Lessons learned from using wild-caught and captive-reared lesser scaup (Aythya affinis) in captive experiments.

Waterfowl are housed in captivity for research studies that are infeasible in the wild. Accommodating the unique requirements of semi-aquatic species in captivity while meeting experimental design criteria for research questions can be challenging and may have unknown effects on animal health. Thus, testing and standardizing best husbandry and care practices for waterfowl is necessary to facilitate proper husbandry and humane care while ensuring reliable and repeatable research results. To inform husbandry practices for captive-reared and wild-caught lesser scaup (Aythya affinis; hereafter, scaup), we assessed body mass and fat composition across two different aspects of husbandry, source population (captive-reared or wild caught), and housing densities (birds/m2). Our results suggest that housing scaup at low densities (≤0.6 m2/bird, P = 0.049) relative to other species can minimize negative health effects. Captive-reared scaup were heavier (P = 0.027) with greater body fat (P < 0.001) and exhibited fewer signs of stress during handling than wild-caught scaup. In our experience, scaup which are captive-reared from eggs collected in the wild were better for long-term captivity studies as they maintained body mass between and recovered lost body mass following trials. Researchers would benefit from carefully evaluating the tradeoffs of using short- and long-term captive methods on their research question before designing projects, husbandry practices, and housing facilities for waterfowl.

CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation.

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.

Expression of IL-37 Induces a Regulatory T-Cell-like Phenotype and Function in Jurkat Cells.

The anti-inflammatory cytokine interleukin-37 (IL-37) plays a key role in inhibiting innate and adaptive immunity. Past results have shown that IL-37 is elevated in human Treg cells compared to other T cell subsets and contributes to enhancing the Treg transcription factor, forkhead box protein P3 (FOXP3). However, it is unknown if ectopic expression of IL-37 in non-Treg CD4+ T cells can lead to the development of Treg phenotype and function. In the present study, we used a PrimeFlow® RNA assay and confirmed elevated IL37 expression in human Treg cells. We then stably transfected the non-Treg CD4+ T cell leukemia cell line, E6 Jurkat cells, with IL37 and found significant induction of the Treg phenotype. These IL-37-expressing Jurkat cells had elevated CTLA-4 and FOXP3 and produced IL-10. In conjunction with the Treg phenotype, IL-37-expressing Jurkat cells suppressed T cell activation/proliferation, comparable to human primary Treg cells. The creation of this stable human Treg-like cell line has the potential to provide further assistance for in vitro studies of human Treg cells, as it is more convenient than the use of primary human Treg cells. Furthermore, it provides insights into Treg cell biology and function.

Citizen science reveals waterfowl responses to extreme winter weather.

Global climate change is increasing the frequency and severity of extreme climatic events (ECEs) which may be especially detrimental during late-winter when many species are surviving on scarce resources. However, monitoring animal populations relative to ECEs is logistically challenging. Crowd-sourced datasets may provide opportunity to monitor species' responses to short-term chance phenomena such as ECEs. We used 14 years of eBird-a global citizen science initiative-to examine distribution changes for seven wintering waterfowl species across North America in response to recent extreme winter polar vortex disruptions. To validate inferences from eBird, we compared eBird distribution changes against locational data from 362 GPS-tagged Mallards (Anas platyrhynchos) in the Mississippi Flyway. Distributional shifts between eBird and GPS-tagged Mallards were similar following an ECE in February 2021. In general, the ECE affected continental waterfowl population distributions; however, responses were variable across species and flyways. Waterfowl distributions tended to stay near wintering latitudes or moved north at lesser distances compared with non-ECE years, suggesting preparedness for spring migration was a stronger "pull" than extreme weather was a "push" pressure. Surprisingly, larger-bodied waterfowl with grubbing foraging strategies (i.e., geese) delayed their northward range shift during ECE years, whereas smaller-bodied ducks were less affected. Lastly, wetland obligate species shifted southward during ECE years. Collectively, these results suggest specialized foraging strategies likely related to resource limitations, but not body size, necessitate movement from extreme late-winter weather in waterfowl. Our results demonstrate eBird's potential to monitor population-level effects of weather events, especially severe ECEs. eBird and other crowd-sourced datasets can be valuable to identify species which are adaptable or vulnerable to ECEs and thus, begin to inform conservation policy and management to combat negative effects of global climate change.

EGCG Inhibits Tumor Growth in Melanoma by Targeting JAK-STAT Signaling and Its Downstream PD-L1/PD-L2-PD1 Axis in Tumors and Enhancing Cytotoxic T-Cell Responses.

Over the last decade, therapies targeting immune checkpoints, such as programmed death-1 (PD-1), have revolutionized the field of cancer immunotherapy. However, low response rates and immune-related adverse events remain a major concern. Here, we report that epigallocatechin gallate (EGCG), the most abundant catechin in green tea, inhibits melanoma growth by modulating an immune response against tumors. In vitro experiments revealed that EGCG treatment inhibited interferon-gamma (IFN-γ)-induced PD-L1 and PD-L2 expression and JAK-STAT signaling. We confirmed that this effect was driven by inhibiting STAT1 gene expression and STAT1 phosphorylation, thereby downregulating the PD-L1/PD-L2 transcriptional regulator IRF1 in both human and mouse melanoma cells. Animal studies revealed that the in vivo tumor-inhibitory effect of EGCG was through CD8+ T cells and that the inhibitory effect of EGCG was comparable to anti-PD-1 therapy. However, their mechanisms of action were different. Dissimilar to anti-PD-1 treatment that blocks PD-1/PD-L1 interaction, EGCG inhibited JAK/STAT signaling and PD-L1 expression in tumor cells, leading to the re-activation of T cells. In summary, we demonstrate that EGCG enhances anti-tumor immune responses by inhibiting JAK-STAT signaling in melanoma. EGCG could be used as an alternative treatment strategy to target the PD-L1/PD-L2-PD-1 axis in cancers.

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.

Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.

Inactivation of Rho GTPases by Burkholderia cenocepacia Induces a WASH-Mediated Actin Polymerization that Delays Phagosome Maturation.

Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV.

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains.

Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.

Interleukin-37 is highly expressed in regulatory T cells of melanoma patients and enhanced by melanoma cell secretome.

Immune suppression is one of the 10 hallmarks of cancer. Interleukin-37 (IL-37), a member of the IL-1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL-37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL-37 in age- and sex-matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL-37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL-37 expression, we discovered that IL-37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (Treg ) cells in healthy individuals, and that IL-37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, Treg cells from melanoma patients exhibited the highest IL-37 gene expression levels. We provided evidence that melanoma-conditioned media induces IL-37 mRNA and protein expression in multiple lymphocyte populations, particularly in Treg cells. We further confirmed that the IL-1-mediated secretome from human melanoma cells, specifically transforming growth factor-ß, induces IL-37 mRNA expression in human Treg cells. Our results suggest a potential immunosuppressive role for IL-1 and IL-37 in melanoma tumorigenesis. Highly elevated IL-37 in specific lymphocyte populations could serve as a biomarker for tumor-induced immunosuppression.

CDK1 Interacts with Sox2 and Promotes Tumor Initiation in Human Melanoma.

: Cancers are composed of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor-initiating capacities and their regulatory mechanisms remain elusive. Here, we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacologic agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions. SIGNIFICANCE: These findings uncover CDK1 as a new regulator of Sox2 during tumor initiation and implicate the CDK1-Sox2 interaction as a potential therapeutic target in cancer.

TCR-CXCR4 signaling stabilizes cytokine mRNA transcripts via a PREX1-Rac1 pathway: implications for CTCL.

As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2(IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.

Investigation of interfacial properties of pure and mixed poloxamers for surfactant-mediated shear protection of mammalian cells.

The Poloxamer family of surfactants are commonly used in the biopharmaceutical industry as cell culture media additives to protect cells from the turbulent environment of sparged bioreactors. Despite the widespread use of poloxamers in cell culture, their performance as cell protectants varies depending on their physical structure, molecular weight, and batch-to-batch composition. In this study, the interfacial properties of Poloxamer 188 (P188), Poloxamer 407 (P407), and a mixture of P188 and P407 were characterized to investigate the mechanism of surfactant-mediated shear protection of mammalian cells. The foam stability and equilibrium surface tension of these surfactant systems correlated with their ability to mitigate physical damage to cells in a turbulent environment. We demonstrate that while P188 can function as highly effective shear protectant, the presence of a surface-active contaminant can greatly hinder its protective characteristics. P407 was found to function as such an interfacially active "impurity," disrupting shear protection when mixed with P188 by preferentially adsorbing to the gas-liquid and membrane-liquid interface. Addition of surface-active impurities altered the interfacial properties of the surfactant system and could be detected using an equilibrium surface tension assay. The mechanism of disruption by P407 was determined to be independent of cell-to-bubble attachment, suggesting that poloxamer adsorption to and subsequent reinforcement of the cell membrane may play a key role in protecting cells in high shear environments. This investigation contributes to our understanding of the mechanism of surfactant-mediated shear protection of cells and demonstrates that a surface tension assay can be utilized as a screening tool to ensure that poloxamer lots are free of surface active impurities.

Structural and mechanistic insights into regulation of the retromer coat by TBC1d5.

Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29-VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.

Monitoring receptor trafficking following retromer and WASH deregulation.

Cell surface receptors that have been internalized and enter the endocytic pathway have multiple fates including entrance into the multivesicular body pathway on their way to lysosomal degradation, recycling back to the cell surface, or retrograde trafficking out of the endolysosomal system back to the Golgi apparatus. Two ubiquitously expressed protein complexes, WASH and the endosomal coat complex retromer, function together to play a central role in directing the fate of receptors into the latter two pathways. In this chapter, we describe fluorescent- and flow cytometry-based methods for analyzing the recycling and retrograde trafficking of two receptors, α5ß1 and CI-M6PR, whose intracellular fates are regulated by WASH and retromer activity. The guidelines presented in this chapter can be applied to the analysis of any cell surface or intracellular membrane protein to determine the impact of WASH or retromer deregulation on its intracellular trafficking route.

SNX17 affects T cell activation by regulating TCR and integrin recycling.

A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and Ag recognition. One protein potentially involved in TCR transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 colocalizes with TCR and localizes to the immune synapse in T- conjugates. Significantly, knockdown of the SNX17 resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared with control T cells. Lastly, we identified the 4.1/ezrin/radixin/moesin domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse.

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A.

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.

Nuclear FAM21 participates in NF-κB-dependent gene regulation in pancreatic cancer cells.

The pentameric WASH complex is best known for its role in regulating receptor trafficking from retromer-rich endosomal subdomains. FAM21 functions to stabilize the WASH complex through its N-terminal head domain and localizes it to endosomes by directly binding the retromer through its extended C-terminal tail. Herein, we used affinity purification combined with mass spectrometry to identify additional FAM21-interacting proteins. Surprisingly, multiple components of the nuclear factor κB (NF-κB) pathway were identified, including the p50 and p65 (RelA) NF-κB subunits. We show that FAM21 interacts with these components and regulates NF-κB-dependent gene transcription at the level of p65 chromatin binding. We further demonstrate that FAM21 contains a functional monopartite nuclear localization signal sequence (NLS) as well as a CRM1/exportin1-dependent nuclear export signal (NES), both of which work jointly with the N-terminal head domain and C-terminal retromer recruitment domain to regulate FAM21 cytosolic and nuclear subcellular localization. Finally, our findings indicate that FAM21 depletion sensitizes pancreatic cancer cells to gemcitabine and 5-fluorouracil. Thus, FAM21 not only functions as an integral component of the cytoplasmic WASH complex, but also modulates NF-κB gene transcription in the nucleus.

Dendritic cells utilize the evolutionarily conserved WASH and retromer complexes to promote MHCII recycling and helper T cell priming.

Immature dendritic cells (DCs) maintain a highly dynamic pool of recycling MHCII that promotes sampling of environmental antigens for presentation to T helper cells. However, the molecular basis of MHCII recycling and the cellular machinery that orchestrates MHCII trafficking are incompletely understood. Using a mouse model we show that WASH, an actin regulatory protein that facilitates retromer function, is essential for MHCII recycling and efficient priming of T helper cells. We further demonstrate that WASH deficiency results in impaired MHCII surface levels, recycling, and an accumulation of polyubiquitinated MHCII complexes, which are subsequently slated for premature lysosomal degradation. Consequently, conditional deletion of the Wash gene in DCs impairs priming of both conventional and autoimmune T helper cells in vivo and attenuates disease progression in a model of experimental autoimmune encephalitis (EAE). Thus, we identify a novel mechanism in which DCs employ the evolutionarily conserved WASH and retromer complex for MHCII recycling in order to regulate T helper cell priming.