Bathyal Research Articles

Therapeutic Targeting of HDAC3 in FOXO1-Mutant DLBCL

Histone deacetylase inhibitors have tremendous potential as cancer therapeutics, yet as single agents they have had limited success in the clinic outside of cutaneous T cell lymphoma and multiple myeloma. One of the major issues is toxicity, as the current compounds are broad-spectrum inhibitors that target all of the class 1 HDACs (HDAC1-3 and 8). Thus, using more selective inhibitors could have a major impact in the right genetic landscape. HDAC3-selective inhibitors are being developed and this deacetylase targets both histone and non-histone substrates in conjunction with its co-factors, NCOR or SMRT. NCOR or SMRT bind to and activate HDAC3, and this association is mediated by an inositol phosphate-4 (IP4) molecule, suggesting that HDAC3 is a sensor of cellular metabolism in the same way that Sirtuins (Class 3 HDACs) sense NAD/NADH levels.Diffuse large B cell lymphoma (DLBCL) is a particularly aggressive form of non-Hodgkin lymphoma (NHL), accounting for approximately 30% of new NHL diagnoses. Recurrent activating mutations in the transcription factor, FOXO1, are present in nearly 10% of DLBCLs at diagnosis. Importantly, these mutations are associated with a poor response to standard therapy, indicating a pressing need for the development of novel treatments for FOXO1 mutant disease. While direct therapeutic targeting of transcription factors has proven largely unsuccessful, indirect disruption of transcription factor function through the inhibition of associated chromatin-modifying enzymes has shown promise. Therefore, a mechanistic understanding of FOXO1 transcriptional complexes relevant for B cell function and DLBCL initiation and/or maintenance will provide insights for the development of new strategies for targeting FOXO1 mutant lymphoma.The germinal center (GC) reaction is critical for the development of high affinity, class-switched antibodies and provides the cell of origin for the majority of diffuse large B cell lymphomas (DLBCLs). We used conditional Hdac3 deletion to assess the requirement of Hdac3 for germinal center formation in mice. In contrast to Bcl6, which is required for germinal center formation and recruits Hdac3, we found that Hdac3 -deficient cells gave rise to germinal centers, albeit in fewer numbers. RNA-seq analysis of Hdac3-/- GC B cells revealed an upregulation of only a subset of Bcl6-regulated genes associated with terminal plasma cell differentiation (e.g. Prdm1, Xbp1) without any changes in other canonical Bcl6 target genes associated with DNA damage checkpoints (e.g. Trp53, Atr). In addition, deletion of Hdac3 resulted in a dramatic reduction in the numbers of GC dark zone B cells and an accumulation of cells as light zone centrocytes. This phenotype, as well as the associated gene expression changes, bore striking similarity to that found upon deletion of Foxo1 from germinal centers. Therefore, we performed further transcriptional analyses in germinal center-derived DLBCL to examine the coordination of FOXO1 and HDAC3 function at shared gene targets.We demonstrate that HDAC3 and FOXO1 associate and disruption of HDAC3 function in DLBLC cells by a selective small molecule led to the upregulation of a FOXO1-regulated gene signature. ChIP-exo revealed localization of FOXO1 at critical BCL6:SMRT-bound enhancers. Furthermore, precision nuclear run-on transcription sequencing (PRO-seq) showed that inhibition of HDAC3 caused an increase in eRNA synthesis that was associated with increased H3K27ac at these enhancers and accompanied an increase in gene body transcription rates. Combined, studies in mouse germinal centers and DLBCL cell lines suggest that HDAC3 is critical for FOXO1-mediated transcriptional control. Therefore, we treated a panel of human DLBCL cell lines with an HDAC3-selective inhibitor, RGFP966, and examined its effect on cell growth and viability. While the response of DLBCL cells to HDAC3 inhibition was highly varied, those cell lines with FOXO1 mutations were the most sensitive to RGFP966 treatment. Thus, HDAC3 regulates FOXO1 gene targets within germinal centers and diffuse large B cell lymphoma, and HDAC3 is a promising therapeutic target for FOXO1-mutant disease. DisclosuresNo relevant conflicts of interest to declare.

A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.

Caribbochiton guadeloupensis n. gen et n. sp. (Mollusca: Polyplacophora) from the Caribbean Sea

ABSTRACT A new genus and new species of the family Callistoplacidae from the bathyal zone near Guadeloupe Island in the Caribbean Sea are described. Caribbochiton guadeloupensis n. gen. et n. sp. is unlike other genera in the family Callistoplacidae: it has no ribs on end valves or on lateral areas of intermediate valves. According to other morphological features (thick shell, relatively narrow valves, noticeably raised lateral areas, similar slit formula, insertion plate teeth thickened at edges of slits, dorsal scales), this genus is closest to the family Callistoplacidae. Given that a number of species of the family do not have at a young age the ribs which appear later, we hypothesise that the new genus could have evolved as a result of paedomorphosis. An emended diagnosis of the family Callistoplacidae is hereby proposed. ZooBank registration: LSIDurn:lsid:zoobank.org:pub:BB94DD8F-C466-4B1B-8868-EEAB75E6930B Carribochiton LSIDurn:lsid:zoobank.org:act:EBD4411B-C39D-446C-AD24-EB5F3A27AB32 Carribochiton guadeloupensis LSIDurn:lsid:zoobank.org:act:19B8AF77-2AB8-4BD1-940A-CA4EAFFCF215

Arthropoda fauna of Zindan Cave (Isparta, Turkey) with notes on new records and some ecological characteristics

The aims of the study were to investigate the Arthropoda fauna of Zindan Cave (Aksu, Isparta Province, Turkey) and to consider some ecological characteristics of the collected species such as feeding habits, cave-dwelling categories, and zone distributions in caves. Twenty-seven species of Arthropoda were determined between May 2015 and December 2016, from Zindan Cave. The species found belong to the following orders: nine to Collembola, six to Coleoptera, five to Araneae, two to Diplopoda and one to Orthoptera, Trichoptera, Diptera, Trombidiformes and Isopoda. Nine species are endemic of Turkey. Traegaardhia distosolenidia Zacharda, 2010 (Acari: Rhagidiidae) and Folsomia asiatica Martynova, 1971 (Collembola: Isotomidae) are newly recorded for the fauna of Turkey. The presence of Heteromurus sexoculatus Brown, 1926 (Collembola: Entomobryidae) in Turkey is confirmed. Twenty-four of the 27 species were from the dark zone and half of the collected species (6 troglobites and 8 troglophiles) are ecologically adapted to cave ecosystems. Species can be divided into three groups according to trophic preferences: 15 scavengers, 10 predators and two omnivores. Food habits, cave-dwelling categories, and zone distributions of collected species are discussed.

Hawaiian Paratachys Casey (Coleoptera, Carabidae): small beetles of sodden summits, stony streams, and stygian voids.

Five Hawaiian species of Paratachys Casey are revised, including four newly described: Paratachys terryli from Kauai; P. perkinsi from Moloka‘i; P. haleakalae from Maui; and P. aaa from Hawai‘i Island. A lectotype is designated for the fifth Hawaiian species currently combined with Paratachys, Tachys arcanicola Blackburn, 1878 of Oahu. Hawaiian Paratachys spp. known from more than one specimen exhibit some degree of ocular polymorphism, that variation being extreme in P. terryli where individuals range in ocular development from macrophthalmic with broadly convex eyes to microphthalmic with small, flat eyes. All Hawaiian Paratachys species comprise individuals with vestigial wings, with the exception of P. terryli, where a single macropterous, macrophthalmic female complements the other 18 brachypterous specimens. Based on a transformation series of characters from the male aedeagus, the biogeographic history of Hawaiian Paratachys is consistent with progressive colonization of the Hawaiian Island chain. Three of the species do not appear to represent species of conservation concern, with P. terryli and P. haleakalae known from terrestrial deep soil, litter, and streamside microhabitats in montane wet rain forest, and the troglobitic P. aaa occupying the dark zone of numerous, recently developed lava tube caves within the Mauna Loa and Kilauea volcanic massifs. The conservation status of the other two species is much more dire, with P. arcanicola of O‘ahu not seen in nature since the early 20th Century, and P. perkinsi known only from a single specimen fortuitously collected in 1894 near sea level on Moloka‘i.

Single Cell Clonal Analysis Identifies an AID-Dependent Pathway of Plasma Cell Differentiation

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody secreting plasma cells. Activation Induced Deaminase (AID) initiates antibody diversification in GCs by somatic hypermutation and class switch recombination. Here we have addressed the role of AID in the terminal differentiation of GC B cells by combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells. We identified 8 transcriptional clusters that include dark zone and light zone GC subsets, plasmablasts/plasma cells (PB), 4 subsets of MBCs and a novel prePB subset, which shares the strongest clonal relationships with PBs. Mice lacking AID have various alterations in the size and expression profiles of these transcriptional clusters. We find that AID deficiency leads to a reduced proportion of prePB cells and severely impairs transitions between the prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

Inequitable protection of multidimensional biogeochemical regions in the Mediterranean Sea

In many cases, protected areas are placed opportunistically and do not fully capture the representation of ecological components for achieving biodiversity conservation goals. In marine systems, where threats and biodiversity are inherently multidimensional and include vertical depth gradients, the designation of protected areas can be more challenging than in the terrestrial realm. Here, we aimed to evaluate the representation equality of biogeochemical regions at different depths in the Mediterranean Sea as a surrogate for biodiversity. We conducted a gap-analysis evaluating the current extent and coverage of protected areas in the Mediterranean Sea biodiversity hotspot to achieve an equitable representation and conservation strategy for the basin. We used biogeochemical regions across different depths, which allow more complex 3D marine spatial planning that includes spatial complexity of marine systems. A total of 60 biogeochemical regions distributed across three bathomes (spaces determined by depth; i.e. epipelagic, mesopelagic, and bathypelagic zones) and the seafloor were assessed. We found that biogeochemical regions in the Mediterranean Sea were not equally protected across bathomes, and that global conservation targets were not met. The bathypelagic bathome had the lowest representation in the Mediterranean Sea marine protected area (MPA) network, as well as the lowest protection equality. The sub-basins with highest protection included the Algero-Provençal basin and the Tyrrhenian Sea. These two sub-basins were the only ones that passed the Aichi protection target of 10%, while all others were lower than the target. The Ionian Sea was identified as the most highly diverse sub-basin regarding biogeochemical regions but with the least protection and with low conservation equality, making this sub-basin a high priority for conservation. Amongst countries, non-European Mediterranean countries had lower values of protection and conservation equality metrics. In general, the bathypelagic bathome was found as the least protected, the most fragmented, and with the lowest protection equality, thus its protection should be enhanced through all the Mediterranean Sea. Decision makers in the Mediterranean can apply the gap-analysis approach presented here as a useful starting point for improving protection equality in the region, incorporating these metrics in the evaluation of new proposed protected areas and sites for conservation.

B cell signatures and tertiary lymphoid structures contribute to outcome in head and neck squamous cell carcinoma

Current immunotherapy paradigms aim to reinvigorate CD8+ T cells, but the contribution of humoral immunity to antitumor immunity remains understudied. Here, we demonstrate that in head and neck squamous cell carcinoma (HNSCC) caused by human papillomavirus infection (HPV+), patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with tertiary lymphoid structures (TLS) with GCs, both of which correlate with favorable outcome. GC TIL-Bs in HPV+ HNSCC are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. Semaphorin 4a expression is enhanced on GC TIL-Bs present in TLS of HPV+ HNSCC and during the differentiation of TIL-Bs. Our study suggests that therapeutics to enhance TIL-B responses in HNSCC should be prioritized in future studies to determine if they can complement current T cell mediated immunotherapies.

Cognitive deficits in episodic ataxia type 2 mouse models.

Episodic ataxia type 2 (EA2) is a rare autosomal dominant disorder characterized by motor incoordination, paroxysmal dystonia, vertigo, nystagmus and more recently cognitive deficits. To date over 100 mutations in the CACNA1A gene have been identified in EA2 patients leading to a loss of P/Q-type channel activity, dysfunction of cerebellar Purkinje cells and motor incoordination. To determine if the cerebellum is contributing to these cognitive deficits, we examined two different EA2 mouse models for cognition impairments where CACNA1A was removed specifically from cerebellar Purkinje or granule cells postnatally. Both mutant mouse models showed anxiolytic behavior to lighted, open areas in the open field and light/dark place preference tests but enhanced anxiousness in the novel suppressed feeding test. However, EA2 mice continued to show augmented latencies in the light/dark preference test and when the arena was divided into two dark zones in the dark/dark preference test. Moreover, increased latencies were also displayed in the novel object recognition test, indicating that EA2 mice are indecisive and anxious to explore new territories and objects and may have memory recognition deficits. Exposure to a foreign mouse led to deficiencies in attention and sniffing as well as in social and genital sniffing. These data suggest that postnatal removal of the P/Q type calcium channel from the cerebellum regulates neuronal activity involved in anxiety, memory, decision making and social interactions. Our EA2 mice will provide a model to identify the mechanisms and therapeutic agents underlying cognitive and psychiatric disorders seen in EA2 patients.

New record of Ectreposebastes niger (Fourmanoir, 1971) (Setarchidae, Scorpaeniformes): a rare bathypelagic fish from La Pérouse Seamount, Western Indian Ocean, and distribution of Ectreposebastes Garman, 1899 in the Indian Ocean

A new record of a rare deep-water scorpionfish Ectreposebastes niger (Fourmanoir, 1971), is described from the south-western Indian Ocean. This is the ninth record in the Indian Ocean and the first documented record of this species in the waters of Reunion Island. It is also the first record of this species in the Indian Ocean since 1982. Its congener, Ectreposebastes imus Garman, 1899 is more common: together with non-published records from museums and online databases the number of verified Indian Ocean records exceeds 15 individuals. Both species are pseudo-oceanic, usually associated with continental and peri-insular slopes and seamounts between 200 and 1300 m depth, occupying a wide range of habitats from demersal to pelagic in the meso- and bathypelagic zones.

Biology of Germinal Center B Cells Relating to Lymphomagenesis.

The germinal center (GC) reaction is a key feature of adaptive humoral immunity. GCs represent the site where mature B cells refine their B-cell receptor (BCR) and are selected based on the newly acquired affinity for the antigen. In the GC, B cells undergo multiple cycles of proliferation, BCR remodeling by immunoglobulin somatic hypermutation (SHM), and affinity-based selection before emerging as effector memory B cells or antibody-secreting plasma cells. At least 2 histologically and functionally distinct compartments are identified in the GC: the dark zone (DZ) and the light zone (LZ). The proliferative burst and immunoglobulin remodeling by SHM occur prevalently in the DZ compartment. In the LZ, GC B cells undergo an affinity-based selection process that requires the interaction with the antigen and accessory cells. GC B cells are also targeted by class switch recombination, an additional mechanism of immunoglobulin remodeling that ensures the expression of diverse isotype classes. These processes are regulated by a complex network of transcription factors, epigenetic modifiers, and signaling pathways that act in concert with mechanisms of intra-GC B-cell trafficking. The same mechanisms underlying the unique ability of GC B cells to generate high affinity antibodies and ensure immunological memory are hijacked during lymphomagenesis and become powerful weapons for malignant transformation. This review will summarize the main processes and transcriptional networks that drive GC B-cell development and are relevant for human B-cell lymphomagenesis.

798-P: Luseogliflozin Suppresses Fine Glycemic Fluctuation Late at Night: New Aspect of SGLT2 Inhibitor

Sodium-glucose cotransporter 2 (SGLT2) inhibitors block reabsorption of glucose by inhibiting SGLT2 in kidney, promotes the renal excretion of glucose. They have been proven to have a good glucose-lowering effect. To evaluating the effect of SGLT2 inhibitor on fine glycemic variability in type 2 diabetes mellitus (T2D) was used a continuous glucose monitoring system (CGMS) Freestyle Libre. The coefficient of variation (CV) of glucose levels every 15 minutes in the midnight zone (11pm-3am), which is less associated with diet and stress, was measured just before and just after the first administration of the SGLT2 inhibitor. Luseogliflozin (2.5mg per day) was administered to 6 patients with T2D. In Addition, fasting plasma glucose, postprandial plasma glucose, C-peptide, other hormones and metabolites were assessed. Luseogliflozin strongly suppressed CV of glucose (11.6±4.8 to 4.1±2.5, p=0.0012) (Figure). SGLT2 inhibitor suppressed fine glycemic fluctuations. There was no correlation with other factors. This inhibitory effect did not appear with sulfonylurea or DPP-4 inhibitors. (data not shown) Although the result is a new aspect of SGLT2 inhibitor, the clinical significance is not clear. Since Risso A et al reported that glycemic fluctuation cause harm to vascular endothelial cells (AJP 2001), SGLT2 inhibitors may reduce damage of blood vessel via regulation of fine glycemic fluctuation.View largeDownload slideView largeDownload slide DisclosureK. Yamauchi: None.

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

SummaryB cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Agricultural Regionalization: Spatial Analysis of Crop Combination Regions

Crop combination regions refer to the number or diversity of crops grown in a particular area during a specific interval of time [1]. These regions provide strong basis for agricultural regionalization. Developmental policies and other planning measures can be taken on the basis of these regions. This research paper is a case study and it is based on Land Record Circles. The method used for delineation of regions is Rafiullah’s maximum positive deviation method. Out of 11 LRC’s 10 are mono crop regions and the prominent crop is Bajra. The results of the paper are analyzed according to the climatic conditions of the study area. This is a semi-arid region and major part of it is a dark zone. So, the rain fed agriculture is done in the study area. The Bajra crop is produced on south west-monsoon.

Fungi associated with aeroponic roots in caves and mines of New Brunswick

We provide the first analysis of the fungi associated with a very special habitat, the aeroponic roots found in caves and mines in New Brunswick, Canada. Fungal diversity was assessed by Illumina sequencing using three complementary primer sets targeting ribosomal RNA genes, and roots were identified using the non-coding trnH - psbA spacer. Early colonizing ectomycorrhizal fungi such as Agaricales, Helotiales, Pezizales, and Thelephorales were predominant. Saprotrophs, endophytes and plant pathogens were also present, but Glomeromycota (arbuscular mycorrhizal fungi) were not detected. Fungal root communities were generally most similar within sites. Fungal diversity was inversely correlated with winter dark zone temperatures and distance from the entrance. By using a combination of three primer sets, we detected more fungal taxa than with any one primer set. This study adds to the understanding of these subterranean ecosystems and suggests that future studies investigate factors limiting the presence of late-stage ectomycorrhizal fungi and Glomeromycota. • We provide a first analysis of fungi associated with aeroponic roots in caves and mines. • We detected ectomycorrhizal fungi, endophytes, saprotrophs and plant pathogens. • Late-stage ectomycorrhizal fungi and arbuscular mycorrhizal fungi were not found. • Similarity of fungal communities was related more to site than host or proximity. • Combining three NGS primer sets detected more fungi than any single primer set.

Sustainability situations for the southern Gulf of Mexico seafloor, based on environmental, benthic, and socioeconomic indicators

Currently, in tropical regions such as the southern Gulf of Mexico (sGM), there are no legal guidelines for assessing the quality of the seafloor. We aim to determine whether the seafloor sustainability of the sGM is evenly distributed, based on pressures that human activities produce upon the marine coastal environment, represented by the water quality, sediment quality, and benthic fauna. We analysed physicochemical characteristics and benthic fauna at 183 sites, sampled during five annual surveys (rainy and dry seasons) in the sublittoral and bathyal zones. Socioeconomic indicators were obtained from official national censuses. We calculated a pressure index (PI) based on water quality, sediment quality, and socioeconomic indicators. PI values ranged from 1 to 2.67, with the highest values being observed at coastal sites. Our approach determined that the benthic quality in the study area was related to depth and oil industry influence and that the sGM's seafloor sustainability was unevenly distributed. For sustainability, we determined specific situations for each site or group of sites: (1) sensitive - sites with high benthic quality and low PI; (2) naturally variable - sites for which the poor benthic quality was not related to the PI; (3) degraded - sites with poor benthic quality and high PI; and (4) resilient - sites with good benthic quality and high PI. This differentiation in sustainability situations might be used as a reference for linking socioeconomic activities in the coasts with the ecological status of marine environments from shallow to deep.

Deep Seabed Mining: A Note on Some Potentials and Risks to the Sustainable Mineral Extraction from the Oceans

The rapidly increasing global populations and socio-economic development in the Global South have resulted in rising demand for natural resources. There are many plans for harvesting natural resources from the ocean floor, especially rare metals and minerals. However, if proper care is not taken, there is substantial potential for long-lasting and even irreversible physical and environmental impacts on the deep-sea ecosystems, including on biodiversity and ecosystem functioning. This paper reviews the literature on some potentials and risks to deep seabed mining (DSM), outlining its legal aspects and environmental impacts. It presents two case studies that describe the environmental risks related to this exploitative process. They include significant disturbance of the seabed, light and noise pollution, the creation of plumes, and negative impacts on the surface, benthic, and meso- and bathypelagic zones. The study suggests some of the issues interested companies should consider in preventing the potential physical and environmental damages DSM may cause. Sustainable mining and the use of minerals are vital in meeting various industrial demands.

The Effects of Climate on the Hypogean Migration of Cave Salamanders, Eurycea lucifuga (Rafinesque, 1822)

Terrestrial migrations of salamanders are influenced by many factors, particularly temperature and moisture availability. Although photoperiod is generally an important migratory cue, it has not been specifically considered for salamanders. We examine the importance of the above abiotic factors at the macro- and microclimatic scales in the subterranean migration of a population of Cave Salamanders (Eurycea lucifuga) in Kentucky. This species exhibited a cyclic, seasonal migratory pattern between the twilight and dark zones in a cave. Our regression analysis of macroclimate indicated this migration was influenced largely by photoperiod, but also highly correlated with temperature and relative humidity; principle components analysis confirmed the importance of similar microclimate variables (illumination, temperature, and substrate wetness), explaining 88% of the variability in the data. These environmental variables fluctuate little in the dark zone of caves and thus likely do not provide cues on seasonal conditions to salamanders. However, these cues do vary in the twilight zone and may inform salamanders of epigean seasonal conditions. This information is critical to these cave-dwelling salamanders, particularly for finding suitable food resources.

The Ubiquitin Ligase Itch Regulates Germinal Center Selection

Abstract More than 23 million Americans suffer from autoimmune diseases driven by somatically mutated autoantibodies. High levels of somatic hypermutation of immunoglobulin genes occurs in germinal center (GC) B cells. Some of these mutations render autoreactivity, but cells bearing autoreactive receptors are eliminated due to strict selection of GC B cells by mechanisms that are still poorly defined. Inappropriate positive selection signals can rescue autoreactive cells from elimination and drive autoimmune disease. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function is not well understood. By studying Itch deficient mice, we have recently shown that Itch directly limits B cell activity to shape antibody responses. While Itch-deficient mice displayed normal numbers of pre-immune B cell populations, they showed elevated numbers of GC B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to skew the proportions of light zone and dark zone GC B cells. Proteomic profiling of GC B cells uncovered that Itch deficient cells exhibit hallmarks of positive selection. These results support a novel role for Itch in limiting positive selection of GC B cells to restrict GC size. In this role, Itch may shape the GC-derived antibody repertoire to prevent autoimmunity.

Costimulatory blockade modifies germinal center B cell function in NZB/W F1 mice

Abstract Systemic lupus erythematosus (SLE) is a complex autoimmune disease with significant morbidity and mortality - demanding further examination of disease reducing treatments. Previously, we showed that a 2-week course of combined CTLA4Ig and anti-CD40L costimulatory blockade prevented loss of tolerance to dsDNA in NZB/W mice while maintaining immune function against exogenous antigen, explained by an altered selection of autoreactive B cells in germinal centers (GC). Here, we examined both B and T cells to clarify if restoration of tolerance is due to a change in GC B cell profile or a decrease in T cell help. 3 months after treatment, large germinal centers persisted in the treated group, but due to smaller spleen size total GC B cells and TFH Cells were decreased compared with controls. The metabolic profile of splenic B and T cells showed a decreased spare respiratory capacity in both subsets and a decrease in B cell glycolysis among the treated group. RNA-seq analysis of GC dark zone and light zone B cells from treated mice showed decreased expression of markers of oxidative phosphorylation and cell proliferation and increased expression of signaling molecules downstream of BCR, including PLCγ, compared with untreated controls. GC B cell stimulation assays showed a delay in phosphorylation of PLCγ in response to anti-IgG in treated mice. These data show a long-lasting effect of short-term costimulatory blockade on both B and T cells with metabolic remodeling of GC B cells and an alteration of BCR signaling. Since both a profound decrease in BCR signaling and an increase in B cell glycolysis have been reported to enhance loss of tolerance in lupus prone mice, our findings help to explain the restoration of tolerance observed after treatment.