Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas

Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. Here, we demonstrate that haploinsufficient deletions of chromatin-bound tumor suppressor NFKB inhibitor alpha (NFKBIA) display distinct patterns of occurrence in relation to other genetic markers and are disproportionately present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation and induce a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wild-type tumors. An externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas confirms that NFKBIA deletions predict comparatively brief survival. Thus, NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into models predicting the disease fate of diffuse gliomas

Lake salinization drives consistent losses of zooplankton abundance and diversity across coordinated mesocosm experiments

Human-induced salinization increasingly threatens inland waters; yet we know little about the multifaceted response of lake communities to salt contamination. By conducting a coordinated mesocosm experiment of lake salinization across 16 sites in North America and Europe, we quantified the response of zooplankton abundance and (taxonomic and functional) community structure to a broad gradient of environmentally relevant chloride concentrations, ranging from 4 to ca. 1400 mg Cl- L-1. We found that crustaceans were distinctly more sensitive to elevated chloride than rotifers; yet, rotifers did not show compensatory abundance increases in response to crustacean declines. For crustaceans, our among-site comparisons indicate: (1) highly consistent decreases in abundance and taxon richness with salinity; (2) widespread chloride sensitivity across major taxonomic groups (Cladocera, Cyclopoida, and Calanoida); and (3) weaker loss of functional than taxonomic diversity. Overall, our study demonstrates that aggregate properties of zooplankton communities can be adversely affected at chloride concentrations relevant to anthropogenic salinization in lakes.Peer reviewe

Widespread variation in salt tolerance within freshwater zooplankton species reduces the predictability of community-level salt tolerance

The salinization of freshwaters is a global threat to aquatic biodiversity. We quantified variation in chloride (Cl-) tolerance of 19 freshwater zooplankton species in four countries to answer three questions: (1) How much variation in Cl- tolerance is present among populations? (2) What factors predict intraspecific variation in Cl- tolerance? (3) Must we account for intraspecific variation to accurately predict community Cl- tolerance? We conducted field mesocosm experiments at 16 sites and compiled acute LC(50)s from published laboratory studies. We found high variation in LC(50)s for Cl- tolerance in multiple species, which, in the experiment, was only explained by zooplankton community composition. Variation in species-LC50 was high enough that at 45% of lakes, community response was not predictable based on species tolerances measured at other sites. This suggests that water quality guidelines should be based on multiple populations and communities to account for large intraspecific variation in Cl- tolerance.Peer reviewe

Current water quality guidelines across North America and Europe do not protect lakes from salinization

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (C-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a >= 50% reduction in cladoceran abundance were at or below Cl thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.Peer reviewe

T Cell Repertoire Maturation Induced by Persistent and Latent Viral Infection Is Insufficient to Induce Costimulation Blockade Resistant Organ Allograft Rejection in Mice

CD28:CD80/86 pathway costimulation blockade (CoB) with the CD80/86-specific fusion protein CTLA4-Ig prevents T cell-mediated allograft rejection in mice. However, in humans, transplantation with CoB has been hampered by CoB-resistant rejection (CoBRR). CoBRR has been attributed in part to pathogen-driven T cell repertoire maturation and resultant heterologous alloreactive memory. This has been demonstrated experimentally in mice. However, prior murine models have used viral pathogens, CoB regimens, graft types, and/or antigen systems atypically encountered clinically. We therefore sought to explore whether CoBRR would emerge in a model of virus-induced memory differentiation designed to more closely mimic clinical conditions. Specifically, we examined mouse homologs of clinically prevalent viruses including murine polyomavirus, cytomegalovirus, and gammaherpesvirus 68 in the presence of clinically relevant maintenance CoB regimens using a fully MHC-mismatched, vascularized allograft model. Infected mice developed a significant, sustained increase in effector memory T cells consistent with that seen in humans, but neither developed heterologous alloreactivity nor rejected primarily vascularized heterotopic heart transplants at an increased rate compared with uninfected mice. These results indicate that memory acquisition alone is insufficient to provoke CoBRR and suggest that knowledge of prior latent or persistent viral infection may have limited utility in anticipating heterologous CoB-resistant alloimmunity

Assessing the role of STAT3 in DC differentiation and autologous DC immunotherapy in mouse models of GBM.

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors

Enhanced proliferation of antigen specific T cells in response to STAT3 deficient DCs.

<p><b>A.</b> Antigen specific MLR assay was used to assess antigen processing and presentation by GM-CSF derived BMDCs. WT and STAT deficient BMDCs were cultured with 1 µg/ml ovalbumin for 18 Hrs before being γ-irradiated. DCs were then washed of excess ovalbumin and cultured 1∶1 with 100,000 CFSE labeled OT-1 T cells for 5 days. Peaks of CFSE fluorescence were analyzed by flow cytometry on CD8⁺ OT-1 T cells. The precursor frequency and proliferation index were quantified from three separate MLR assays using non-related mice. Student's t-test was used to determine statistical significance (*, <i>p</i><0.05 versus wild type).</p

Cytokine secretion by WT and STAT3 deficient BMDCs.

<p>Secretion of IL-12p70, IL-10, TNFα, and IL-6 was measured by ELISA from supernatants of 1×10⁶ WT and STAT3^−/− GM-CSF BMDCs stimulated with CpG 1668 (500 ng/ml) for 18 hours in 1 ml of RPMI-10. Cytokine secretion was evaluated from 5 WT and 5 STAT3 KO mice in triplicate wells (*, <i>p</i><0.05; two-tail students t-test).</p

Deletion of STAT3 in DCs enhances the proliferation of allogeneic T cells.

<p><b>A.</b> An allogeneic mixed lymphocyte reaction (MLR) was used to determine if DCs (stimulator) could induce allogeneic T cell (responder) proliferation. WT and STAT3^−/− DCs were derived using GM-CSF as previously outlined. BMDCs were stimulated with CpG 1668 (500 ng/ml) for 12 hours to increase cell surface expression of MHC-II prior to being γ-irradiated. To induce proliferation, 100,000 DCs were cultured with CFSE-labeled allogeneic T cells at a 1∶1 ratio in 96-well flat bottom wells for 5 days. CFSE intensity of CD8⁺ T cells at day 5 is presented as histograms. The precursor frequency and proliferation index were derived using the proliferation analysis wizard in Modfit LT computer software. <b>B.</b> CpG-matured DCs were irradiated and cultured with 100,000 allogeneic T cells at decreasing ratios of stimulator to responder in 96-well flat bottom wells. Cells were allowed to proliferate for 4 days prior to addition of the nucleotide analogue BrdU (18 hours incubation). Incorporation of BrdU into dividing DNA was determined using a colorimetric ELISA kit. 2-way ANOVA test followed by Tukey-Kramer multiple comparison test were employed to determine statistical significance (*, <i>p</i><0.05 versus wild type).</p

Induction of anti-tumor immunity in response to DC vaccination is independent of STAT3 signaling.

<p><b>A.</b> Diagram illustrating the culture and priming of WT and STAT3 knockout GM-CSF derived BMDCs. Micrograph captured at day 6 of GM-CSF culture demonstrates the formation of loosely adherent cDC clusters. Tumor cell lysate was generated by subjecting GL26 cells to repeated freeze-thaw cycles in liquid nitrogen and a 37°C water bath. DCs were primed with GL26 tumor cell lysate at a 2∶1 ratio of tumor cells to DCs in RPMI-10 for 12 hours at 37°C. After loading, DCs were washed three times with PBS to remove residual tumor lysate. Loaded DCs were mixed with 30 µg of CpG 1668 immediately prior to subcutaneous vaccination. <b>B.</b> DC vaccinations were administered before (prophylactic model) tumor challenge. C57BL/6J mice were vaccinated subcutaneously with 1×10⁶ primed WT DCs (blue line, n = 5), STAT3 KO DCs (green line, n = 5), PBS-control or CpG-control on the indicated days. On day 0, mice were intracranially injected with 20,000 Gl26 glioma cells and followed for survival. <b>C.</b> Animals were monitored daily and euthanized upon signs of morbidity. Survival data is depicted as a Kaplan-Meyer curve and analyzed statistically using the Mantel log-rank test (*, <i>p</i><0.05 versus PBS and CpG control). <b>D.</b> IFNγ ELISPOT assay was used to assess T cell IFNγ secretion from splenocytes of mice at 12 days post tumor implantation. The ELISPOT data showed a significant increase in mice treated WT DCs or STAT3 null DCs treated with CpG compared to CpG treated control mice. <b>E.</b> Quantification of CD3e immunohistochemistry showed significantly more CD3e+ T cells within both DC treatment groups compared to CpG control mice; there was no significant difference between WT DC and STAT3 null DC treated mice. <b>F.</b> Quantification of Iba1⁺ cells within the tumor showed no significant difference Iba1+ microglia between any groups. <b>G.</b> Therapeutic model of tumor bearing mouse vaccination. Mice were vaccinated subcutaneously with 1×10⁶ primed WT DCs (blue line, n = 5), STAT3 KO DCs (green line, n = 5), PBS-control on the indicated days. On day 0, mice were intracranially injected with 20,000 Gl26 glioma cells and followed for survival. <b>H.</b> Mice were monitored for survival and presented as Kaplan-Meyer survival curves. Mantel log-rank test was used to determine statistical significance (*, p<0.05 versus PBS and CpG control).</p