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Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3′UTR with two regions of AID-dependent Ago2-binding sites (5′- and 3′dABs). Top1 3′UTR knockout (3′UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3′UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3′UTR–mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5′- and 3′dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3′UTRKO cells to wild-type cells whereas it does not impact on 3′UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3′UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage is the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR/ELAVL1, a protein that binds to AU-rich elements in RNA. HuR-knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR-KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR-KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR-KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR-KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR and hypermutation in HuR-KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis.

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Although PD-1 blockade cancer immunotherapy has shown potential for a wide range of patients with cancer, its efficacy is limited, in part, due to the loss of effector cytotoxic T lymphocytes (CTLs) via terminal differentiation–induced apoptosis. We previously demonstrated that mitochondrial activation, by the agonists of peroxisome proliferator–activated receptor γ (PPARγ) coactivator 1-α (PGC-1α)/transcription factor complexes, had synergistic effects with a PD-1–blocking monoclonal antibody in a mouse tumor model. In the current study, we examined the molecular mechanism of the synergistic effects of bezafibrate, an agonist of PGC-1α/ PPAR complexes, which enhanced the tumoricidal effects of PD-1 blockade. Bezafibrate activated CTL mitochondria and upregulated oxidative phosphorylation as well as glycolysis, resulting in more proliferation of naïve T cells and improved effector function in CTLs. Bezafibrate also increased fatty acid oxidation (FAO) and mitochondrial respiratory capacity, which supports the extra energy demands of cells in emergencies, allowing cell survival. Carnitine palmitoyl transferase 1 (Cpt1), which is needed for FAO, and Bcl2 were both upregulated. Cpt1 and Bcl2 can form a complex to prevent apoptosis of CTLs. Together, these results indicate that bezafibrate increases or maintains the number of functional CTLs by activating mitochondrial and cellular metabolism, leading in turn to enhanced antitumor immunity during PD-1 blockade. Cancer Immunol Res; 6(11); 1375–87. ©2018 AACR.

The PD-1 receptor triggers a negative immunoregulatory mechanism that prevents overactivation of immune cells and subsequent inflammatory diseases. Because of its biological significance, PD-1 has been a drug target for modulating immune responses. Immunoenhancing anti–PD-1 blocking antibodies have become a widely used cancer treatment; however, little is known about the required characteristics for anti–PD-1 antibodies to be capable of stimulating immunosuppressive activity. Here, we show that PD-1 agonists exist in the group of anti–PD-1 antibodies recognizing the membrane-proximal extracellular region in sharp contrast to the binding of the membrane-distal region by blocking antibodies. This trend was consistent in an analysis of 81 anti-human PD-1 monoclonal antibodies. Because PD-1 agonist antibodies trigger immunosuppressive signaling by cross-linking PD-1 molecules, Fc engineering to enhance FcγRIIB binding of PD-1 agonist antibodies notably improved human T cell inhibition. A PD-1 agonist antibody suppressed inflammation in murine disease models, indicating its clinical potential for treatment of various inflammatory disorders, including autoimmune diseases.

Despite the success of cancer immunotherapies such as programmed cell death–1 (PD-1) and PD-1 ligand 1 (PD-L1) inhibitors, patients often develop resistance. New combination therapies with PD-1/PD-L1 inhibitors are needed to overcome this issue. Bezafibrate, a ligand of peroxisome proliferator–activated receptor–γ coactivator 1α/peroxisome proliferator–activated receptor complexes, has shown a synergistic antitumor effect with PD-1 blockade in mice that is mediated by activation of mitochondria in T cells. We have therefore now performed a phase 1 trial (UMIN000017854) of bezafibrate with nivolumab in previously treated patients with advanced non–small cell lung cancer. The primary end point was the percentage of patients who experience dose-limiting toxicity, and this combination regimen was found to be well tolerated. Preplanned comprehensive analysis of plasma metabolites and gene expression in peripheral cytotoxic T cells indicated that bezafibrate promoted T cell function through up-regulation of mitochondrial metabolism including fatty acid oxidation and may thereby have prolonged the duration of response. This combination strategy targeting T cell metabolism thus has the potential to maintain antitumor activity of immune checkpoint inhibitors and warrants further validation.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8⁺ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses., GABAを標的とする抗腫瘍免疫機構 --代謝産物を介した免疫細胞間制御の一端を解明--. 京都大学プレスリリース. 2021-11-10.

Neoantigen production is a determinant of cancer immunotherapy. However, the expansion of neoantigen abundance for cancer therapeutics is technically challenging. Here, we report that the synthetic compound RECTAS can induce the production of splice-neoantigens that could be used to boost antitumor immune responses. RECTAS suppressed tumor growth in a CD8 + T cell– and tumor major histocompatibility complex class I–dependent manner and enhanced immune checkpoint blockade efficacy. Subsequent transcriptome analysis and validation for immunogenicity identified six splice-neoantigen candidates whose expression was induced by RECTAS treatment. Vaccination of the identified neoepitopes elicited T cell responses capable of killing cancer cells in vitro, in addition to suppression of tumor growth in vivo upon sensitization with RECTAS. Collectively, these results provide support for the further development of splice variant–inducing treatments for cancer immunotherapy.

The transcriptional co-activator Med12 regulates gene expression through the function of its kinase module and by interacting with the larger Mediator complex and associated RNA Polymerase II (RNAPII). Here, we show a kinase module-independent function of Med12 in antibody class switching recombination (CSR). Med12 is essential for IgH 3’ regulatory region (3’RR) or super-enhancer activation and functions with p300 and Jmjd6/Carm1 coactivator complexes. Med12 deficiency leads to a dramatic decrease in H3K27 acetylation and enhancer RNA (eRNA) transcription at 3’RR, with concomitant impairment of AID-induced DNA double strand breaks, long-range S-S synapse formation, and 3’RR-Eμ interaction. CRISPR/dCas9-mediated enhancer activation re-establishes the epigenomic and transcriptional hallmarks of the 3’RR super-enhancer, fully restoring Med12 depletion defects. Notably, we find that 3’RR derived eRNAs are critical for promoting proper S region epigenetic regulation, S-S synapse formation and recruitment of Med12 and AID to the IgH locus. We find specific X-Linked intellectual disability syndrome associated Med12 mutations are defective in both 3’RR eRNA transcription and CSR, suggesting B and neuronal cells may have cell-specific super-enhancer dysfunctions. We conclude Med12 is essential for IgH3’RR activation and eRNA transcription and plays a central role in AID-induced antibody gene diversification and genomic instability in B cells.

Spermidine (SPD) delays age-related pathologies in various organisms. SPD supplementation overcame the impaired immunotherapy against tumors in aged mice by increasing mitochondrial function and activating CD8 + T cells. Treatment of naïve CD8 + T cells with SPD acutely enhanced fatty acid oxidation. SPD conjugated to beads bound to the mitochondrial trifunctional protein (MTP). In the MTP complex, synthesized and purified from Escherichia coli , SPD bound to the α and β subunits of MTP with strong affinity and allosterically enhanced their enzymatic activities. T cell–specific deletion of the MTP α subunit abolished enhancement of programmed cell death protein 1 (PD-1) blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.

Although immune checkpoint blockade therapies have dramatically improved the survival rate of patients with cancer compared with other treatment approaches, a substantial number of patients show poor response or resistance to these therapies. Meta-analyses for programmed cell death 1 (PD-1) blockade responses highlight the importance of neoantigens, derived from non-synonymous mutations in cancer cells, for antitumor immune responses. Intriguingly, recent transcriptome and genomics studies predict the presence of putative neoantigens as a result of an irregular pre-mRNA splicing regulation in cancer cells, indicating that alterations in RNA splicing in cancer cells might induce antitumor immune responses. Here, we report that induction of serine/arginine-rich splicing factor (SRSF)-dependent splicing boosts the production of splicing-associated neoantigens (splice-neoantigens) and potentiates the response to PD-1 blockade. Administration of a synthetic SRSF activator RECTAS suppressed tumor growth in a host CD8+ T cell- and tumor major histocompatibility complex class I-dependent manner and promoted the antitumor effect of anti-PD-L1 antibody without detectable autoimmunity. Subsequent transcriptome analysis and validation for immunogenicity identified six splice-neoantigen candidates whose expression was induced by RECTAS treatment. Importantly, vaccination of the identified neoepitopes elicited T cell responses capable of killing cancer cells in vitro, in addition to suppression of tumor growth in vivo upon sensitization with RECTAS. Collectively, these results provide support for the further development of splice variant-inducing treatments for cancer immunotherapy. Citation Format: Shingo Matsushima, Masahiko Ajiro, Kei Iida, Kenji Chamoto, Tasuku Honjo, Masatoshi Hagiwara. Chemically inducible splice-neoantigens for cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5093.

The gut microbiome has garnered attention as an effective target to boost immunity and improve cancer immunotherapy. We found that B cell-defective (BCD) mice, such as µ-membrane targeted deletion (µMT) and activation-induced cytidine deaminase (AID) knockouts (KOs), have elevated antitumor immunity under specific pathogen-free but not germ-free conditions. Microbial dysbiosis in these BCD mice enriched the type I IFN (IFN) signature in mucosal CD8+ T cells, resulting in up-regulation of the type I IFN-inducible protein stem cell antigen-1 (Sca-1). Among CD8+ T cells, naive cells predominantly circulate from the gut to the periphery, and those that had migrated from the mesenteric lymph nodes (mLNs) to the periphery had significantly higher expression of Sca-1. The gut-educated Sca-1+ naive subset is endowed with enhanced mitochondrial activity and antitumor effector potential. The heterogeneity and functional versatility of the systemic naive CD8+ T cell compartment was revealed by single-cell analysis and functional assays of CD8+ T cell subpopulations. These results indicate one of the potential mechanisms through which microbial dysbiosis regulates antitumor immunity.

Programmed cell death 1 (PD-1) signal receptor blockade has revolutionized the field of cancer therapy. Despite their considerable potential for treating certain cancers, drugs targeting PD-1 still present two main drawbacks: the substantial number of unresponsive patients and/or patients showing recurrences, and side effects associated with the autoimmune response. These drawbacks highlight the need for further investigation of the mechanisms underlying the therapeutic effects, as well as the need to develop novel biomarkers to predict the lack of treatment response and to monitor potential adverse events. Combination therapy is a promising approach to improve the efficacy of PD-1 blockade therapy. Considering the increasing number of patients with cancer worldwide, solving the above issues is central to the field of cancer immunotherapy. In this review, we discuss these issues and clinical perspectives associated with PD-1 blockade cancer immunotherapy.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1–3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8+ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses., A paper in Nature demonstrates that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages able to limit anti-tumour T cell cytotoxicity.

CD8(+) T cells play a central role in antitumor immune responses that kill cancer cells directly. In aged individuals, CD8(+) T cell immunity is strongly suppressed, which is associated with cancer and other age-related diseases. The mechanism underlying this age-related decrease in immune function remains largely unknown. This study investigated the role of T cell function in age-related unresponsiveness to PD-1 blockade cancer therapy. We found inefficient generation of CD44(low)CD62L(low) CD8(+) T cell subset (P4) in draining lymph nodes of tumor-bearing aged mice. In vitro stimulation of naive CD8(+) T cells first generated P4 cells, followed by effector/memory T cells. The P4 cells contained a unique set of genes related to enzymes involved in one-carbon (1C) metabolism, which is critical to antigen-specific T cell activation and mitochondrial function. Consistent with this finding, 1C-metabolism–related gene expression and mitochondrial respiration were down-regulated in aged CD8(+) T cells compared with young CD8(+) T cells. In aged OVA-specific T cell receptor (TCR) transgenic mice, ZAP-70 was not activated, even after inoculation with OVA-expressing tumor cells. The attenuation of TCR signaling appeared to be due to elevated expression of CD45RB phosphatase in aged CD8(+) T cells. Surprisingly, strong stimulation by nonself cell injection into aged PD-1–deficient mice restored normal levels of CD45RB and ameliorated the emergence of P4 cells and 1C metabolic enzyme expression in CD8(+) T cells, and antitumor activity. These findings indicate that impaired induction of the P4 subset may be responsible for the age-related resistance to PD-1 blockade, which can be rescued by strong TCR stimulation.

Programmed cell death-1 (PD-1) is a co-inhibitory receptor that dampens immune responses upon interaction with PD-L1 and PD-L2. Although PD-1 expression on T cells is known to be activation-dependent, how cytokines modify its regulation is not fully resolved. Using polyclonal T-cell activation to study cytokine-dependent PD-1 regulation, we found that IL-2 inhibited transcriptional up-regulation of PD-1 despite the promotion of T-cell activation. The IL-2-mediated reduction in PD-1 expression augmented CD8+ T-cell activities against PD-L1-expressing target cells. To study the mechanism of PD-1 reduction, we focused on STAT5 activation in the IL-2 signaling pathway. Bioinformatic analysis suggested a novel conserved PD-1 promoter domain where NFAT and STAT5 can potentially compete with each other for binding. NFAT1 interaction with this domain revealed substantial potency in PD-1 transcription compared to STAT5A, and STAT5A overexpression could quench NFAT1-dependent PD-1 up-regulation in a sequence-specific manner. Chromatin immunoprecipitation analysis of activated T cells showed that IL-2 treatment significantly diminished the binding of NFAT1 and NFAT2 in the hypothesized competition site, while STAT5 binding to the same region was increased. These results raise the possibility that the competition of transcriptional factors might be involved in the fine-tuning of PD-1 expression by cytokines such as IL-2.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient’s leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient’s lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4−CD8− double-negative αβ T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade. Dysregulated immune features in a patient with a homozygous loss-of-function mutation in PDCD1 suggest that IL-6, IL-23, STAT3 and RORγT might be potential targets for treatment of PD-1 blockade-induced autoimmunity.

The gut microbiome has garnered attention as an effective target to boost immunity and improve cancer immunotherapy. We found that B cell-defective (BCD) mice, such as µ-membrane targeted deletion (µMT) and activation-induced cytidine deaminase (AID) knockouts (KOs), have elevated antitumor immunity under specific pathogen-free but not germ-free conditions. Microbial dysbiosis in these BCD mice enriched the type I IFN (IFN) signature in mucosal CD8(+) T cells, resulting in up-regulation of the type I IFN-inducible protein stem cell antigen-1 (Sca-1). Among CD8(+) T cells, naïve cells predominantly circulate from the gut to the periphery, and those that had migrated from the mesenteric lymph nodes (mLNs) to the periphery had significantly higher expression of Sca-1. The gut-educated Sca-1(+) naïve subset is endowed with enhanced mitochondrial activity and antitumor effector potential. The heterogeneity and functional versatility of the systemic naïve CD8(+) T cell compartment was revealed by single-cell analysis and functional assays of CD8(+) T cell subpopulations. These results indicate one of the potential mechanisms through which microbial dysbiosis regulates antitumor immunity.

Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.

BACKGROUND: Current clinical biomarkers for the programmed cell death 1 (PD-1) blockade therapy are insufficient because they rely only on the tumor properties, such as programmed cell death ligand 1 expression frequency and tumor mutation burden. Identifying reliable, responsive biomarkers based on the host immunity is necessary to improve the predictive values. METHODS: We investigated levels of plasma metabolites and T cell properties, including energy metabolism markers, in the blood of patients with non-small cell lung cancer before and after treatment with nivolumab (n = 55). Predictive values of combination markers statistically selected were evaluated by cross-validation and linear discriminant analysis on discovery and validation cohorts, respectively. Correlation between plasma metabolites and T cell markers was investigated. RESULTS: The 4 metabolites derived from the microbiome (hippuric acid), fatty acid oxidation (butyrylcarnitine), and redox (cystine and glutathione disulfide) provided high response probability (AUC = 0.91). Similarly, a combination of 4 T cell markers, those related to mitochondrial activation (PPARγ coactivator 1 expression and ROS), and the frequencies of CD8(+)PD-1(hi) and CD4(+) T cells demonstrated even higher prediction value (AUC = 0.96). Among the pool of selected markers, the 4 T cell markers were exclusively selected as the highest predictive combination, probably because of their linkage to the abovementioned metabolite markers. In a prospective validation set (n = 24), these 4 cellular markers showed a high accuracy rate for clinical responses of patients (AUC = 0.92). CONCLUSION: Combination of biomarkers reflecting host immune activity is quite valuable for responder prediction. FUNDING: AMED under grant numbers 18cm0106302h0003, 18gm0710012h0105, and 18lk1403006h0002; the Tang Prize Foundation; and JSPS KAKENHI grant numbers JP16H06149, 17K19593, and 19K17673.

Although PD-1 blockade cancer immunotherapy has shown potential for a wide range of patients with cancer, its efficacy is limited, in part, due to the loss of effector cytotoxic T lymphocytes (CTLs) via terminal differentiation–induced apoptosis. We previously demonstrated that mitochondrial activation, by the agonists of peroxisome proliferator–activated receptor γ (PPARγ) coactivator 1-α (PGC-1α)/transcription factor complexes, had synergistic effects with a PD-1–blocking monoclonal antibody in a mouse tumor model. In the current study, we examined the molecular mechanism of the synergistic effects of bezafibrate, an agonist of PGC-1α/ PPAR complexes, which enhanced the tumoricidal effects of PD-1 blockade. Bezafibrate activated CTL mitochondria and upregulated oxidative phosphorylation as well as glycolysis, resulting in more proliferation of naïve T cells and improved effector function in CTLs. Bezafibrate also increased fatty acid oxidation (FAO) and mitochondrial respiratory capacity, which supports the extra energy demands of cells in emergencies, allowing cell survival. Carnitine palmitoyl transferase 1 (Cpt1), which is needed for FAO, and Bcl2 were both upregulated. Cpt1 and Bcl2 can form a complex to prevent apoptosis of CTLs. Together, these results indicate that bezafibrate increases or maintains the number of functional CTLs by activating mitochondrial and cellular metabolism, leading in turn to enhanced antitumor immunity during PD-1 blockade. Cancer Immunol Res; 6(11); 1375–87. ©2018 AACR.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes. Studies on in vitro mutagenized AID as well as its mutations in human patients with hyper-IgM (HIGM)-syndrome type II revealed that C-terminal AID mutations were defective in CSR whereas their DNA cleavage and SHM activities remained intact. The C-terminal mutants of AID were speculated to exert the dominant negative effect on wild-type (WT) AID whereas its mechanism remains unknown. We generated the JP41 (R190X) mutation in one allele and a null mutation on the other allele in a mouse B cell line (CH12F3-2A) using CRISPR/Cas9 genome-editing tools and studied the effect of JP41 expression on the function of exogenously introduced WT AID fused with estrogen receptor (AIDER) in AIDJP41/∆/AIDER CH12F3-2A cells. We found that JP41 expression strongly suppressed not only CSR but also Igh/c-Myc chromosomal translocations by AIDER. We showed that the dominant negative effect is not evident at the DNA cleavage step but obvious at both deletional and inversional recombination steps. We also confirmed the dominant negative effect of other C-terminal mutants, JP8Bdel (R183X) and P20 (34-aa insertion at residue 182) in AID-deficient spleen B cells. Finally, we showed that the expression of JP41 reduced the binding of AIDER with its cofactors (hnRNP L, SERBP1 and hnRNP U). Together, these data indicate that dominant negative effect of JP41 on CSR is likely due to the depletion of the CSR-specific RNA-binding proteins from WT AID.

PD-1 blockade therapy has revolutionized cancer treatments. However, a substantial population of patients is unresponsive. To rescue unresponsive patients, the mechanism of unresponsiveness to PD-1 blockade therapy must be elucidated. Using a ‘bilateral tumor model’ where responsive and unresponsive tumors were inoculated into different sides of the mouse belly, we demonstrated that unresponsive tumors can be categorized into two groups: with and without systemic immunosuppressive property (SIP). The SIP-positive tumors released uncharacterized, non-proteinaceous small molecules that inhibited mitochondrial activation and T cell proliferation. By contrast, the SIP-negative B16 tumor escaped from immunity by losing MHC class I expression. Unresponsiveness of SIP-positive tumors was partially overcome by improving the mitochondrial function with a mitochondrial activator; this was not successful for B16, which employs immune ignorance. These results demonstrated that the ‘bilateral tumor model’ was useful for stratifying tumors to investigate the mechanism of unresponsiveness and develop a strategy for proper combination therapy., eLife digest Immunotherapy is a fast-emerging treatment area that turns the body’s own immune system against cancer. One powerful group of treatments are the PD-1 blockers. PD-1 is an inducible protein that is sometimes found on healthy immune cells called T cells and normally acts to stop T cells mistakenly attacking healthy cells. However, it can also prevent T cells attacking cancer. This happens when cancer cells make a protein called PD-1 ligand, which interacts with PD-1 to switch off nearby T cells. Antibodies that block PD-1 or PD-1 ligand can reactivate T cells, allowing them to destroy the cancer, but this PD-1 blocking therapy currently works in less than half of all patients who receive the treatment. To mount a successful defense against cancer, a T cell needs to be able to perform two key tasks: recognize cancer cells and prepare to attack. T cells are alerted to the presence of the disease by MHC class I proteins on the surface of cancer cells holding up small fragments of molecules that are tell-tale sign that the cell is cancerous. To prepare to attack, a T cell depends on its mitochondria – the powerhouses of the cell – to send a cascade of signals inside the T cell that help it to activate and multiply. It is possible that cancer cells escape PD-1 blocking treatments by interfering with either one of these two tasks. They may either hide their MHC class I proteins to become invisible to passing T cells – a phenomenon known as “local immune ignorance”; or they may release long-range molecules to stop T cells preparing to attack – “systemic immune suppression”. To explore these options further, Kumar, Chamoto et al. developed a new tumor model in mice. Each mouse had two tumors, one that responded to PD-1 blocking treatment and one that did not. The idea was that, if the unresponsive tumor was simply hiding from passing T cells, its presence should not affect the other tumor. But, if it was releasing molecules to block T-cell activation, the other tumor could become unresponsive to PD-1 blocking treatment too. Kumar, Chamoto et al. examined different types of unresponsive tumor in this model system and found that they fell into two groups. The first group simply hid themselves from nearby T cells, while the second group released molecules to dampen all T cells. The identity of these molecules is unknown, but further experiments suggested that they likely work by blocking the mitochondria in T cells. In mice with these tumors, drugs that boosted mitochondria activity made anti-PD-1 treatment more effective. If the findings in this mouse model parallel those in humans, it could open a new research area for immunotherapy. The next step is for researchers need to identify the molecule responsible for systemic immune suppression. This could help to make PD-1 blocking treatments more effective in people who do not currently respond.

Current clinically approved biomarkers for the PD-1 blockade cancer immunotherapy are based entirely on the properties of tumour cells. With increasing awareness of clinical responses, more precise biomarkers for the efficacy are required based on immune properties. In particular, expression levels of immune checkpoint-associated molecules such as PD-1, PD-L1, and CTLA-4 would be critical to evaluate the immune state of individuals. Although quantification of their soluble form leased from the membrane will provide quick evaluation of patients’ immune status, available methods such as enzyme-linked immunosorbent assays to measure these soluble factors have limitations in sensitivity and reproducibility for clinical use. To overcome these problems, we developed a rapid and sensitive immunoassay system based on chemiluminescent magnetic technology. The system is fully automated, providing high reproducibility. Application of this system to plasma of patients with several types of tumours demonstrated that soluble PD-1, PD-L1, and CTLA-4 levels were increased compared to those of healthy controls and varied among tumour types. The sensitivity and detection range were sufficient for evaluating plasma concentrations before and after the surgical ablation of cancers. Therefore, our newly developed system shows potential for accurate detection of soluble PD-1, PD-L1, and CTLA-4 levels in the clinical practice.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutieres syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

PD-1 (programmed-death 1), an immune-inhibitory receptor required for immune self-tolerance whose deficiency causes autoimmunity with variable severity and tissue specificity depending on other genetic factors, is expressed on activated T cells, including the transcription factor FoxP3(+) Treg cells known to play critical roles in maintaining immune tolerance. However, whether PD-1 expression by the Treg cells is required for their immune regulatory function, especially in autoimmune settings, is still unclear. We found that mice with partial FoxP3 insufficiency developed early-onset lympho-proliferation and lethal autoimmune pancreatitis only when PD-1 is absent. The autoimmune phenotype was rescued by the transfer of FoxP3-sufficient T cells, regardless of whether they were derived from WT or PD-1-deficient mice, indicating that Treg cells dominantly protect against development of spontaneous autoimmunity without intrinsic expression of PD-1. The absence of PD-1 combined with partial FoxP3 insufficiency, however, led to generation of ex-FoxP3 T cells with proinflammatory properties and expansion of effector/memory T cells that contributed to the autoimmune destruction of target tissues. Altogether, the results suggest that PD-1 and FoxP3 work collaboratively in maintaining immune tolerance mostly through nonoverlapping pathways. Thus, PD-1 is modulating the activation threshold and maintaining the balance between regulatory and effector T cells, whereas FoxP3 is sufficient for dominant regulation through maintaining the integrity of the Treg function. We suggest that genetic or environmental factors that even moderately affect the expression of both PD-1 and FoxP3 can cause life-threatening autoimmune diseases by disrupting the T-cell homeostasis., 免疫のブレーキPD-1は、制御性T細胞との役割分担によって自己免疫性膵炎を抑制する. 京都大学プレスリリース. 2016-07-20.

Purpose Programmed death-1 (PD-1), a coinhibitory immune signal receptor expressed in T cells, binds to PD-1 ligand and regulates antitumor immunity. Nivolumab is an anti–PD-1 antibody that blocks PD-1 signaling. We assessed the safety and antitumor activity of nivolumab in patients with platinum-resistant ovarian cancer. Patients and Methods Twenty patients with platinum-resistant ovarian cancer were treated with an intravenous infusion of nivolumab every 2 weeks at a dose of 1 or 3 mg/kg (constituting two 10-patient cohorts) from October 21, 2011. This phase II trial defined the primary end point as the best overall response. Patients received up to six cycles (four doses per cycle) of nivolumab treatment or received doses until disease progression occurred. Twenty nivolumab-treated patients were evaluated at the end of the trial on December 7, 2014. Results Grade 3 or 4 treatment-related adverse events occurred in eight (40%) of 20 patients. Two patients had severe adverse events. In the 20 patients in whom responses could be evaluated, the best overall response was 15%, which included two patients who had a durable complete response (in the 3-mg/kg cohort). The disease control rate in all 20 patients was 45%. The median progression-free survival time was 3.5 months (95% CI, 1.7 to 3.9 months), and the median overall survival time was 20.0 months (95% CI, 7.0 months to not reached) at study termination. Conclusion This study, to our knowledge, is the first to explore the effects of nivolumab against ovarian cancer. The encouraging safety and clinical efficacy of nivolumab in patients with platinum-resistant ovarian cancer indicate the merit of additional large-scale investigations (UMIN Clinical Trials Registry UMIN000005714).