SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens

The baseline composition of T cells directly impacts later response to a pathogen, but the complexity of precursor states remains poorly defined. Here we examined the baseline state of SARS-CoV-2 specific T cells in unexposed individuals. SARS-CoV-2 specific CD4+ T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2 specific T cells that expressed memory phenotype markers, including memory cells with gut homing receptors. T cell clones generated from tetramer-labeled cells cross-reacted with bacterial peptides and responded to stool lysates in a MHC-dependent manner. Integrated phenotypic analyses revealed additional precursor diversity that included T cells with distinct polarized states and trafficking potential to other barrier tissues. Our findings illustrate a complex pre-existing memory pool poised for immunologic challenges and implicate non-infectious stimuli from commensal colonization as a factor that shapes pre-existing immunity.

Atomic scale symmetry and polar nanoclusters in the paraelectric phase of ferroelectric materials

The nature of the “forbidden” local- and long-range polar order in nominally non-polar paraelectric phases of ferroelectric materials has been an open question since the discovery of ferroelectricity in oxide perovskites, ABO3. A currently considered model suggests locally correlated displacements of B-site atoms along a subset of <111> cubic directions. Such off-site displacements have been confirmed experimentally; however, being essentially dynamic in nature they cannot account for the static nature of the symmetry-forbidden polarization implied by the macroscopic experiments. Here, in an atomically resolved study by aberration-corrected scanning transmission electron microscopy complemented by Raman spectroscopy, we reveal, directly visualize and quantitatively describe static, 2–4 nm large polar nanoclusters in the nominally non-polar cubic phases of (Ba,Sr)TiO3 and BaTiO3. These results have implications on understanding of the atomic-scale structure of disordered materials, the origin of precursor states in ferroelectrics, and may help answering ambiguities on the dynamic-versus-static nature of nano-sized clusters.

The spatial landscape of progression and immunoediting in primary melanoma at single cell resolution

Cutaneous melanoma is a highly immunogenic disease, surgically curable at early stages, but life-threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis from precursor states to melanoma in situ to invasive tumor. Hallmarks of immunosuppression are detectable by the precursor stage, and when tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary. This environment is established by cytokine gradients that promote expression of MHC-II and IDO1 and by PDL1-expressing macrophages and dendritic cells engaging activated T cells. However, a few mm away, T cells synapse with melanoma cells in fields of tumor regression. Thus, invasion and immunoediting can co-exist within a few millimeters of each other in a single specimen.

CHIP, CCUS, and Other Acronyms: Definition, Implications, and Impact on Practice

Unexplained blood cytopenias can be a clinical challenge for patients and clinicians alike. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes (MDS) is currently an area of active research. There have been marked developments in our understanding of clonal hematopoiesis based on findings of somatic mutations in genes known to be associated with MDS. This has led to newer terms to describe precursor states to MDS, such as clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). These conditions may allow earlier diagnosis, modify surveillance for MDS, and guide additional therapies. This review summarizes recent updates in the field for affected patients.

Molecular Characterization By Immune Profiling of Paired Blood and Bone Marrow in Multiple Myeloma and Its Precursor States

INTRODUCTION: Progression from precursor states, MGUS and smoldering multiple myeloma (SMM), to multiple myeloma (MM) is dependent upon adaptive and innate immune contexture shaped by cross-talk between malignant plasma cells and bone marrow (BM) milieu. The complexity and heterogeneity of interactions between the immune system and plasma cells in BM triggers alterations in peripheral blood (PB) immune cell subsets. The advantage of using PB as a surrogate is that dynamic changes in the immune cells can be measured at various time points during disease progression or therapeutic intervention. Here, we performed a comprehensive analysis of immune repertoire to identify immune signatures in PB and BM associated with MM or its precursor states. We also performed T cell receptor (TCR) clonotyping to quantify clonal expansion specific to each immunotype. METHODS: Paired PB and BM specimens were collected from patients with MGUS/SMM (n=12) and MM (n=16) through an IRB-approved biospecimen protocol. PB mononuclear cells and BM mononuclear cells were isolated for immune profiling. A total of 59 immune variables were analyzed by flow cytometry surveying 6 cell lineages' [NK, NK-T, Th, CTL, Treg and ɣδ T cells] distribution and functional status [activation, differentiation and anergy]. In addition, ArcherDx Immunoverse TCR αδ-βɣ CDR3 targeted NGS assay was performed to study clonal distributions of Vα24Jα18 NK-T, βα and ɣδ T cell. Univariate analyses (ANOVA) were performed using p<0.15 cutoff. Each set of variables (PB or BM) was then validated by multivariate analyses (Wilk's lambda) and used for unsupervised hierarchical analysis by WPGMA methods. Innate (NK-T, ɣδ T) and adaptive (βα T) mobilization for each cluster were finally confirmed by calculating Shannon's TCR clonal diversity index (SI). RESULTS: PB immunotyping identified 1 marker of innate inflammation and 9 markers of adaptive T mobilization that differentiated precursor states and MM (p=0.005). This model generated 3 PB immune clusters (Figure): cluster #1 [8 precursor states, 1 MM] showed a lack of innate inflammation and low Th/CTL mobilization, cluster #2 [2MGUS, 5MM] showed low innate inflammation and, cluster #3 [2SMM, 10MM] showed strong innate inflammation (Vɣ9-Vδ2-NKG2D+), Th terminal differentiation (central memory phenotype) and CTL anergy (Tim3+). TCR clonotyping confirmed increased innate inflammation (TCRδ SI 3.99±0.3 vs 4.75±0.15, p<0.05) and T cell mobilization (TCRα SI 7.12±0.3 vs. 8.20± 0.2, p<0.05) in PB cluster #3 compared with PB cluster #1. BM immunotyping identified 3 markers of innate inflammation and 2 markers of adaptive T mobilization (p=0.0274) distinguishing precursor states from MM. This model generated 3 BM immune clusters: cluster #1 [6 precursor states, 6 MM] showed innate inflammation (ɣδ T) and CTL terminal differentiation (central memory phenotype); cluster #2 [4 SMM, 8 MM] showed innate inflammation (NK-T, ɣδ T) and CTL effector anergy; and cluster #3 [2 MGUS, 2 MM] showed low NK cell cytotoxicity (KIR3DL1+) and CTL terminal differentiation. TCR clonotyping confirmed qualitative differences in innate inflammation between BM cluster #1 and #2 with higher NK-T (%Vα24Jα18 p<0.01) but lower ɣδ T (TCRδ SI 3.36±0.2 vs 4.57±0.2, p<0.05). In addition, CTL mobilization whether resulting in terminal differentiation or anergy in BM cluster #1 and #2, respectively was associated with similar clonal expansion of T cells (TCRα SI 7.21±0.26 vs. 7.87± 0.4, ns). Comparisons showed associations between PB and BM ɣδ T cell involvement in 13/13 patients. High PB Th/CTL mobilization (terminal differentiation) was associated with high T cell anergy in BM in 9/12 patients; conversely low PB Th/CTL mobilization was associated with low BM T cell involvement in 6/7 patients. CONCLUSION: This pilot study shows immune clustering of MGUS, SMM and MM patients based on BM and PB immunotypes. This is the first study to demonstrate two very distinct MM immunotypes based on low vs. high inflammatory states. We also show a high correlation between innate immune inflammation status in both PB and BM, specifically pertaining to ɣδ T cell, conventional T cell mobilization or lack thereof. Additional studies including a larger cohort for validation and longer follow up to establish correlation with clinical outcomes are currently underway. Figure. Figure. Disclosures Foureau: Teneobio Inc.: Research Funding. Berlin:ArcherDx: Employment. Johnson:ArcherDx: Employment. Williams:ArcherDx: Employment. Voorhees:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Other: served on an IRC; Amgen Inc.: Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; TeneoBio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Usmani:Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding; Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy.

Dissecting the Epigenetic Landscape of Smoldering, Newly Diagnosed and Relapsed Multiple Myeloma Revealed IRAK3 As a Marker of Disease Progression

Introduction. Multiple myeloma (MM) is a complex and heterogeneous malignancy of plasma cells that has two precursor states: monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). MGUS and SMM are asymptomatic states that eventually give rise to overt MM, with some patients progressing, while others do not. Recent studies in MM pathobiology have highlighted epigenetic alterations that contribute to the onset, progression and heterogeneity of MM. Global hypomethylation of DNA, including tumor suppressor genes, and hypermethylation of B-cell specific enhancers, abnormal histone methylation patterns due to the overexpression of histone methyltransferases such as MMSET, and deregulation of non-coding RNAs along with mutations in different classes of chromatin modulators underline a potential for epigenetic biomarkers in disease prognosis and treatment. This study aimed to define epigenetic pathways that lead to the dynamic regulation of gene expression in MM pathogenesis. Methods. We performed ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq on 10 MM cell lines and CD138+ plasma cells isolated from bone marrow aspirates of 3 healthy donors, 9 SMM, 8 newly diagnosed MM (NDMM) and 9 relapsed (RRMM) patients. ATAC-seq reads were trimmed of adapters, aligned to hg19 using bowtie2, and filtered for mapping quality >=Q30 using the ENCODE ATAC-seq pipeline. Reads mapping to promoter regions, defined as -400 to +250 bases from a refseq transcription start site, were counted using bedtools for each sample. Promoter read counts were then normalized by the total number of reads in promoters in the sample, scaled to 1 million total reads, and converted to log10(x+1) space. Results. To characterize the epigenetic contribution to disease progression in MM, we first identified accessible promoter regions in normal plasma cells (NPC), SMM, NDMM and RRMM patients and found regions displaying differential accessibility in MM progression. Next, we intersected the list of differential accessible regions (DARs) with matched transcriptome data and observed two main clusters: genes with unaltered transcription profiles and genes in which the dynamics of open chromatin regions (OCRs) correlated with gene expression. Transcriptomic analysis revealed that a large portion of the differentially expressed (DE) genes in SMM remain DE in NDMM as compared to NPCs (882 genes out of 1642 and 1150 DE genes in SMM and NDMM, respectively). Those genes were significantly enriched for pathways like epithelial mesenchymal transition, cell cycle checkpoints and mitosis, KRAS signaling and interleukin-JAK-STAT pathways. To investigate the genes that behaved differently among the stages of disease, we looked at differential accessibility and expression in NDMM and SMM samples, and integrated them with Whole-Genome Bisulfite-Sequencing and 450K DNA-methylation data from MM patients and healthy donors (BLUEPRINT). This analysis led to the identification of novel genes in MM progression, such as the transcriptional repressor ZNF254 and IRAK3, a negative regulator of the TLR/IL1R signaling pathway. Although gene expression data for these genes showed comparable mRNA levels in SMM and NPCs, followed by a significant decrease in NDMM/ RRMM, ATAC-seq revealed a striking drop in promoter accessibility in SMM, NDMM and RRMM cases. Comparison of ATAC-seq peaks to DNA methylation and ChIP-seq data revealed that the altered OCR of IRAK3 is actually hypermethylated in MM patients and marked by H3K4me3, a marker of active promoters, in MM cell lines. Hypermethylation of IRAK3 has been described in hepatocellular carcinoma, where it is associated with poor prognosis. Together, our data suggest that the identified IRAK3 OCR may act as a bivalent domain that loses accessibility in the precursor states and gains DNA methylation in MM progression. Hence, IRAK3 methylation could be a novel prognostic marker in MM. Conclusion. We have generated a global epigenetic map of primary tumors from patients at the smoldering, newly diagnosed and relapsed/refractory stage of multiple myeloma. Integrative analysis of ATAC-seq data with DNA methylome, transcriptome and whole-genome map of active and repressive histone marks in our study led to the identification of IRAK3 as a novel epigenetic biomarker of disease progression. Disclosures Licht: Celgene: Research Funding. Ghobrial:Takeda: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy.