Diversity of Intratumoral Regulatory T Cells in Non-Hodgkin Lymphoma

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity. Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015). Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL. Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. Disclosures Holte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Contactless Detection of Natural Bending Frequencies using Embedded Metallic-Glass Ribbons inside Plastic Beams made of 3-D Printing

In the current work, to identify the bending mode harmonics, 30 microns thin magnetoelastic ribbons made of metallic glass are embedded inside 6 mm thick PLA plastic cantilever beams made by 3-D printing. This is possible because the ribbons are of magnetoelastic nature and thus change their mechanical state inducing a corresponding change in their magnetic state. The ribbons are placed at four different depths, starting with zero depth at the beam’s external surface all the way inside to the beam’s mid-plane. This technique is capable of detecting seven harmonics, and remarkably, these frequencies remain the same within a marginal error of 1% for all the depths. The amplitude of the modes drops with the increase in depth but is still strong enough, except at the midplane, to be used as a sensing signal. The harmonics spectrum is the unique signature of the structure’s state; this is a proof of concept that in a contactless fashion, the embedded ribbons provide useful information about the mechanical health of a structure.

Genomic and Transcriptomic Characterization of IgM Multiple Myeloma Identifies a Pre-Germinal Center Plasma Cell Disorder with Immature B-Cell Transcription-Factor Signature

Multiple myeloma (MM) is a proliferation of terminally differentiated plasma cells (PC) producing monoclonal immunoglobulins (Ig), most commonly IgG and IgA (50% and 25% respectively), and less frequently, light-chain only disease, non-secretory, and IgD. IgM-MM is a rare entity (<0.5%), and its differentiation from common IgM producing PC disorders like Waldenström's macroglobulinemia (WM) is essential considering their distinct treatments and prognoses. Recent advancements in molecular techniques have shed light on the genomic characteristics and unique alterations in MM and WM, however, comprehensive profiling is still lacking for IgM-MM. We performed deep whole-genome sequencing on five IgM samples as well as 211 MM and 34 WM samples, and transcriptome sequencing on the IgM samples as well as 30 MM, 35 WM, and 3 PC. All IgM-MM samples harbored t(11;14) which combines super enhancers in Ig genes with CCND1. All translocations involved VHDHJH regions (Figure 1A) at the immunoglobulin heavy chain (IGH) locus, compared to IgG/IgA MM samples that had predominantly switch-region translocations (Figure 1B/C). Switch-region translocations are generated through class-switch recombination (CSR) in mature B-cells in germinal centers (GC), and VHDHJH translocations occur during recombination at the early pro-B-cell stage in the bone marrow (BM). While IgG/IgA-MM displayed evidence of CSR with deletions between IGHM switch-region and IGHG/IGHA switch regions, IgM-MM had no such events. IgM-MM therefore appears to undergo malignant transformation prior to late-stage B-cell maturation, after which CSR is unlikely, which is supported by a lack of progression of IgM-monoclonal gammopathy of undetermined significance (MGUS) to non-IgM-MM. IgM-MM also displayed similar copy number variation (CNV) patterns and driver mutations compared to non-IgM-MM suggesting similar progression events. Unsupervised hierarchical clustering using differentially expressed genes between non-IgM-MM and WM separated the IgM-MM samples within non-IgM-MM. This indicates a closer molecular homology to MM compared to WM with a unique signature for this group not accounted for by the t(11;14) translocation. Running the same analysis using only B-cell specific transcription factors (TFs) yielded similar results, with separation of WM and MM and preferential clustering of IgM-MM within the latter while also exhibiting a unique signature (Figure 1D). Some noteworthy examples were the upregulation of PBX3, PAX5, BCL11A, and ATF2, and the downregulation of PRDM1 and BCL3 compared to non-IgM-MM. The loss of PAX5 and upregulation of PRDM1 in B-cells has been implicated in promoting commitment to PC differentiation, while BCL11A was found essential for early B-cell progenitor development through the GC but extinguished in terminally differentiated PC. It appears that IgM-MM has therefore a more immature phenotype compared with non-IgM-MM, which further supports the previously discussed findings of its pre-GC origin and lack of terminal development. Three clinically relevant targets were noted to be upregulated in IgM-MM, Bruton's tyrosine kinase (BTK), CD20 and BCL-2. BTK was significantly higher in IgM-MM compared to non-IgM-MM (log2fold=1.3; FDR<10-3) with no difference between IgM-MM and WM (log2fold =-0.4; FDR>0.2). This could elucidate a more prominent role for BTK-inhibition in the IgM-MM subgroup. Furthermore, as documented in t(11;14)-MM, IgM-MM had elevated transcript levels of CD20 with possible targeting using anti-CD20 antibodies. Finally, elevated levels of BCL-2 in both IgM and non-IgM-t(11;14)-MM were observed, an established target for both single-agent and combination therapy. Interestingly, although not significant, IgM-MM had lower transcript levels of BCL-XL and MCL-1 compared to non-IgM-t(11;14)-MM, believed to be a predictor of higher sensitivity to venetoclax, and therefore an important guide for treatment choice. Clinical data however is lacking, and further investigations are needed to fully understand the potential role of these drugs in treating IgM-MM. In summary we describe a unique genomic and transcriptomic profile of IgM-MM, compared to both non-IgM-MM and WM, that describes its cellular origin and provides the rationale for potential therapeutic intervention. Disclosures Fulciniti: NIH: Research Funding. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Parmigiani:Phaeno Biotehnologies: Current equity holder in publicly-traded company; CRA Health: Current equity holder in publicly-traded company; Foundation Medicine Institute: Consultancy; Delphi Diagnostics: Consultancy; BayesMendel Laboratory: Other: Co-lead. Treon:Bristol-Meyer-Squibb: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding. Mohty:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Munshi:Takeda: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy.

Exploring the Applications of the Photoprotective Properties of Anthocyanins in Biological Systems

Due to their physical and chemical characteristics, anthocyanins are amongst the most versatile groups of natural compounds. Such unique signature makes these compounds a focus in several different areas of research. Anthocyanins have well been reported as bioactive compounds in a myriad of health disorders such as cardiovascular diseases, cancer, and obesity, among others, due to their anti-inflammatory, antioxidant, anti-diabetic, anti-bacterial, and anti-proliferative capacities. Such a vast number of action mechanisms may be also due to the number of structurally different anthocyanins plus their related derivatives. In this review, we highlight the recent advances on the potential use of anthocyanins in biological systems with particular focus on their photoprotective properties. Topics such as skin aging and eye degenerative diseases, highly influenced by light, and the action of anthocyanins against such damages will be discussed. Photodynamic Therapy and the potential role of anthocyanins as novel photosensitizers will be also a central theme of this review.

Nudging the N170 forward with prior stimulation – Bridging the gap between N170 and recognition potential

Some neural responses are classified by the stimulus conditions leading up to that response while other neural responses are also classified by the morphology of the responses themselves. However, morphology-based classification may not be appropriate if one can nudge a neural response into looking like another neural response. Morphology-based classification occurs with the N170 and RP (Recognition Potential), ERP components that are studied in separate literatures and yet share much in common in terms of functionality. In this study, we demonstrate a gradual transformation in the morphology of the N170 to the RP using a simple parametric manipulation of forward masks that is unlikely to cause a change in the underlying processing. Both the N170 and RP are N1 components, meaning that they are the first negative deflection of the evoked response. However, the RP is often measured with a forward mask that ends at stimulus onset whereas the N170 is often measured with no masking at all. This study investigates how ISI may delay and distort the N170 into an RP by manipulating the temporal gap (ISI) between forward mask and target. The results revealed reverse relationships between the ISI on the one hand, and the N170 latency, single-trial N1 jitter (an approximation of N1 width) and reaction time on the other hand. Importantly, we find that scalp topographies have a unique signature at the N1 peak across all conditions, from the longest gap (N170) to the shortest (RP). These findings prove that the mask-delayed N1 is still the same N170, even under conditions that are normally associated with a different component like the RP. In general, our results suggest that greater caution should be taken to interpret the time course of a measured effect when forward masks are employed.