Counterproductive effects of anti-CD38 and checkpoint inhibitor for the treatment of NK/T cell lymphoma.

Introduction: Natural killer/T cell lymphoma (NKTL) is an aggressive malignancy associated with poor prognosis. This is largely due to limited treatment options, especially for relapsed patients. Immunotherapies like immune checkpoint inhibitors (ICI) and anti-CD38 therapies have shown promising but variable clinical efficacies. Combining these therapies has been suggested to enhance efficacy. Methods: We conducted a case study on a relapsed NKTL patient treated sequentially with anti-CD38 followed by ICI (anti-PD1) using cytometry analyses. Results and Discussion: Our analysis showed an expected depletion of peripheral CD38+ B cells following anti-CD38 treatment. Further analysis indicated that circulating anti-CD38 retained their function for up to 13 weeks post-administration. Anti-PD1 treatment triggered re-activation and upregulation of CD38 on the T cells. Consequently, these anti-PD1-activated T cells were depleted by residual circulating anti-CD38, rendering the ICI treatment ineffective. Finally, a meta-analysis confirmed this counterproductive effect, showing a reduced efficacy in patients undergoing combination therapy. In conclusion, our findings demonstrate that sequential anti-CD38 followed by anti-PD1 therapy leads to a counterproductive outcome in NKTL patients. This suggests that the treatment sequence is antithetic and warrants re-evaluation for optimizing cancer immunotherapy strategies.

Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma.

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

Analysis of archaic human haplotypes suggests that 5hmC acts as an epigenetic guide for NCO recombination.

BACKGROUND: Non-crossover (NCO) refers to a mechanism of homologous recombination in which short tracks of DNA are copied between homologue chromatids. The allelic changes are typically restricted to one or few SNPs, which potentially allow for the gradual adaptation and maturation of haplotypes. It is assumed to be a stochastic process but the analysis of archaic and modern human haplotypes revealed a striking variability in local NCO recombination rates. METHODS: NCO recombination rates of 1.9 million archaic SNPs shared with Denisovan hominids were defined by a linkage study and correlated with functional and genomic annotations as well as ChIP-Seq data from modern humans. RESULTS: We detected a strong correlation between NCO recombination rates and the function of the respective region: low NCO rates were evident in introns and quiescent intergenic regions but high rates in splice sites, exons, 5'- and 3'-UTRs, as well as CpG islands. Correlations with ChIP-Seq data from ENCODE and other public sources further identified epigenetic modifications that associated directly with these recombination events. A particularly strong association was observed for 5-hydroxymethylcytosine marks (5hmC), which were enriched in virtually all of the functional regions associated with elevated NCO rates, including CpG islands and 'poised' bivalent regions. CONCLUSION: Our results suggest that 5hmC marks may guide the NCO machinery specifically towards functionally relevant regions and, as an intermediate of oxidative demethylation, may open a pathway for environmental influence by specifically targeting recently opened gene loci.

A genomic-augmented multivariate prognostic model for the survival of natural-killer/T-cell lymphoma patients from an international cohort.

With lowering costs of sequencing and genetic profiling techniques, genetic drivers can now be detected readily in tumors but current prognostic models for Natural-killer/T cell lymphoma (NKTCL) have yet to fully leverage on them for prognosticating patients. Here, we used next-generation sequencing to sequence 260 NKTCL tumors, and trained a genomic prognostic model (GPM) with the genomic mutations and survival data from this retrospective cohort of patients using LASSO Cox regression. The GPM is defined by the mutational status of 13 prognostic genes and is weakly correlated with the risk-features in International Prognostic Index (IPI), Prognostic Index for Natural-Killer cell lymphoma (PINK), and PINK-Epstein-Barr virus (PINK-E). Cox-proportional hazard multivariate regression also showed that the new GPM is independent and significant for both progression-free survival (PFS, HR: 3.73, 95% CI 2.07-6.73; p < .001) and overall survival (OS, HR: 5.23, 95% CI 2.57-10.65; p = .001) with known risk-features of these indices. When we assign an additional risk-score to samples, which are mutant for the GPM, the Harrell's C-indices of GPM-augmented IPI, PINK, and PINK-E improved significantly (p < .001, χ2 test) for both PFS and OS. Thus, we report on how genomic mutational information could steer toward better prognostication of NKTCL patients.

Parkinson's Disease-Specific Autoantibodies against the Neuroprotective Co-Chaperone STIP1.

Parkinson's disease (PD) is a debilitating movement disorder characterised by the loss of dopaminergic neurons in the substantia nigra. As neuroprotective agents mitigating the rate of neurodegeneration are unavailable, the current therapies largely focus only on symptomatic relief. Here, we identified stress-inducible phosphoprotein 1 (STIP1) as a putative neuroprotective factor targeted by PD-specific autoantibodies. STIP1 is a co-chaperone with reported neuroprotective capacities in mouse Alzheimer's disease and stroke models. With human dopaminergic neurons derived from induced pluripotent stem cells, STIP1 was found to alleviate staurosporine-induced neurotoxicity. A case-control study involving 50 PD patients (average age = 62.94 ± 8.48, Hoehn and Yahr >2 = 55%) and 50 age-matched healthy controls (HCs) (average age = 63.1 ± 8) further revealed high levels of STIP1 autoantibodies in 20% of PD patients compared to 10% of HCs. Using an overlapping peptide library covering the STIP1 protein, we identified four PD-specific B cell epitopes that were not recognised in HCs. All of these epitopes were located within regions crucial for STIP1's chaperone function or prion protein association. Our clinical and neuro-immunological studies highlight the potential of the STIP1 co-chaperone as an endogenous neuroprotective agent in PD and suggest the possible involvement of autoimmune mechanisms via the production of autoantibodies in a subset of individuals.

Assessment of T-cell Reactivity to the SARS-CoV-2 Omicron Variant by Immunized Individuals.

Importance: The emergence of the highly contagious Omicron variant of SARS-CoV-2 and the findings of a significantly reduced neutralizing potency of sera from individuals with previous SARS-CoV-2 infection or vaccination highlights the importance of studying cellular immunity to estimate the degree of immune protection to the new SARS-CoV-2 variant. Objective: To determine T-cell reactivity to the Omicron variant in individuals with established (natural and/or vaccine-induced) immunity to SARS-CoV-2. Design, Setting, and Participants: This was a cohort study conducted between December 20 and 21, 2021, at the Santa Lucia Foundation Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy, among health care worker and scientist volunteers. Lymphocytes from freshly drawn blood samples were isolated and immediately tested for reactivity to the spike protein of SARS-CoV-2. Main Outcomes and Measures: The main outcomes were the measurement of T-cell reactivity to the mutated regions of the spike protein of the Omicron BA.1 SARS-CoV-2 variant and the assessment of remaining T-cell immunity to the spike protein by stimulation with peptide libraries. Results: A total of 61 volunteers (mean (range) age, 41.62 (21-62) years; 38 women [62%]) with different vaccination and SARS-CoV-2 infection backgrounds were enrolled. The median (range) frequency of CD4+ T cells reactive to peptides covering the mutated regions in the Omicron variant was 0.039% (0%-2.356%), a decrease of 64% compared with the frequency of CD4+ cells specific for the same regions of the ancestral strain (0.109% [0%-2.376%]). Within CD8+ T cells, a median (range) of 0.02% (0%-0.689%) of cells recognized the mutated spike regions, while 0.039% (0%-3.57%) of cells were reactive to the equivalent unmutated regions, a reduction of 49%. However, overall reactivity to the peptide library of the full-length protein was largely maintained (estimated 87%). No significant differences in loss of immune recognition were identified between groups of participants with different vaccination or infection histories. Conclusions and Relevance: This cohort study of immunized adults in Italy found that despite the mutations in the spike protein, the SARS-CoV-2 Omicron variant was recognized by the cellular component of the immune system. It is reasonable to assume that protection from hospitalization and severe disease will be maintained.

Histone acetylome-wide associations in immune cells from individuals with active Mycobacterium tuberculosis infection.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of an infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of host response to pathogens.

Data-Driven Analysis of COVID-19 Reveals Persistent Immune Abnormalities in Convalescent Severe Individuals.

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of "long COVID-19", and defines key cells and cytokines that delineate true and quasi-convalescent states.

Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma.

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1ß inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

FUT6 deficiency compromises basophil function by selectively abrogating their sialyl-Lewis x expression.

Sialyl-Lewis x (sLex, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLex expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLex+ and sLex- basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts.

Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2.

The immune responses and mechanisms limiting symptom progression in asymptomatic cases of SARS-CoV-2 infection remain unclear. We comprehensively characterized transcriptomic profiles, cytokine responses, neutralization capacity of antibodies, and cellular immune phenotypes of asymptomatic patients with acute SARS-CoV-2 infection to identify potential protective mechanisms. Compared to symptomatic patients, asymptomatic patients had higher counts of mature neutrophils and lower proportion of CD169+ expressing monocytes in the peripheral blood. Systemic levels of pro-inflammatory cytokines were also lower in asymptomatic patients, accompanied by milder pro-inflammatory gene signatures. Mechanistically, a more robust systemic Th2 cell signature with a higher level of virus-specific Th17 cells and a weaker yet sufficient neutralizing antibody profile against SARS-CoV-2 was observed in asymptomatic patients. In addition, asymptomatic COVID-19 patients had higher systemic levels of growth factors that are associated with cellular repair. Together, the data suggest that asymptomatic patients mount less pro-inflammatory and more protective immune responses against SARS-CoV-2 indicative of disease tolerance. Insights from this study highlight key immune pathways that could serve as therapeutic targets to prevent disease progression in COVID-19.

T-Cell Lymphoma Clonality by Copy Number Variation Analysis of T-Cell Receptor Genes.

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

Viral Dynamics and Immune Correlates of Coronavirus Disease 2019 (COVID-19) Severity.

BACKGROUND: Key knowledge gaps remain in the understanding of viral dynamics and immune response of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. METHODS: We evaluated these characteristics and established their association with clinical severity in a prospective observational cohort study of 100 patients with PCR-confirmed SARS-CoV-2 infection (mean age, 46 years; 56% male; 38% with comorbidities). Respiratory samples (n = 74) were collected for viral culture, serum samples for measurement of IgM/IgG levels (n = 30), and plasma samples for levels of inflammatory cytokines and chemokines (n = 81). Disease severity was correlated with results from viral culture, serologic testing, and immune markers. RESULTS: Fifty-seven (57%) patients developed viral pneumonia, of whom 20 (20%) required supplemental oxygen, including 12 (12%) with invasive mechanical ventilation. Viral culture from respiratory samples was positive for 19 of 74 patients (26%). No virus was isolated when the PCR cycle threshold (Ct) value was >30 or >14 days after symptom onset. Seroconversion occurred at a median (IQR) of 12.5 (9-18) days for IgM and 15.0 (12-20) days for IgG; 54/62 patients (87.1%) sampled at day 14 or later seroconverted. Severe infections were associated with earlier seroconversion and higher peak IgM and IgG levels. Levels of IP-10, HGF, IL-6, MCP-1, MIP-1α, IL-12p70, IL-18, VEGF-A, PDGF-BB, and IL-1RA significantly correlated with disease severity. CONCLUSIONS: We found virus viability was associated with lower PCR Ct value in early illness. A stronger antibody response was associated with disease severity. The overactive proinflammatory immune signatures offer targets for host-directed immunotherapy, which should be evaluated in randomized controlled trials.

Atopic dermatitis microbiomes stratify into ecologic dermotypes enabling microbial virulence and disease severity.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease affecting up to 20% of the global population, with significant clinical heterogeneity and limited information about molecular subtypes and actionable biomarkers. Although alterations in the skin microbiome have been described in subjects with AD during progression to flare state, the prognostic value of baseline microbiome configurations has not been explored. OBJECTIVE: Our aim was to identify microbial signatures on AD skin that are predictive of disease fate. METHODS: Nonlesional skin of patients with AD and healthy control subjects were sampled at 2 time points separated by at least 4 weeks. Using whole metagenome analysis of skin microbiomes of patients with AD and control subjects (n = 49 and 189 samples), we identified distinct microbiome configurations (dermotypes A and B). Blood was collected for immunophenotyping, and skin surface samples were analyzed for correlations with natural moisturizing factors and antimicrobial peptides. RESULTS: Dermotypes were robust and validated across 2 additional cohorts (63 individuals), with strong enrichment of subjects with AD in dermotype B. Dermotype B was characterized by reduced microbial richness, depletion of Cutibacterium acnes, Dermacoccus and Methylobacterium species, individual-specific outlier abundance of Staphylococcus species (eg, S epidermidis, S capitis, S aureus), and enrichment in metabolic pathways (eg, branched chain amino acids and arginine biosynthesis) and virulence genes (eg, ß-toxin, δ-toxin) that defined a pathogenic ecology. Skin surface and circulating host biomarkers exhibited a distinct microbial-associated signature that was further reflected in more severe itching, frequent flares, and increased disease severity in patients harboring the dermotype B microbiome. CONCLUSION: We report distinct clusters of microbial profiles that delineate the role of microbiome configurations in AD heterogeneity, highlight a mechanism for ongoing inflammation, and provide prognostic utility toward microbiome-based disease stratification.