The circadian system temporally regulates physiology to maintain homeostasis. Co-opting and disrupting circadian signals appear to be distinct attributes that are functionally important for the development of a tumor and can enable or give rise to the hallmarks that tumors use to facilitate their initiation, growth and progression. Because circadian signals are also strong regulators of immune cell proliferation, trafficking and exhaustion states, they play a role in how tumors respond to immune-based cancer therapeutics. While immuno-oncology has heralded a paradigm shift in cancer therapeutics, greater accuracy is needed to increase our capability of predicting who will respond favorably to, or who is likely to experience the troubling adverse effects of, immunotherapy. Insights into circadian signals may further refine our understanding of biological determinants of response and help answer the fundamental question of whether certain perturbations in circadian signals interfere with the activity of immune checkpoint inhibitors. Here we review the body of literature highlighting circadian disruption as a cancer promoter and synthesize the burgeoning evidence suggesting circadian signals play a role in how tumors respond to immune-based anti-cancer therapeutics. The goal is to develop a framework to advance our understanding of the relationships between circadian markers, cancer biology, and immunotherapeutics. Bolstered by this new understanding, these relationships may then be pursued in future clinical studies to improve our ability to predict which patients will respond favorably to, and avoid the adverse effects of, traditional and immune-based cancer therapeutics.

LPS delays neutrophil apoptosis by a process generally assumed to involve cell-intrinsic TLR4 signaling. However, neutrophil survival responses to LPS have been reported to be monocyte-dependent, which would indicate more complexity than is currently appreciated. We compared the survival responses of conventionally purified vs highly purified neutrophils to confirm or refute the need for secondary cell-types and to identify the cellular or molecular mechanisms involved. Direct stimulation of TLR4 failed to extend the survival of highly purified neutrophils, but survival activity was retained in less pure neutrophil preparations containing low numbers of eosinophils, monocytes, platelets and CD3+ lymphocytes. Sequential depletions identified monocytes as the only cell type required. Transfer of culture supernatants after lipid A-conditioning revealed that purified monocytes were sufficient for production of nearly all of the survival activity observed in mixed populations. The survival factors secreted upon TLR4 stimulation remain unidentified, but were not correlated with IL-1β, IL-6 or TNF-α nor could survival activity be inhibited by Ab blockade of IL-8 or of several other candidate factors other than endogenously produced GM-CSF, which was responsible for about one-tenth of the survival activity present in conditioned supernatants. These observations confirm that ex vivo neutrophil survival responses to TLR4 agonists are not cell intrinsic and involve potentially novel factors secreted by TLR4-stimulated monocytes.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5′ RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5′ RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

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The Fc receptor for IgM, FcMR, is unusual in that it is preferentially expressed by cells of the adaptive immune system. It is, moreover, the only constitutively expressed Fc receptor on human T cells. Efforts to decipher the normal functions of FcMR have been complicated by species-specific expression patterns in lymphocytes from mice (B cells) versus humans (B, NK, and T cells). In human cells, FcMR cellsurface expression has been reported to be low at baseline ex vivo, with one suggested contribution being ligand-induced internalization by serum IgM. Indeed, preincubation overnight in IgM-free culture medium is recommended for studies of FcMR because surface display is increased under these conditions. We investigated FcMR display on human lymphocytes in PBMCs and found that, surprisingly, cell-surface FcMR was unaffected by IgM abundance and was instead downregulated in high-cell density cultures by a yet undefined mechanism. We further found that ex vivo processing of whole blood decreased surface FcMR, supporting the idea that FcMR expression is likely to be greater on circulating lymphocytes than previously appreciated. Collectively, these findings prompt new predictions of where and when FcMR might be available for functional interactions in vivo. ImmunoHorizons, 2022, 6: 47-63.