research and strategies to better assess the complex pathways triggered by adjuvant candidates that can increase adjuvanticity and vaccine efficacy while minimizing reactogenicity. We propose a more systematic use of broad immunoprofiling, coupled with data integration using computational and mathematical modeling. This comprehensive evaluation of the host immune response will facilitate the selection of the most appropriate adjuvant for a vaccine, ultimately leading to the expeditious evaluation of novel adjuvants for vaccines against emerging infectious diseases, which will prove especially valuable during a pandemic where speed is of the essence when developing vaccines.

The fifth "Melanoma Bridge Meeting" took place in Naples, December 1-5th, 2015. The main topics discussed at this meeting were: Molecular and Immuno advances, Immunotherapies and Combination Therapies, Tumor Microenvironment and Biomarkers and Immunoscore. The natural history of cancer involves interactions between the tumor and the immune system of the host. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient's clinical outcome. Moreover, incredible progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors. It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. The road ahead is still very long, but the knowledge of the mechanisms of immune escape, the study of tumor neo-antigens as well as of tumor microenvironment and the development of new immunotherapy strategies, will make cancer a more and more treatable disease. [ABSTRACT FROM AUTHOR]

Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a "signature" set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy. [ABSTRACT FROM AUTHOR]

Parallel studies of primary breast carcinomas and corresponding distant metastases samples reveal considerable differences. Our aim was to highlight this issue from another perspective and provide further data based on 98 patient samples: 69 primary breast carcinoma and 85 distant metastases from bone, central nervous system (CNS) and lung (56 paired). Two independent series of immunohistochemical reactions with different antibodies for estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (Her2), along with HER2 fluroscence in situ hybridization (FISH) were performed on tissue microarrays to classify breast carcinoma and distant metastases samples into Luminal A, Luminal B-proliferating, Luminal B-HER2+, HER2+ and triple negative (TNBC) surrogate breast cancer groups. Correlation and agreement between the two assessments of ER and PgR were fair-to-moderate, and almost perfect for HER2 and Ki67. There was 40% discordance concerning immunophenotype between breast carcinomas and distant metastases. Most common metastatic site of ER+ breast carcinoma was the skeletal system (59.2%), whereas that of TNBCs was the CNS (58.8%) and lungs (23.5%). Distant metastases in bones were mostly luminal (54.3%), in the CNS, Luminal B (53.2%), and in the lung, TNBC (37.5%). The change of drugable properties of primary breast cancers in the respective bone and CNS metastases suggests that characterization of the metastasis is necessary for appropriate treatment planning. [ABSTRACT FROM AUTHOR]

The genetic loci influencing individual resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are still largely unknown. In the current study, we searched for genetic loci associated with resistance to MAP infection by evaluating the performance of monocyte-derived macrophages (MDMs) isolated from the peripheral blood of 75 healthy Holsteins cows and infected ex vivo with MAP. Bacterial load (log colony-forming units, log CFUs) within MDMs was quantified at 2 h and 7 days p. i. using a BACTEC MGIT 960 instrument. In addition, the expression levels of some genes with important roles in the innate immune response including epiregulin (EREG), complement component C3 (C3), galectin-9 (Gal9), and nitric oxide (NO - ) were measured in the supernatant of the infected cells. DNA from peripheral blood samples of the animals included in the study was isolated and genotyped with the EuroG MD bead Chip (44,779 single nucleotide-polymorphisms, SNPs). Linear mixed models were used to calculate the heritability ( h 2 ) estimates for each indicator of MDM performance, MAP load within MDMs and EREG, C3, Gal9, and NO - expression. After performing a genome-wide association study, the only phenotypes that showed SNPs with a significant association were the bacterial load within MDMs at 2 h ( h 2 = 0. 87) and 7 days ( h 2 = 0.83) p.i. A total of 6 SNPs, 5 candidate genes, and one microRNA on the Bos taurus chromosomes BTA2, BTA17, BTA18, and BTA21 were associated with MAP load at 2 h p.i. Overlap was seen in two SNPs associated with the log CFUs at 2 h and 7 d p.i. The identified SNPs had negative regression coefficients, and were, therefore, associated with a low bacterial load within MDMs. Some of the identified SNPs were located within QTLs previously associated with longevity, reproductive, and udder health traits. Some of the identified candidate genes; Oxysterol Binding Protein Like 6, Cysteine and Serine Rich Nuclear Protein 3, and the Coiled-Coil Domain Containing 92 regulate cellular cholesterol trafficking and efflux, apoptosis, and interferon production, respectively. Taken together, our results define a heritable and distinct immunogenetic profile in MAP-infected macrophages designed to limit bacterial load early after infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Badia-Bringué, Canive and Alonso-Hearn.)

Snakebite envenoming is a public health issue linked to high mortality and morbidity rates worldwide. Although antivenom has been the mainstay treatment for envenomed victims receiving medical care, the diverse therapeutic efficacy of the produced antivenom is a major limitation. Deinagkistrodon acutus is a venomous snake that poses significant concern of risks to human life in Taiwan, and successful production of antivenom against D. acutus envenoming remains a considerable challenge. Among groups of horses subjected to immunization schedules, few or none subsequently meet the quality required for further scale-up harvesting. The determinants underlying the variable immune responses of horses to D. acutus venom are currently unknown. In this study, we assessed the immunoprofiles of high-potency and low-potency horse plasma against D. acutus venom and explored the conspicuous differences between these two groups. Based on the results of liquid chromatography with tandem mass spectrometry (LC-MS/MS), acutolysin A was identified as the major component of venom proteins that immunoreacted differentially with the two plasma samples. Our findings indicate underlying differences in antivenoms with variable neutralization efficacies, and may provide valuable insights for improvement of antivenom production in the future.

Background: Adoptive T cell therapy (ACT) has shown great promise in melanoma, with over 50% response rate in patients where autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) can be cultured and expanded. A major limitation of ACT is the inability to generate or expand autologous tumor-reactive TIL in 25-45% of patients tested. Methods that successfully identify tumors that are not suitable for TIL generation by standard methods would eliminate the costs of fruitless expansion and enable these patients to receive alternate therapy immediately. Methods: Multispectral fluorescent immunohistochemistry with a panel including CD3, CD8, FoxP3, CD163, PD-L1 was used to analyze the tumor microenvironment in 17 patients with melanoma among our 36-patient cohort to predict successful TIL generation. Additionally, we compared tumor fragments and enzymatic digestion of tumor samples for efficiency in generating tumor-reactive TIL. Results: Tumor-reactive TIL were generated from 21/36 (58%) of melanomas and for 12/13 (92%) tumors where both enzymatic and fragment methods were compared. TIL generation was successful in 10/13 enzymatic preparations and in 10/13 fragment cultures; combination of both methods resulted in successful generation of autologous tumor-reactive TIL in 12/13 patients. In 17 patients for whom tissue blocks were available, IHC analysis identified that while the presence of CD8+ T cells alone was insufficient to predict successful TIL generation, the CD8+ to FoxP3+ ratio was predictive with a positive-predictive value (PPV) of 91% and negative-predictive value (NPV) of 86%. Incorporation of CD163+ macrophage numbers and CD8:PD-L1 ratio did not improve the PPV. However, the NPV could be improved to 100% by including the ratio of CD8+:PD-L1+ expressing cells. Conclusion: This is the first study to apply 7-color multispectral immunohistochemistry to analyze the immune environment of tumors from patients with melanoma. Assessment of the data using unsupervised hierarchical clustering identified tumors from which we were unable to generate TIL. If substantiated, this immune profile could be applied to select patients for TIL generation. Additionally, this biomarker profile may also indicate a pre-existing immune response, and serve as a predictive biomarker of patients who will respond to checkpoint blockade. We postulate that expanding the spectrum of inhibitory cells and molecules assessed using this technique could guide combination immunotherapy treatments and improve response rates. [ABSTRACT FROM AUTHOR]

Here we present the case of a 73-year-old woman with an ulcerated, advanced, hepatoid, and α-fetoprotein-producing poorly differentiated (G3) primary gastric adenocarcinoma pT3 N3a M1 with multinucleated cells and evident neuroendocrine component. This tumor was consistent with giant cell tumor type gastric carcinoma with osteoclast-like giant cells (OGCs). The cancer was HER2 and E-cadherin negative, chromogranin A dispersedly and moderately positive, and strongly α-fetoprotein-positive with evident CK AE1/AE3 immunoreactivity, while OGCs expressed CD68. To provide an insight into the molecular background of this peculiar neoplasm, next-generation sequencing (NGS) was performed to analyze the 50 most frequently mutated oncogenes and tumor suppressors. We detected mutations in the primary tumor in the following genes: KIT, EGFR, PTEN, ATM, and RB1. In the liver metastasis, we revealed mutations in 3 genes: PIK3CA, KIT, and CDKN2A. [ABSTRACT FROM AUTHOR]

Background: Although AJCC/TNM staging remains the gold standard for prognostic assessment of colon cancer, stage-specific outcomes vary. We therefore prospectively evaluated the prognostic role of immunoprofiling. Methods: Our cohort included 35 patients from an ongoing prospective trial of ultrastaging for colon cancer. Specimens were analyzed for T cell markers (CD3, CD4, CD8, and FoxP3). The number of tumor-infiltrating lymphocytes was analyzed at the tumor's margin and center and correlated with AJCC/TNM stage, clinicopathologic variables, and disease-free survival. Results: There was a significant inverse association between number of CD3 cells in the tumor center and tumor stage ( P = 0.05). The tumor center/margin ratio of CD3 cells also showed an inverse but non-significant relationship with nodal involvement ( P = 0.07). Body mass index was inversely associated with numbers of CD3( P = 0.04) and CD8( P = 0.02) cells. Longer disease-free survival was correlated with higher CD8+ counts ( P = 0.07), lower CD4/CD8 ratios ( P = 0.008), and higher CD8/FoxP3 ratios ( P = 0.02). Conclusions: This is the first prospective validation of immunoprofiling in patients whose colon cancer is staged with strict surgical and pathology quality measures. The apparent correlation between immunophenotypic response and clinical outcome warrants evaluation in a larger prospective trial. [ABSTRACT FROM AUTHOR]