Characterization of an orthotopic mouse transplant model reveals early changes in the tumor microenvironment of lung cancer.

To understand the cellular and molecular dynamics in the early stages of lung cancer, we explored a mouse model of orthotopic tumor transplant created from the Lewis Lung Carcinoma (LLC) cell line. Employing single-cell RNA sequencing, we analyzed the cellular landscape during tumor engraftment, focusing particularly on LLC cells harboring the Kras G12C mutation. This allowed us to identify LLC tumor cells via the detection of mutant Kras transcripts and observe elevated levels of Myc and mesenchymal gene expression. Moreover, our study revealed significant alterations in the lung microenvironment, including the activation of tissue remodeling genes in a fibroblast and the downregulation of MHC class II genes in myeloid subsets. Additionally, T/NK cell subsets displayed more regulatory phenotypes, coupled with reduced proliferation in CD8+ T cells. Collectively, these findings enhance our understanding of lung cancer progression, particularly in a tumor microenvironment with low immunogenicity.

Single-cell RNA sequencing reveals myeloid and T cell co-stimulation mediated by IL-7 anti-cancer immunotherapy.

BACKGROUND: Immune checkpoint inhibitors unleash inhibitory signals on T cells conferred by tumors and surrounding stromal cells. Despite the clinical efficacy of checkpoint inhibitors, the lack of target expression and persistence of immunosuppressive cells limit the pervasive effectiveness of the therapy. These limitations may be overcome by alternative approaches that co-stimulate T cells and the immune microenvironment. METHODS: We analyzed single-cell RNA sequencing data from multiple human cancers and a mouse tumor transplant model to discover the pleiotropic expression of the Interleukin 7 (IL-7) receptor on T cells, macrophages, and dendritic cells. RESULTS: Our experiment on the mouse model demonstrated that recombinant IL-7 therapy induces tumor regression, expansion of effector CD8 T cells, and pro-inflammatory activation of macrophages. Moreover, spatial transcriptomic data support immunostimulatory interactions between macrophages and T cells. CONCLUSION: These results indicate that IL-7 therapy induces anti-tumor immunity by activating T cells and pro-inflammatory myeloid cells, which may have diverse therapeutic applicability.

Altered tumor signature and T-cell profile after chemotherapy reveal new therapeutic opportunities in high-grade serous ovarian carcinoma.

Chemotherapy combined with debulking surgery is the standard treatment protocol for high-grade serous ovarian carcinoma (HGSOC). Nonetheless, a significant number of patients encounter relapse due to the development of chemotherapy resistance. To better understand and address this resistance, we conducted a comprehensive study investigating the transcriptional alterations at the single-cell resolution in tissue samples from patients with HGSOC, using single-cell RNA sequencing and T-cell receptor sequencing techniques. Our analyses unveiled notable changes in the tumor signatures after chemotherapy, including those associated with epithelial-mesenchymal transition and cell cycle arrest. Within the immune compartment, we observed alterations in the T-cell profiles, characterized by naïve or pre-exhausted populations following chemotherapy. This phenotypic change was further supported by the examination of adjoining T-cell receptor clonotypes in paired longitudinal samples. These findings underscore the profound impact of chemotherapy on reshaping the tumor landscape and the immune microenvironment. This knowledge may provide clues for the development of future therapeutic strategies to combat treatment resistance in HGSOC.

PARIS undergoes liquid-liquid phase separation and poly(ADP-ribose)-mediated solidification.

ZNF746 was identified as parkin-interacting substrate (PARIS). Investigating its pathophysiological properties, we find that PARIS undergoes liquid-liquid phase separation (LLPS) and amorphous solid formation. The N-terminal low complexity domain 1 (LCD1) of PARIS is required for LLPS, whereas the C-terminal prion-like domain (PrLD) drives the transition from liquid to solid phase. In addition, we observe that poly(ADP-ribose) (PAR) strongly binds to the C-terminus of PARIS near the PrLD, accelerating its LLPS and solidification. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PAR formation leads to PARIS oligomerization in human iPSC-derived dopaminergic neurons that is prevented by the PARP inhibitor, ABT-888. Furthermore, SDS-resistant PARIS species are observed in the substantia nigra (SN) of aged mice overexpressing wild-type PARIS, but not with a PAR binding-deficient PARIS mutant. PARIS solidification is also found in the SN of mice injected with preformed fibrils of α-synuclein (α-syn PFF) and adult mice with a conditional knockout (KO) of parkin, but not if α-syn PFF is injected into mice deficient for PARP1. Herein, we demonstrate that PARIS undergoes LLPS and PAR-mediated solidification in models of Parkinson's disease.

Single-cell mapping of combinatorial target antigens for CAR switches using logic gates.

Identification of optimal target antigens that distinguish cancer cells from normal surrounding tissue cells remains a key challenge in chimeric antigen receptor (CAR) cell therapy for tumors with intratumoral heterogeneity. In this study, we dissected tissue complexity to the level of individual cells through the construction of a single-cell expression atlas that integrates ~1.4 million tumor, tumor-infiltrating normal and reference normal cells from 412 tumors and 12 normal organs. We used a two-step screening method using random forest and convolutional neural networks to select gene pairs that contribute most to discrimination between individual malignant and normal cells. Tumor coverage and specificity are evaluated for the AND, OR and NOT logic gates based on the combinatorial expression pattern of the pairing genes across individual single cells. Single-cell transcriptome-coupled epitope profiling validates the AND, OR and NOT switch targets identified in ovarian cancer and colorectal cancer.

CTLA-4 inhibition facilitates follicular T and B cell interaction and the production of tumor-specific antibodies.

Immune checkpoint inhibitors (ICIs) induce activation and expansion of cytotoxic T cells. To depict a comprehensive immune cell landscape reshaped by the CTLA-4 checkpoint inhibitor, we performed single-cell RNA sequencing in a mouse syngeneic tumor transplant model. After CTLA-4 inhibition, tumor regression was accompanied by massive immune cell expansion, especially in T and B cells. We found that B cells in tumor transplant represented follicular, germinal center and plasma B cells, some of which shared identical B cell receptor clonotypes and possessed tumor reactivity. Furthermore, the posttreatment tumor contained a tertiary lymphoid-like structure with intermingled T and B cells. These data suggest germinal center formation within the tumor mass and in situ differentiation of tumor-specific plasma cells. Taken together, our data provide a panoramic view of the immune microenvironment after CTLA-4 inhibition and suggest a role for tumor-specific B cells in antitumor immunity.

Prescreening of tumor samples for tumor-centric transcriptome analyses of lung adenocarcinoma.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) enables the systemic assessment of intratumoral heterogeneity within tumor cell populations and in diverse stromal cells of the tumor microenvironment. Gain of treatment resistance during tumor progression or drug treatment are important subjects of tumor-centric scRNA-seq analyses, which are hampered by scarce tumor cell portions. To guarantee the inclusion of tumor cells in the data analysis, we developed a prescreening strategy for lung adenocarcinoma. METHODS: We obtained candidate genes that were differentially expressed between normal and tumor cells, excluding stromal cells, from the scRNA-seq data. Tumor cell-specific expression of the candidate genes was assessed via real-time reverse transcription-polymerase chain reaction (RT-PCR) using lung cancer cell lines, normal vs. lung cancer tissues, and lymph node biopsy samples with or without metastasis. RESULTS: We found that CEA cell adhesion molecule 5 (CEACAM5) and high mobility group box 3 (HMGB3) were reliable markers for RT-PCR-based prescreening of tumor cells in lung adenocarcinoma. CONCLUSIONS: The prescreening strategy using CEACAM5 and HMGB3 expression facilitates tumor-centric scRNA-seq analyses of lung adenocarcinoma.

Loss of zinc-finger protein 212 leads to Purkinje cell death and locomotive abnormalities with phospholipase D3 downregulation.

Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. To identify genes related to the ataxia-like phenotype of Zfp212-KO mice, 39 ataxia-associated genes in the Cb were monitored. Substantial alterations in the expression of ataxin 10, protein phosphatase 2 regulatory subunit beta, protein kinase C gamma, and phospholipase D3 (Pld3) were observed. Among them, Pld3 alone was tightly regulated by Flag-tagged ZNF212 overexpression or Zfp212 knockdown in the HT22 cell line. The Cyclic Amplification and Selection of Targets assay identified the TATTTC sequence as a recognition motif of ZNF212, and these motifs occurred in both human and mouse PLD3 gene promoters. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp212 and Pld3 in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212-PLD3 relationship is important for Purkinje cell survival.

Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

Aberrant activation of the non-receptor kinase c-Abl is implicated in the development of pathogenic hallmarks of Parkinson's disease, such as α-synuclein aggregation and progressive neuronal loss. c-Abl-mediated phosphorylation and inhibition of parkin ligase function lead to accumulation of parkin interacting substrate (PARIS) that mediates α-synuclein pathology-initiated dopaminergic neurodegeneration. Here we show that, in addition to PARIS accumulation, c-Abl phosphorylation of PARIS is required for PARIS-induced cytotoxicity. c-Abl-mediated phosphorylation of PARIS at Y137 (within the Krüppel-associated box domain) drives its association with KAP1 and the repression of genes with diverse functions in pathways such as chromatin remodelling and p53-dependent cell death. One phosphorylation-dependent PARIS target, MDM4 (a p53 inhibitor that associates with MDM2; also known as MDMX), is transcriptionally repressed in a histone deacetylase-dependent manner via PARIS binding to insulin response sequence motifs within the MDM4 promoter. Virally induced PARIS transgenic mice develop c-Abl activity-dependent Parkinson's disease features such as motor deficits, dopaminergic neuron loss and neuroinflammation. PARIS expression in the midbrain resulted in c-Abl activation, PARIS phosphorylation, MDM4 repression and p53 activation, all of which are blocked by the c-Abl inhibitor nilotinib. Importantly, we also observed aberrant c-Abl activation and PARIS phosphorylation along with PARIS accumulation in the midbrain of adult parkin knockout mice, implicating c-Abl in recessive Parkinson's disease. Inhibition of c-Abl or PARIS phosphorylation by nilotinib or Y137F-PARIS expression in adult parkin knockout mice blocked MDM4 repression and p53 activation, preventing motor deficits and dopaminergic neurodegeneration. Finally, we found correlative increases in PARIS phosphorylation, MDM4 repression and p53 activation in post-mortem Parkinson's disease brains, pointing to clinical relevance of the c-Abl-PARIS-MDM4-p53 pathway. Taken together, our results describe a novel mechanism of epigenetic regulation of dopaminergic degeneration downstream of pathological c-Abl activation in Parkinson's disease. Since c-Abl activation has been shown in sporadic Parkinson's disease, PARIS phosphorylation might serve as both a useful biomarker and a potential therapeutic target to regulate neuronal loss in Parkinson's disease.

α-Synuclein A53T Binds to Transcriptional Adapter 2-Alpha and Blocks Histone H3 Acetylation.

α-Synuclein (α-syn) is a hallmark amyloidogenic protein component of Lewy bodies in dopaminergic neurons affected by Parkinson's disease (PD). Despite the multi-faceted gene regulation of α-syn in the nucleus, the mechanism underlying α-syn crosstalk in chromatin remodeling in PD pathogenesis remains elusive. Here, we identified transcriptional adapter 2-alpha (TADA2a) as a novel binding partner of α-syn using the BioID system. TADA2a is a component of the p300/CBP-associated factor and is related to histone H3/H4 acetylation. We found that α-syn A53T was more preferentially localized in the nucleus than the α-syn wild-type (WT), leading to a stronger disturbance of TADA2a. Indeed, α-syn A53T significantly reduced the level of histone H3 acetylation in SH-SY5Y cells; its reduction was also evident in the striatum (STR) and substantia nigra (SN) of mice that were stereotaxically injected with α-syn preformed fibrils (PFFs). Interestingly, α-syn PFF injection resulted in a decrease in TADA2a in the STR and SN of α-syn PFF-injected mice. Furthermore, the levels of TADA2a and acetylated histone H3 were significantly decreased in the SN of patients with PD. Therefore, histone modification through α-syn A53T-TADA2a interaction may be associated with α-syn-mediated neurotoxicity in PD pathology.

Tumor-promoting macrophages prevail in malignant ascites of advanced gastric cancer.

Gastric cancer (GC) patients develop malignant ascites as the disease progresses owing to peritoneal metastasis. GC patients with malignant ascites have a rapidly deteriorating clinical course with short survival following the onset of malignant ascites. Better optimized treatment strategies for this subset of patients are needed. To define the cellular characteristics of malignant ascites of GC, we used single-cell RNA sequencing to characterize tumor cells and tumor-associated macrophages (TAMs) from four samples of malignant ascites and one sample of cerebrospinal fluid. Reference transcriptomes for M1 and M2 macrophages were generated by in vitro differentiation of healthy blood-derived monocytes and applied to assess the inflammatory properties of TAMs. We analyzed 180 cells, including tumor cells, macrophages, and mesothelial cells. Dynamic exchange of tumor-promoting signals, including the CCL3-CCR1 or IL1B-IL1R2 interactions, suggests macrophage recruitment and anti-inflammatory tuning by tumor cells. By comparing these data with reference transcriptomes for M1-type and M2-type macrophages, we found noninflammatory characteristics in macrophages recovered from the malignant ascites of GC. Using public datasets, we demonstrated that the single-cell transcriptome-driven M2-specific signature was associated with poor prognosis in GC. Our data indicate that the anti-inflammatory characteristics of TAMs are controlled by tumor cells and present implications for treatment strategies for GC patients in which combination treatment targeting cancer cells and macrophages may have a reciprocal synergistic effect.

Single-cell RNA sequencing reveals the tumor microenvironment and facilitates strategic choices to circumvent treatment failure in a chemorefractory bladder cancer patient.

BACKGROUND: Tumor cell-intrinsic mechanisms and complex interactions with the tumor microenvironment contribute to therapeutic failure via tumor evolution. It may be possible to overcome treatment resistance by developing a personalized approach against relapsing cancers based on a comprehensive analysis of cell type-specific transcriptomic changes over the clinical course of the disease using single-cell RNA sequencing (scRNA-seq). METHODS: Here, we used scRNA-seq to depict the tumor landscape of a single case of chemo-resistant metastatic, muscle-invasive urothelial bladder cancer (MIUBC) addicted to an activating Harvey rat sarcoma viral oncogene homolog (HRAS) mutation. In order to analyze tumor evolution and microenvironmental changes upon treatment, we also applied scRNA-seq to the corresponding patient-derived xenograft (PDX) before and after treatment with tipifarnib, a HRAS-targeting agent under clinical evaluation. RESULTS: In the parallel analysis of the human MIUBC and the PDX, diverse stromal and immune cell populations recapitulated the cellular composition in the human and mouse tumor microenvironment. Treatment with tipifarnib showed dramatic anticancer effects but was unable to achieve a complete response. Importantly, the comparative scRNA-seq analysis between pre- and post-tipifarnib-treated PDX revealed the nature of tipifarnib-refractory tumor cells and the tumor-supporting microenvironment. Based on the upregulation of programmed death-ligand 1 (PD-L1) in surviving tumor cells, and the accumulation of multiple immune-suppressive subsets from post-tipifarnib-treated PDX, a PD-L1 inhibitor, atezolizumab, was clinically applied; this resulted in a favorable response from the patient with acquired resistance to tipifarnib. CONCLUSION: We presented a single case report demonstrating the power of scRNA-seq for visualizing the tumor microenvironment and identifying molecular and cellular therapeutic targets in a treatment-refractory cancer patient.

Alterations in the Transcriptional Programs of Myeloma Cells and the Microenvironment during Extramedullary Progression Affect Proliferation and Immune Evasion.

PURPOSE: In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites. EXPERIMENTAL DESIGN: Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed. RESULTS: Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells. CONCLUSIONS: These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion.

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells.

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.

Identification of transketolase as a target of PARIS in substantia nigra.

Recently, PARIS (ZNF746) is introduced as authentic substrate of parkin and transcriptionally represses PGC-1α by binding to insulin responsive sequences (IRSs) in the promoter of PGC-1α. The overexpression of PARIS selectively leads to the loss of dopaminergic neurons (DN) and mitochondrial abnormalities in the substantia nigra (SN) of Parkinson's disease (PD) models. To identify PARIS target molecules altered in SN region-specific manner, LC-MS/MS-based quantitative proteomic analysis is employed to investigate proteomic alteration in the cortex, striatum, and SN of AAV-PARIS injected mice. Herein, we find that the protein and mRNA of transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) of glucose metabolism, is exclusively decreased in the SN of AAV-PARIS mice. PARIS overexpression suppresses TKT transcription via IRS-like motif in the TKT promoter. Moreover, the reduction of TKT by PARIS is found in primary DN but not in cortical neurons, suggesting that PARIS-medicated TKT suppression is cell type-dependent. Interestingly, we observe the reduced level of TKT in the SN of PD patients but not in the cortex. These findings indicate that TKT might be a SN-specific target of PARIS, providing new clues to understand the mechanism underlying selective DNs death in PD.

Activation of the ATF2/CREB-PGC-1α pathway by metformin leads to dopaminergic neuroprotection.

Progressive dopaminergic neurodegeneration is responsible for the canonical motor deficits in Parkinson's disease (PD). The widely prescribed anti-diabetic medicine metformin is effective in preventing neurodegeneration in animal models; however, despite the significant potential of metformin for treating PD, the therapeutic effects and molecular mechanisms underlying dopaminergic neuroprotection by metformin are largely unknown.In this study, we found that metformin induced substantial proteomic changes, especially in metabolic and mitochondrial pathways in the substantia nigra (SN). Consistent with this data, metformin increased mitochondrial marker proteins in SH-SY5Y neuroblastoma cells. Mitochondrial protein expression by metformin was found to be brain region specific, with metformin increasing mitochondrial proteins in the SN and the striatum, but not the cortex. As a potential upstream regulator of mitochondria gene transcription by metformin, PGC-1α promoter activity was stimulated by metformin via CREB and ATF2 pathways. PGC-1α and phosphorylation of ATF2 and CREB by metformin were selectively increased in the SN and the striatum, but not the cortex. Finally, we showed that metformin protected dopaminergic neurons and improved dopamine-sensitive motor performance in an MPTP-induced PD animal model. Together these results suggest that the metformin-ATF2/CREB-PGC-1α pathway might be promising therapeutic target for PD.

Estrogen receptor activation contributes to RNF146 expression and neuroprotection in Parkinson's disease models.

RNF146 is an E3 ubiquitin ligase that specifically recognizes and polyubiquitinates poly (ADP-ribose) (PAR)-conjugated substrates for proteasomal degradation. RNF146 has been shown to be neuroprotective against PAR polymerase-1 (PARP1)-induced cell death during stroke. Here we report that RNF146 expression and RNF146 inducers can prevent cell death elicited by Parkinson's disease (PD)-associated and PARP1-activating stimuli. In SH-SY5Y cells, RNF146 expression conferred resistance to toxic stimuli that lead to PARP1 activation. High-throughput screen using a luciferase construct harboring the RNF146 promoter identified liquiritigenin as an RNF146 inducer. We found that RNF146 expression by liquiritigenin was mediated by estrogen receptor activation and contributed to cytoprotective effect of liquiritigenin. Finally, RNF146 expression by liquiritigenin in mouse brains provided dopaminergic neuroprotection in a 6-hydroxydopamine PD mouse model. Given the presence of PARP1 activity and RNF146 deficits in PD, it could be a potential therapeutic strategy to restore RNF146 expression by natural compounds or estrogen receptor activation.

Long-term exposure to triphenylphosphate alters hormone balance and HPG, HPI, and HPT gene expression in zebrafish (Danio rerio).

With the global decline in the use of polybrominated diphenyl ethers, the demand for alternative flame retardants, such as triphenylphosphate (TPP), has increased substantially. Triphenylphosphate is now detected in various environments including aquatic ecosystems worldwide. However, studies on the toxicological consequences of chronic TPP exposure on aquatic organisms are scarce. The zebrafish model was used to investigate the effects of long-term TPP exposure on the endocrine system. Zebrafish embryos were exposed to 5 µg/L, 50 µg/L, or 500 µg/L TPP for 120 d, and hormonal and transcriptional responses were measured along the hypothalamic-pituitary-gonad (HPG) axis, the hypothalamic-pituitary-interrenal (HPI) axis, and the hypothalamic-pituitary-thyroid (HPT) axis. Exposure to TPP significantly increased plasma 17ß-estradiol, but decreased 11-ketotestosterone in both sexes. Gene expression data support these changes. In the HPI axis, plasma cortisol and proopiomelanocortin (pomc) and mineralocorticoid receptor transcripts increased in females, but in males cortisol decreased whereas pomc increased (p < 0.05). Thyroxine and triiodothyronine increased, and thyrotrophin-releasing hormone receptor 2 (trhr2) and trh expression were affected only in females (p < 0.05). In summary, long-term exposure to TPP enhanced estrogenicity in both males and females, potentially through influencing the HPG axis, but modulated the HPI, and HPT axes differently by sex, suggesting that both genomic and nongenomic responses might be involved. Environ Toxicol Chem 2016;35:2288-2296. © 2016 SETAC.

Brca2 deficiency leads to T cell loss and immune dysfunction.

Germline mutations in the breast cancer type 2 susceptibility gene (BRCA2) are linked to familial breast cancer and the progressive bone marrow failure syndrome Fanconi anaemia. Established Brca2 mouse knockout models show embryonic lethality, but those with a truncating mutation at the C-terminus survive to birth and develop thymic lymphoma at an early age. To overcome early lethality and investigate the function of BRCA2, we used T cell-specific conditional Brca2 knockout mice, which were previously shown to develop thymic lymphoma at a low penetrance. In the current study we showed that the number of peripheral T cells, particularly naïve pools, drastically declined with age. This decline was primarily ascribed to improper peripheral maintenance. Furthermore, heterozygous mice with one wild-type Brca2 allele manifested reduced T cell numbers, suggesting that Brca2 haploinsufficiency might also result in T cell loss. Our study reveals molecular events occurring in Brca2-deficient T cells and suggests that both heterozygous and homozygous Brca2 mutation may lead to dysfunction in T cell populations.