Cytokine release syndrome and cancer immunotherapies - historical challenges and promising futures.

Cancer is the leading cause of death worldwide. Cancer immunotherapy involves reinvigorating the patient\u27s own immune system to fight against cancer. While novel approaches like Chimeric Antigen Receptor (CAR) T cells, bispecific T cell engagers, and immune checkpoint inhibitors have shown promising efficacy, Cytokine Release Syndrome (CRS) is a serious adverse effect and remains a major concern. CRS is a phenomenon of immune hyperactivation that results in excessive cytokine secretion, and if left unchecked, it may lead to multi-organ failure and death. Here we review the pathophysiology of CRS, its occurrence and management in the context of cancer immunotherapy, and the screening approaches that can be used to assess CRS and de-risk drug discovery earlier in the clinical setting with more predictive pre-clinical data. Furthermore, the review also sheds light on the potential immunotherapeutic approaches that can be used to overcome CRS associated with T cell activation

Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighborhoods

Typically, eukaryotic nuclei contain 10–30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain “choice” appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3–4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments (∼5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic “neighborhoods.

Folding and organization of a contiguous chromosome region according to the gene distribution pattern in primary genomic sequence

Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization

Replication-dependent Histone Gene Expression Is Related to Cajal Body (CB) Association but Does Not Require Sustained CB Contact

Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3′ end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3′ end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci

Spectral karyotyping reveals pervasive aneuploidy in <i>Lmna^Dhe/+</i> fibroblasts.

<p>(A) Histogram of chromosome numbers in <i>Lmna^Dhe/+</i> (red bars) and <i>Lmna^+/+</i> (blue bars) dermal fibroblasts, as determined by metaphase spreading and SKY. Over 90% of mutant cells possessed an abnormal number of chromosomes ranging from 38 to 104. (B and C) Representative spectral karyotypes of <i>Lmna^+/+</i> (B) and <i>Lmna^Dhe/+</i> (C) cells. The wild-type cell exhibited a normal karyotype of 40XY (B), while the mutant cell was aneuploid with a 104XXXXX karyotype (C).</p

Mitotic progression in <i>Lmna^+/+</i> and <i>Lmna^Dhe/+</i> cells.

1<p>Mitotic stages determined in fibroblasts immunofluorescently labeled with anti-Lamin A and anti-α-Tubulin antibodies and DAPI counterstain.</p>2<p>Total number of cells from two experiments.</p>3<p>p-values were computed using the χ²-test.</p

Cell cycling properties of <i>Lmna^Dhe/+</i> fibroblasts.

1<p>Means and standard deviations for cultures from three mice are reported.</p>2<p>Culture growth over four days measured by static adhesion assay.</p>3<p>Proportion of all cells positive for SA-β-gal fluorescence versus total cells counted by flow cytometry.</p>4<p>Cycling cells identified by Ki-67 immunostaining.</p>5<p>Apoptosis measured by Annexin V labeling and flow cytometry.</p>6<p>Mitotic indices determined from cells stained with anti-Lamin A and anti-α-Tubulin antibodies and DAPI.</p>7<p>p-values were computed using Student's t-test.</p

<i>Lmna^Dhe/+</i> fibroblasts exhibit lower Lamin A levels, but no change in Lamin B expression.

<p>(A–D) Western blot analysis of insoluble Lamin B (A), insoluble Lamin A (B), soluble Lamin A (C) and soluble Prelamin A (D). These data indicate no difference in Lamin B protein levels between <i>Lmna^+/+</i>and mutant cells, but less soluble Lamin A/C, insoluble Lamin A/C and Prelamin A protein in mutant cells. α-Tubulin was used as a loading control for all western blots.</p