Immunological Heterogeneity Research Articles

Immunological tumor heterogeneity and diagnostic profiling for advanced and immune therapies

Immunotherapies have changed the way how we treat cancer at all stages. The understanding of the immune system in individual tumor specimens guides the selection of immune-modulating agents such as immune checkpoint inhibitors alone or in combination with other therapeutic agents that target, modulate or unleash the patient's immune system. Despite the similar histopathological diagnosis, each tumor is unique at its primary site and site of metastasis, also depending on previous treatment regimens or genetic alterations, such as chromosomal instability or acquired mutations. The clinically well-established use of PD-1/PD-L1 inhibitors already requires the assessment of its target molecules in different cells (viable tumor cells alone or in combination with immune cells or immune cells alone) with different thresholds in various indications. Anyhow, checkpoint inhibitors show the best overall response rate when immune effector cells like tumor-infiltrating lymphocytes are in close spatial proximity without being suppressed by other humoral or cellular regulatory mechanisms. Therefore, immune cell-rich tumors (“hot tumors”) are usually quite reactive to immune-modulating agents, whereas other immune-depleted or immune-excluded tumor areas are less responsive and require alternative treatment regimens such as modified immune effectors cells or immune-stimulating agents, for example, oncolytic viruses. Here, we summarize the relevance to understand the entire tumor heterogeneity and its environment, the contextual relationship and spatial quantification of all immune and tumor cells along with the genetic background of the individual cancer through the application of multiplex in-situ technologies and the application of machine learning tools.

Differential Proinflammatory Responses to Aspergillus fumigatus by Airway Epithelial Cells In Vitro Are Protease Dependent.

Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall–epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner.

60-LB: Dendritic Cells Engineered with Tailored Multiepitopes-Encoding mRNA as Precision Medicine for Immunotherapy of Type 1 Diabetes

Type 1 diabetes (T1D) is a complex autoimmune disease characterized by heterogeneity in genetic risk factors and immune profiles of patients and for which a cure or approved treatment is not yet available. Antigen-specific immunotherapy (ASIT) offers a targeted treatment of T1D that selectively inhibits autoreactive T cells. So far, ASIT trials showed limited efficacy, as only a subset of patients is responsive. Thus, approaches that consider the genetic and immunological heterogeneity of T1D patients to provide more tailored treatments are needed. Using mRNA encoding multiple epitopes tailored to the NOD mouse (model of T1D), we engineered bone marrow-derived dendritic cells (DCs) by mRNA electroporation (mRNA-DCs). The mRNA is designed to enable optimal presentation of epitopes to both CD4 and CD8 T cells. Antigen presentation persisted at least three days in vivo. Markers of anergy as well as IL-10 expression were found to be upregulated in antigen-specific CD4 and CD8 T cells after treating NOD mice with mRNA-DCs. T1D development was significantly suppressed with 3 biweekly treatments with 106 DCs containing 2 µg of antigen mRNA. Moreover, efficacy of the mRNA-DCs was enhanced when mRNAs encoding antigens and IL-27 were co-delivered into DCs by electroporation or when DCs were treated with rapamycin during differentiation. Endogenous expression of antigens may enable post-translational modifications and more prolonged presentation. Thus, we developed a promising and customizable ASIT platform to deliver multiple epitopes to exogenous tolerogenic DCs (or to endogenous APCs using a nanoparticle-based platform not covered here) that will consider the patient’s immune profile for a precision medicine treatment of T1D. Disclosure R. Fite: None. R. J. Creusot: None. Funding American Diabetes Association (1-19-PMF-022 to R.F.F.)

Identification of Precise Therapeutic Targets and Characteristic Prognostic Genes Based on Immune Gene Characteristics in Uveal Melanoma.

The tumor microenvironment is an important factor for the immunotherapy of tumor patients. The sequenced transcriptome data can be used to describe the tumor microenvironment and various immune subtypes. We exploited published data on patients with uveal melanoma (UVM) to identify immune subtypes. Based on the immune-related gene sets of 80 patients with UVM in the TCGA database, we used consensus clustering to identify two immune subgroups. In the two immune subtypes, we analyzed clinical characteristics and immune infiltration. Class1 has low immune infiltration, contains memory B cells, Th2 cells, Th17 cells, eosinophils, natural killer cells, and has a better prognosis. Class2 has higher immune infiltration. CD8+ T cells, Th1 cells, MDSCs, and Dendritic cells are enriched in class2, which has strong cytolytic activity, high expression of immune checkpoint genes, and poor outcome. Moreover, we have developed and verified an immune characteristic model that can predict the prognosis of patients well. Through this model, we screened prostaglandin-endoperoxide synthase 2 (PTGS2) as the therapeutic target of UVM. Treatment of choroidal melanoma cell line (OCM1) cells with celecoxib (an inhibitor of PTGS2) effectively inhibits cell growth, proliferation, and promotes apoptosis. Our results show the immunological heterogeneity of UVM patients and also provide an ideal therapeutic target for the future treatment design of patients.

Updated Diagnostic Criteria for Paraneoplastic Neurologic Syndromes.

ObjectiveThe contemporary diagnosis of paraneoplastic neurologic syndromes (PNSs) requires an increasing understanding of their clinical, immunologic, and oncologic heterogeneity. The 2004 PNS criteria are partially outdated due to advances in PNS research in the last 16 years leading to the identification of new phenotypes and antibodies that have transformed the diagnostic approach to PNS. Here, we propose updated diagnostic criteria for PNS.MethodsA panel of experts developed by consensus a modified set of diagnostic PNS criteria for clinical decision making and research purposes. The panel reappraised the 2004 criteria alongside new knowledge on PNS obtained from published and unpublished data generated by the different laboratories involved in the project.ResultsThe panel proposed to substitute “classical syndromes” with the term “high-risk phenotypes” for cancer and introduce the concept of “intermediate-risk phenotypes.” The term “onconeural antibody” was replaced by “high risk” (>70% associated with cancer) and “intermediate risk” (30%–70% associated with cancer) antibodies. The panel classified 3 levels of evidence for PNS: definite, probable, and possible. Each level can be reached by using the PNS-Care Score, which combines clinical phenotype, antibody type, the presence or absence of cancer, and time of follow-up. With the exception of opsoclonus-myoclonus, the diagnosis of definite PNS requires the presence of high- or intermediate-risk antibodies. Specific recommendations for similar syndromes triggered by immune checkpoint inhibitors are also provided.ConclusionsThe proposed criteria and recommendations should be used to enhance the clinical care of patients with PNS and to encourage standardization of research initiatives addressing PNS.

Immunological and clinical heterogeneity in cutaneous lupus erythematosus.

Immunological and clinical heterogeneity in cutaneous lupus erythematosus.

English

BACKGROUND Breast cancer is now the most common cancer in Indian women, having recently surpassed cervical cancer in incidence. Triple negative breast cancer (TNBC), which accounts for 15 % of all the breast cancers is an aggressive type seen in younger women with early signs of metastasis, has a poor prognosis due to systemic recurrence and its refractoriness to conventional adjuvant therapy. The purpose of this study was to look into the various prognostic factors associated with 5 years disease-free survival (DFS) and overall survival (OS) in TNBC. METHODS This retrospective study included 67 patients with complete treatment and followup (median 57 months) presented and treated in the Department of Radiotherapy, Kottayam, between January 2011 and December 2012. The Kaplan-Meier approach was used to analyse survival. Using the log-rank test, univariate analysis of prognostic factors was completed. Using the Cox regression process, multivariate analysis was performed on IBM SPSS version 20. RESULTS The average age was 51.36 ± 11.393 (median, 51.36 years; range 30.0 – 80.0 years), with a median of 50 months, the five-year OS was 65.7 % and DFS was found to be 59.7 % with a median of 45 months, suggesting aggressive nature and poor TNBC survival. Univariate analysis of prognostic factor, clinical stage (cN) and positive nodes (pN) status, clinical tumour size, lympho-vascular invasion (LVI), grade, and nodal density were found to have a significant impact on DFS. Except tumour grade and LVI all were found to be associated with OS. Multivariate analysis, clinical tumour size and pathological nodal status had a significant impact on OS and DFS. CONCLUSIONS TNBC is an aggressive subtype of breast cancer in younger patients with a high risk of metastasis to visceral organs with inherent molecular subtypes and immunological heterogeneity. For treatment of TNBC, targeted estimated glomerular filtration rate (EGFR), fibroblast growth factor receptor 2 (FGFR2), vascular endothelial growth factor (VEGF), and mechanistic target of rapamycin (mTOR) receptor based initial treatment setting will improve the outcome dramatically and will fill the unmet clinical needs. KEYWORDS TNBC, Recurrence, OS, DFS, Nodal Density

The design of multiepitope vaccines from plasmids of diarrheagenic Escherichia coli against diarrhoea infection: Immunoinformatics approach.

Diarrhoea infection is a major global health public problem and is caused by many organisms including diarrheagenic Escherichia coli pathotypes. The common problem with diarrhoea is the drug resistance of pathogenic bacteria, the most promising alternative means of preventing drug resistance is vaccination. However, there has not been any significant success in the prevention of diarrhoea caused by E. coli through vaccination. Epitope-based vaccine is gaining more attention due to its safety and specificity. Sequence variation of protective antigens of the pathogen has posed a new challenge in the development of epitope-based vaccines against the infection, leading to the necessity of multiepitope based design. In this study, immunoinformatics tools were used to design multiepitope vaccine candidates from plasmid genome sequences of multiple pathotypes of E. coli species involved in diarrhoea infections. The ability of the identified epitopes to be used as a cross-protect multiepitope vaccine was achieved by identifying conserved, immunogenic and antigenic peptides that can elicit CD4+ T-cell, CD8+ T-cell and B-cell and bind to MHC I and II HLA alleles. The molecular docking results of T-cell epitopes showed their well binding affinity to receptive protein and with a wider population coverage. The different multiepitope-based vaccines (MEVCs) candidates were constructed and based on the types of epitope linker they contained. The MEVCs exhibited very good binding interactions with the human immune receptor. Among multiepitope vaccines constructed, MEVC6, MEVCA and MEVCB are more promising as potential vaccine candidates for cross-protection against gastrointestinal infections according to the computational study. It is also hoped that after validation and testing, the predicted multiepitope-based vaccine candidates will probably resolve the challenge of immunological heterogeneity facing enteric vaccine development.

Single-Cell Profiling to Explore Immunological Heterogeneity of Tumor Microenvironment in Breast Cancer.

Immune infiltrates in the tumor microenvironment (TME) of breast cancer (BRCA) have been shown to play a critical role in tumorigenesis, progression, invasion, and therapy resistance, and thereby will affect the clinical outcomes of BRCA patients. However, a wide range of intratumoral heterogeneity shaped by the tumor cells and immune cells in the surrounding microenvironment is a major obstacle in understanding and treating BRCA. Recent progress in single-cell technologies such as single-cell RNA sequencing (scRNA-seq), mass cytometry, and digital spatial profiling has enabled the detailed characterization of intratumoral immune cells and vastly improved our understanding of less-defined cell subsets in the tumor immune environment. By measuring transcriptomes or proteomics at the single-cell level, it provides an unprecedented view of the cellular architecture consist of phenotypical and functional diversities of tumor-infiltrating immune cells. In this review, we focus on landmark studies of single-cell profiling of immunological heterogeneity in the TME, and discuss its clinical applications, translational outlook, and limitations in breast cancer studies.

IPEX Syndrome: Genetics and Treatment Options.

(1) Background: IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome characterizes a complex autoimmune reaction beginning in the perinatal period, caused by a dysfunction of the transcription factor forkhead box P3 (FOXP3). (2) Objectives: Studies have shown the clinical, immunological, and molecular heterogeneity of patients with IPEX syndrome. The symptoms, treatment, and survival were closely connected to the genotype of the IPEX syndrome. Recognition of the kind of mutation is important for the diagnostics of IPEX syndrome in newborns and young infants, as well as in prenatal screening. The method of choice for treatment is hematopoietic stem cell transplantation and immunosuppressive therapy. In children, supportive therapy for refractory diarrhea is very important, as well as replacement therapy of diabetes mellitus type 1 (DMT1) and other endocrinopathies. In the future, genetic engineering methods may be of use in the successful treatment of IPEX syndrome. (3) Conclusions: The genetic defects determine a diagnostic approach and prognosis, making the knowledge of the genetics of IPEX syndrome fundamental to introducing novel treatment methods.

Clinical and Immunological Heterogeneity in Japanese Patients with Gain-of-Function Variants in STAT3.

Germline loss-of-function variants in the signal transducer and activator of transcription 3 (STAT3) gene result in autosomal dominant hyper IgE syndrome, whereas somatic gain-of-function (GOF) variants in STAT3 are associated with some malignancies. In addition, germline GOF variants in STAT3 are linked to disorders involving autoimmunity and lymphoproliferation. In this study, we describe five Japanese families with germline GOF variants in STAT3, including three novel variants. We also present the clinical and immunological characteristics of these patients. Eight patients from five families were enrolled in this study. We performed genetic and immunological analyses, and collected the associated clinical information. We identified five heterozygous variants in STAT3 using whole-exome sequencing and target gene sequencing. Two of these (E286G and T716M) were previously reported and three (K348E, E415G, and G618A) were novel. A STAT3 reporter assay revealed that all of the variants were GOF. However, the immunological and clinical characteristics among the patients were highly variable. Patients with STAT3 GOF variants exhibited clinical and immunological heterogeneity with incomplete penetrance.

Identification of subtypes correlated with tumor immunity and immunotherapy in cutaneous melanoma

Because immune checkpoint inhibitors (ICIs) are effective for a subset of melanoma patients, identification of melanoma subtypes responsive to ICIs is crucial. We performed clustering analyses to identify immune subtypes of melanoma based on the enrichment levels of 28 immune cells using transcriptome datasets for six melanoma cohorts, including four cohorts not treated with ICIs and two cohorts treated with ICIs. We identified three immune subtypes (Im-H, Im-M, and Im-L), reproducible in these cohorts. Im-H displayed strong immune signatures, low stemness and proliferation potential, genomic stability, high immunotherapy response rate, and favorable prognosis. Im-L showed weak immune signatures, high stemness and proliferation potential, genomic instability, low immunotherapy response rate, and unfavorable prognosis. The pathways highly enriched in Im-H included immune, MAPK, apoptosis, calcium, VEGF, cell adhesion molecules, focal adhesion, gap junction, and PPAR. The pathways highly enriched in Im-L included Hippo, cell cycle, and ErbB. Copy number alterations correlated inversely with immune signatures in melanoma, while tumor mutation burden showed no significant correlation. The molecular features correlated with favorable immunotherapy response included immune-promoting signatures and pathways of PPAR, MAPK, VEGF, calcium, and glycolysis/gluconeogenesis. Our data recapture the immunological heterogeneity in melanoma and provide clinical implications for the immunotherapy of melanoma.

Immunological Heterogeneity

Ease of access to circulating peripheral blood cells (PBMCs) can offer unique insights into human immune function, as well as responses to vaccination and infection. Nevertheless, PBMC heterogeneity has been under-appreciated since results obtained from mixed populations may reflect changes in subset abundance as opposed to true age-related changes involving a specific subset. Technological advances have allowed for the examination of age-related heterogeneity with regards to systemic cytokine levels, immune cell frequencies and chemokine receptor expression by peripheral lymphocytes. Furthermore, introduction of sex as a variable in the examination of human PBMCs adds additional dimorphism to the study of aging and immunity including differences in epigenetic modifications, levels of pro-inflammatory activity and adaptive immunity.

TBIO-02. IMMUNE PROFILING OF RARE EMBRYONAL BRAIN TUMORS REVEAL EVIDENCE OF DYSREGULATED INTERFERON SIGNALLING AS A POTENTIAL DETERMINANT OF IMMUNOLOGICAL HETEROGENEITY

Embryonal brain tumors (EBTs) remain the most common malignant pediatric brain tumors. Despite recent advances and improved understanding of the molecular biology of EBTs, clinical outcomes remain poor for rare EBTs. Previous large-scale genomic studies of rare EBTs have shed light on distinct genomic, transcriptomic and epigenomic profiles. Interestingly, these studies have revealed prominent tumor heterogeneity that provides opportunity to develop novel treatment strategies to improve patient outcomes. To examine the tumor microenvironment and identify tumor- specific biological dependencies, we performed deconvolution analysis of bulk gene expression (171 RNA-seq, 236 microarrays) and 586 methylation arrays, which revealed significant intra and inter-tumoral heterogeneity and implicated interferon (IFN)-mediated signalling as a determinant of a distinct immunological profile in rare EBTs. To further elucidate the importance of IFN signalling, we performed scRNA-seq on 20 primary samples, which provided evidence of a spectrum of IFN-immunological responses that vary from immunosuppressive to immunologically exhaustive that occur in a host dependent manner. To further validate our findings, we utilised a genetically engineered murine model of Atypical Teratoid Rhabdoid Tumor and primary xenografts in humanised mice to corroborate our in-silico profiles in vivo. Through amalgamation of our in-silico data with our in vivo data, we have identified evidence that dysregulated IFN responses represent a core element of the immunological heterogeneity present within subsets of rare EBTs. An improved understanding of the immune milieu in rare EBTs will provide avenues to develop specific onco-immune targets to address this clinical need.

Global differences in atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder, with a highly variable prevalence worldwide. Recent evidence, however, has shown an increase in prevalence in the Asia Pacific region. Nevertheless, most of the published literature has focused mainly on Western populations, and only few clinical trials have included subgroups of other ethnic populations. Reasons for the observed ethnic and geographical differences in AD are not well established. This calls into question the need for a better understanding of AD pathogenesis and inter-ethnic differences in clinical and immuno-phenotypes. These differences may reflect inherent variability in disease mechanisms between populations, which in turn may impact upon treatment responses such as biologics that are currently tailored mainly to a specific immuno-phenotype (T-helper type 2 dominant). In this article, we reviewed existing literature on the prevalence of AD globally, highlighting differences, if any, in the clinical and immuno-phenotypes of AD between different ethnicities. We discussed genetic and environmental factors that affect AD in different populations and therapeutic considerations. Our review highlights AD as a disease with ethnic-dependent clinical and immunological heterogeneity and calls for greater inclusion of ethnic diversity in future research in order to develop targeted treatments.

Beyond monogenetic rare variants: tackling the low rate of genetic diagnoses in predominantly antibody deficiency.

Predominantly antibody deficiency (PAD) is the most prevalent form of primary immunodeficiency, and is characterized by broad clinical, immunological and genetic heterogeneity. Utilizing the current gold standard of wholeexome sequencing for diagnosis, pathogenic gene variants are only identified in less than 20% of patients. While elucidation of the causal genes underlying PAD has provided many insights into the cellular and molecular mechanisms underpinning disease pathogenesis, many other genes may remain as yet undefined to enable definitive diagnosis, prognostic monitoring and targeted therapy of patients. Considering that many patients display a relatively late onset of disease presentation in their 2nd or 3rd decade of life, it is questionable whether a single genetic lesion underlies disease in all patients. Potentially, combined effects of other gene variants and/or non-genetic factors, including specific infections can drive disease presentation. In this review, we define (1) the clinical and immunological variability of PAD, (2) consider how genetic defects identified in PAD have given insight into B-cell immunobiology, (3) address recent technological advances in genomics and the challenges associated with identifying causal variants, and (4) discuss how functional validation of variants of unknown significance could potentially be translated into increased diagnostic rates, improved prognostic monitoring and personalized medicine for PAD patients. A multidisciplinary approach will be the key to curtailing the early mortality and high morbidity rates in this immune disorder.

Why the Outcome of Anti-Tumor Immune Responses is Heterogeneous: A Novel Idea in the Context of Immunological Heterogeneity in Cancers.

The question as to why some hosts can eradicate their tumors while others succumb to tumor-progression remains unanswered. Here, a provocative concept is proposed that intrinsic differences in the T cell receptor (TCR) repertoire of individuals may influence the outcome of anti-tumor immunity by affecting the frequency and/or variety of tumor-reactive CD8 and/or CD4 tumor-infiltrating lymphocytes. This idea implicates that the TCR repertoire in a given patient might not provide sufficiently different TCR clones that can recognize tumor antigens, namely, "a hole in the TCR repertoire" might exist. This idea may provide a novel perspective to further dissect the mechanisms underlying heterogeneous anti-tumor immune responses in different hosts. Besides tumor-intrinsic heterogeneity and host microbiome, the various factors that may constantly shape the dynamic TCR repertoire are also discussed. Elucidating mechanistic differences in different individuals' immune systems will allow to better harness immune system to design new personalized cancer immunotherapy.

A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaning diarrhea

Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG–FedF–FanC–FasA–Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG–FedF–FanC–FasA–Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

Neutrophil Extracellular Traps Generation Relates with Early Stage and Vascular Complications in Systemic Sclerosis.

Systemic sclerosis (SSc) is a systemic disease characterized by a great clinical and immunological heterogeneity whose pathophysiology is still being unraveled. Recently, innate immunity has been proposed to participate to the pathogenesis of SSc. In this study, we investigated the release of neutrophil extracellular traps (NETs) according to patient phenotype. Polymorphonuclear neutrophils (PMN) from 34 SSc patients and 26 healthy controls were stimulated by serum from SSc or healthy subject. NETs were visualized using epifluorescence microscope after DNA, myeloperoxidase, and Histone H3 tagging. Area of NETs were quantified using an original macro running in ImageJ® software. PMN from SSc patients were significantly more prone to releasing NETs than control PMN after autologous stimulation. PMN from patients with severe vascular complications (pulmonary arterial hypertension, digital ulcers) produced more NETs than PMN from other SSc patients and their aberrant NET production appeared to be sustained over time. In patients with pulmonary interstitial disease or extensive cutaneous fibrosis, NET production was high at an early stage of the disease before progressively decreasing. Both serum factors and PMN activation status were involved in the enhanced production of NETs in SSc. Consequently, neutrophils and especially NETosis represent new physiopathological and therapeutic fields in SSc.

Classification of diffuse lower‐grade glioma based on immunological profiling

Transcriptomic data derived from bulk sequencing have been applied to delineate the tumor microenvironment (TME) and define immune subtypes in various cancers, which may facilitate the design of immunotherapy treatment strategies. We herein gathered published gene expression data from diffuse lower‐grade glioma (LGG) patients to identify immune subtypes. Based on the immune gene profiles of 402 LGG patients from The Cancer Genome Atlas, we performed consensus clustering to determine robust clusters of patients, and evaluated their reproducibility in three Chinese Glioma Genome Atlas cohorts. We further integrated immunogenomics methods to characterize the immune environment of each subtype. Our analysis identified and validated three immune subtypes—Im1, Im2, and Im3—characterized by differences in lymphocyte signatures, somatic DNA alterations, and clinical outcomes. Im1 had a higher infiltration of CD8+ T cells, Th17, and mast cells. Im2 was defined by elevated cytolytic activity, exhausted CD8+ T cells, macrophages, higher levels of aneuploidy, and tumor mutation burden, and these patients had worst outcome. Im3 displayed more prominent T helper cell and APC coinhibition signatures, with elevated pDCs and macrophages. Each subtype was associated with distinct somatic alterations. Moreover, we applied graph structure learning‐based dimensionality reduction to the immune landscape and revealed significant intracluster heterogeneity with Im2 subtype. Finally, we developed and validated an immune signature with better performance of prognosis prediction. Our results demonstrated the immunological heterogeneity within diffuse LGG and provided valuable stratification for the design of future immunotherapy.