SHARPIN controls regulatory T cells by negatively modulating the T cell antigen receptor complex.

SHARPIN forms a linear-ubiquitin-chain-assembly complex that promotes signaling via the transcription factor NF-κB. SHARPIN deficiency leads to progressive multi-organ inflammation and immune system malfunction, but how SHARPIN regulates T cell responses is unclear. Here we found that SHARPIN deficiency resulted in a substantial reduction in the number of and defective function of regulatory T cells (Treg cells). Transfer of SHARPIN-sufficient Treg cells into SHARPIN-deficient mice considerably alleviated their systemic inflammation. SHARPIN-deficient T cells displayed enhanced proximal signaling via the T cell antigen receptor (TCR) without an effect on the activation of NF-κB. SHARPIN conjugated with Lys63 (K63)-linked ubiquitin chains, which led to inhibition of the association of TCRζ with the signaling kinase Zap70; this affected the generation of Treg cells. Our study therefore identifies a role for SHARPIN in TCR signaling whereby it maintains immunological homeostasis and tolerance by regulating Treg cells.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation.

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

The deubiquitinase CYLD controls protective immunity against helminth infection by regulation of Treg cell plasticity.

BACKGROUND: Type 2 immunity can be modulated by regulatory T (Treg) cell activity. It has been suggested that the deubiquitinase cylindromatosis (CYLD) plays a role in the development or function of Treg cells, implying that it could be important for normal protective immunity, where type 2 responses are prevalent. OBJECTIVE: We sought to investigate the role of CYLD in Treg cell function and TH2 cell immune responses under steady-state conditions and during helminth infection. METHODS: Foxp3-restricted CYLD conditional knockout (KO) mice were examined in mouse models of allergen-induced airway inflammation and Nippostrongylus brasiliensis infection. We performed multiplex magnetic bead assays, flow cytometry, and quantitative PCR to understand how a lack of CYLD affected cytokine production, homing, and suppression in Treg cells. Target genes regulated by CYLD were identified and validated by microarray analysis, coimmunoprecipitation, short hairpin RNA knockdown, and transfection assays. RESULTS: Treg cell-specific CYLD KO mice showed severe spontaneous pulmonary inflammation with increased migration of Treg cells into the lung. CYLD-deficient Treg cells furthermore produced high levels of IL-4 and failed to suppress allergen-induced lung inflammation. Supporting this, the conditional KO mice displayed enhanced protection against N brasiliensis infection by contributing to type 2 immunity. Treg cell conversion into IL-4-producing cells was due to augmented mitogen-activated protein kinase and nuclear factor κB signaling. Moreover, Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, and controlled IL-4 production through forming complexes with mitogen-activated protein kinase kinase/extracellular receptor kinase. Correspondingly, both excessive IL-4 production in vivo and the protective role of CYLD-deficient Treg cells against N brasiliensis were reversed by Scinderin ablation. CONCLUSIONS: Our findings indicate that CYLD controls type 2 immune responses by regulating Treg cell conversion into TH2 cell-like effector cells, which potentiates parasite resistance.

Neoadjuvant modified FOLFIRINOX followed by postoperative gemcitabine in borderline resectable pancreatic adenocarcinoma: a Phase 2 study for clinical and biomarker analysis.

BACKGROUND: Patients with borderline resectable pancreatic cancer (BRPC) have poor prognosis with upfront surgery. METHODS: This was a single-arm Phase 2 trial for clinical and biomarker analysis. The primary endpoint is 1-year progression-free survival (PFS) rate. Patients received 8 cycles of neoadjuvant modified (m) FOLFIRINOX. Up to 6 cycles of gemcitabine were given for patients who underwent surgery. Plasma immune cell subsets were measured for analysing correlations with overall survival (OS). RESULTS: Between May 2016 and March 2018, 44 chemotherapy- and radiotherapy-naïve patients with BRPC were included. With neoadjuvant mFOLFIRINOX, the objective response rate was 34.1%, and curative-intent surgery was done in 27 (61.4%) patients. With a median follow-up duration of 20.6 months (95% confidence interval [CI], 19.7-21.6 months), the median PFS and OS were 12.2 months (95% CI, 8.9-15.5 months) and 24.7 months (95% CI, 12.6-36.9), respectively. The 1-year PFS rate was 52.3% (95% CI, 37.6-67.0%). Higher CD14+ monocyte (quartile 4 vs 1-3) and lower CD69+ γδ T cell (γδ TCR+/CD69+) levels (quartiles 1-3 vs 4) were significantly associated with poor OS (p = 0.045 and p = 0.043, respectively). CONCLUSIONS: Neoadjuvant mFOLFIRINOX followed by postoperative gemcitabine were feasible and effective in BRPC patients. Monocyte and γδ T cells may have prognostic implications for patients with pancreatic cancer. ClinicalTrials.gov identifier: NCT02749136.

Neddylation pathway regulates T-cell function by targeting an adaptor protein Shc and a protein kinase Erk signaling.

NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is a ubiquitin-like molecule whose action on modifying protein substrates is critical in various cellular functions but whose importance in the immune system is not well understood. Here we investigated the role of protein neddylation in regulating T-cell function using an in vivo knockdown technique. We found that reduced expression of Ubc12 in CD4(+) T cells led to impaired T-cell receptor/CD28-induced proliferation and cytokine production both in vitro and in vivo, accompanied by reduced Erk activation. These findings were recapitulated by treatment with MLN4924, an inhibitor of NEDD8-activating enzyme. Furthermore, Shc, an adaptor molecule between antigen receptors and the Ras/Erk pathway, was identified as a target for neddylation. Importantly, mice adoptively transferred with Ubc12 knockdown CD4(+) T cells showed markedly ameliorated allergic responses. This study thus identifies an important role for protein neddylation in T-cell function, which may serve as a therapeutic target for inflammatory diseases.

Regulation of T cell function by the ubiquitin-specific protease USP9X via modulating the Carma1-Bcl10-Malt1 complex.

The ubiquitin conjugation system plays an important role in immune regulation; however, the ubiquitin-specific proteases (USPs) that carry out deubiquitination of cellular substrates are poorly understood. Here we show that in vivo knockdown of the deubiquitinating enzyme USP9X attenuates T-cell proliferation. In addition, naïve CD4(+) T cells from USP9X knockdown chimeric mice display decreased cytokine production and T helper cell differentiation in vitro, which we confirmed in vivo by performing adoptive transfer of transgenic T cells and subsequent immunization. USP9X silencing in both a human T-cell line and mouse primary T cells reduced T-cell receptor (TCR) signaling-induced NF-κB activation. Mechanistically, USP9X interacts with Bcl10 of the Carma1-Bcl10-Malt1 (CBM) complex and removes the TCR-induced ubiquitin chain from Bcl10, which facilitates the association of Carma1 with Bcl0-Malt1. These results demonstrate that USP9X is a crucial positive regulator of the TCR signaling pathway and is required for T-cell function through the modulation of CBM complex formation.

Enhancing adoptive T-cell therapy with fucoidan-based IL-2 delivery microcapsules.

Adoptive cell therapy (ACT) with antigen-specific T cells is a promising treatment approach for solid cancers. Interleukin-2 (IL-2) has been utilized in boosting the efficacy of ACT. However, the clinical applications of IL-2 in combination with ACT is greatly limited by short exposure and high toxicities. Herein, a complex coacervate was designed to intratumorally deliver IL-2 in a sustained manner and protect against proteolysis. The complex coacervate consisted of fucoidan, a specific IL-2 binding glycosaminoglycan, and poly-l-lysine, a cationic counterpart (FPC2). IL-2-laden FPC2 exhibited a preferential bioactivity in ex vivo expansion of CD8+T cells over Treg cells. Additionally, FPC2 was embedded in pH modulating injectable gel (FPC2-IG) to endure the acidic tumor microenvironment. A single intratumoral administration of FPC2-IG-IL-2 increased expansion of tumor-infiltrating cytotoxic lymphocytes and reduced frequencies of myeloid populations. Notably, the activation and persistency of tumor-reactive T cells were observed only in the tumor site, not in the spleen, confirming a localized effect of FPC2-IG-IL-2. The immune-favorable tumor microenvironment induced by FPC2-IG-IL-2 enabled adoptively transferred TCR-engineered T cells to effectively eradicate tumors. FPC2-IG delivery system is a promising strategy for T-cell-based immunotherapies.

A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity.

BACKGROUND: Immunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies. METHODS: We conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity. RESULTS: Our genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors. CONCLUSIONS: These findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.

PBK/TOPK Is a Favorable Prognostic Biomarker Correlated with Antitumor Immunity in Colon Cancers.

Immune checkpoint inhibitor therapy has proven efficacy in a subset of colon cancer patients featuring a deficient DNA mismatch repair system or a high microsatellite instability profile. However, there is high demand for more effective biomarkers to expand the colon cancer population responding to ICI therapy. PBK/TOPK, a serine/threonine kinase, plays a role in cell cycle regulation and mitotic progression. Here, we investigated the correlation between PBK/TOPK expression and tumor immunity and its prognostic value in colon cancer. Based on large-scale bioinformatics analysis, we discovered that elevated PBK/TOPK expression predicted a favorable outcome in patients with colon cancer and was positively associated with immune infiltration levels of CD8+ T cells, CD4+ T cells, natural killer cells, and M1 macrophages. In contrast, a negative correlation was found between PBK/TOPK expression and immune suppressor cells, including regulatory T cells and M2 macrophages. Furthermore, the expression of PBK/TOPK was correlated with the expression of T-cell cytotoxicity genes in colon cancer. Additionally, high PBK/TOPK expression was associated with mutations in DNA damage repair genes, and thus with increased tumor mutation and neoantigen burden. These findings suggest that PBK/TOPK may serve as a prognostic and predictive biomarker for immunotherapy in colon cancer.

A novel inhibitor of apoptosis protein (IAP)-interacting protein, Vestigial-like (Vgl)-4, counteracts apoptosis-inhibitory function of IAPs by nuclear sequestration.

The inhibitors of apoptosis proteins (IAP), which include cIAP1, cIAP2 and XIAP, suppress apoptosis through the inhibition of caspases, and the activity of IAPs is regulated by a variety of IAP-binding proteins. Herein, we report the identification of a Vestigial-like 4 (Vgl-4), which functions as a transcription cofactor in cardiac myocytes, as a new IAP binding protein. Vgl-4 is expressed predominantly in the nucleus and its overexpression triggers a relocalization of IAPs from the cytoplasm to the nucleus. cIAP1/2-interacting protein TRAF2 (TNF receptor-associated factor 2) prevented the Vgl-4-driven nuclear localization of cIAP2. Accordingly, the forced relocation of IAPs to the nucleus by Vgl-4 significantly reduced their ability to prevent Bax- and TNFα-induced apoptosis, which can be recovered by co-expression with TRAF2. Our results suggest that Vgl-4 may play a role in the apoptotic pathways by regulating translocation of IAPs between different cell compartments.

Hitting the complexity of the TIGIT-CD96-CD112R-CD226 axis for next-generation cancer immunotherapy.

Antibody-based therapeutics targeting the inhibitory receptors PD-1, PD-L1, or CTLA-4 have shown remarkable clinical progress on several cancers. However, most patients do not benefit from these therapies. Thus, many efforts are being made to identify new immune checkpoint receptor-ligand pathways that are alternative targets for cancer immunotherapies. Nectin and nectin-like molecules are widely expressed on several types of tumor cells and play regulatory roles in T- and NK-cell functions. TIGIT, CD226, CD96 and CD112R on lymphoid cells are a group of immunoglobulin superfamily receptors that interact with Nectin and nectin-like molecules with different affinities. These receptors transmit activating or inhibitory signals upon binding their cognate ligands to the immune cells. The integrated signals formed by their complex interactions contribute to regulating immune-cell functions. Several clinical trials are currently evaluating the efficacy of anti-TIGIT and anti-CD112R blockades for treating patients with solid tumors. However, many questions still need to be answered in order to fully understand the dynamics and functions of these receptor networks. This review addresses the rationale behind targeting TIGIT, CD226, CD96, and CD112R to regulate T- and NK-cell functions and discusses their potential application in cancer immunotherapy. [BMB Reports 2021; 54(1): 2-11].

Mucin1 and Mucin16: Therapeutic Targets for Cancer Therapy.

The mucin (MUC) family is a group of highly glycosylated macromolecules that are abundantly expressed in mammalian epithelial cells. MUC proteins contribute to the formation of the mucus barrier and thus have protective functions against infection. Interestingly, some MUC proteins are aberrantly expressed in cancer cells and are involved in cancer development and progression, including cell growth, proliferation, the inhibition of apoptosis, chemoresistance, metabolic reprogramming, and immune evasion. With their unique biological and structural features, MUC proteins have been considered promising therapeutic targets and also biomarkers for human cancer. In this review, we discuss the biological roles of the transmembrane mucins MUC1 and MUC16 in the context of hallmarks of cancer and current efforts to develop MUC1- and MUC16-targeted therapies.

TIGIT/CD226 Axis Regulates Anti-Tumor Immunity.

Tumors escape immune surveillance by inducing various immunosuppressive pathways, including the activation of inhibitory receptors on tumor-infiltrating T cells. While monoclonal antibodies (mAbs) blocking programmed cell death 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have been approved for multiple cancer indications, only a subset of patients benefit from immune checkpoint blockade therapies, highlighting the need for additional approaches. Therefore, the identification of new target molecules acting in distinct or complementary pathways in monotherapy or combination therapy with PD-1/PD-L1 blockade is gaining immense interest. T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) has received considerable attention in cancer immunotherapy. Recently, anti-TIGIT mAb (tiragolumab) has demonstrated promising clinical efficacy in non-small cell lung cancer treatment when combined with an anti-PD-L1 drug (Tecentriq), leading to phase III trial initiation. TIGIT is expressed mainly on T and natural killer cells; it functions as an inhibitory checkpoint receptor, thereby limiting adaptive and innate immunity. CD226 competes for binding with the same ligands with TIGIT but delivers a positive stimulatory signal to the immune cells. This review discusses the recent discoveries regarding the roles of TIGIT and CD226 in immune cell function and their potential application in cancer immunotherapy.

Harnessing NK cells for cancer immunotherapy: immune checkpoint receptors and chimeric antigen receptors.

Natural killer (NK) cells, key antitumor effectors of the innate immune system, are endowed with the unique ability to spontaneously eliminate cells undergoing a neoplastic transformation. Given their broad reactivity against diverse types of cancer and close association with cancer prognosis, NK cells have gained considerable attention as a promising therapeutic target for cancer immunotherapy. NK cell-based therapies have demonstrated favorable clinical efficacies in several hematological malignancies but limited success in solid tumors, thus highlighting the need to develop new therapeutic strategies to restore and optimize anti-tumor activity while preventing tumor immune escape. The current therapeutic modalities yielding encouraging results in clinical trials include the blockade of immune checkpoint receptors to overcome the immune-evasion mechanism used by tumors and the incorporation of tumor-directed chimeric antigen receptors to enhance NK cell anti-tumor specificity and activity. These observations, together with recent advances in the understanding of NK cell activation within the tumor microenvironment, will facilitate the optimal design of NK cell-based therapy against a broad range of cancers and, more desirably, refractory cancers. [BMB Reports 2021; 54(1): 44-58].

Genome wide CRISPR screening reveals a role for sialylation in the tumorigenesis and chemoresistance of acute myeloid leukemia cells.

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.

CD226hiCD8+ T Cells Are a Prerequisite for Anti-TIGIT Immunotherapy.

Clinical trials are evaluating the efficacy of anti-TIGIT for use as single-agent therapy or in combination with programmed death 1 (PD-1)/programmed death-ligand 1 blockade. How and whether a TIGIT blockade will synergize with immunotherapies is not clear. Here, we show that CD226loCD8+ T cells accumulate at the tumor site and have an exhausted phenotype with impaired functionality. In contrast, CD226hiCD8+ tumor-infiltrating T cells possess greater self-renewal capacity and responsiveness. Anti-TIGIT treatment selectively affects CD226hiCD8+ T cells by promoting CD226 phosphorylation at tyrosine 322. CD226 agonist antibody-mediated activation of CD226 augments the effect of TIGIT blockade on CD8+ T-cell responses. Finally, mFOLFIRINOX treatment, which increases CD226hiCD8+ T cells in patients with pancreatic ductal adenocarcinoma, potentiates the effects of TIGIT or PD-1 blockade. Our results implicate CD226 as a predictive biomarker for cancer immunotherapy and suggest that increasing numbers of CD226hiCD8+ T cells may improve responses to anti-TIGIT therapy.

Extracellular pH modulating injectable gel for enhancing immune checkpoint inhibitor therapy.

Clinical data from diverse cancer types shows that the increased T cell infiltration in tumors correlates with improved patient prognosis. Acidic extracellular pH is a major attribute of the tumor microenvironment (TME) that promotes immune evasion and tumor progression. Therefore, antagonizing tumor acidity can be a powerful approach in cancer immunotherapy. Here, Pluronic F-127 is used as a NaHCO3 releasing carrier to focally alleviate extracellular tumor acidity. In a mouse tumor model, intratumoral treatment with pH modulating injectable gel (pHe-MIG) generates immune-favorable TME, as evidenced by the decrease of immune-suppressive cells and increase of tumor infiltrating CD8+T cells. The combination of pHe-MIG with immune checkpoint inhibitors, anti-PD-1 and anti-TIGIT antibodies, increases intratumoral T cell function, which leads to tumor clearance. Mechanistically, extracellular acidity was shown to upregulate co-inhibitory immune checkpoint receptors and inhibit mTOR signaling pathways in memory CD8+T cells, which impaired effector functions. Furthermore, an acidic pH environment increased the expression and engagement of TIGIT and its ligand CD155, which suggested that the extracellular pH can regulate the suppressive function of TIGIT pathway. Collectively, these findings suggest that pHe-MIG holds potential as a new TME modulator for effective immune checkpoint inhibitor therapies.

Cell cycle-dependent expression of cIAP2 at G2/M phase contributes to survival during mitotic cell cycle arrest.

cIAP2 (cellular inhibitor of apoptosis protein 2) is induced by NF-kappaB (nuclear factor kappaB) when cells need to respond quickly to different apoptotic stimuli. A recent study using cDNA microarray technology has suggested that cIAP2 transcription is regulated in a cell cycle-dependent manner, although the mechanism for such regulation is unknown. In this study, we confirmed the cell cycle-dependent regulation of cIAP2 expression at both the mRNA and protein levels. Additionally, we found that a bipartite CDE (cell cycle-dependent element)/CHR (cell cycle gene homology region) element in the cIAP2 promoter mediates cIAP2 gene activation in G2/M phase. Cell cycle-dependent G2/M-phase-specific cIAP2 expression is enhanced by NF-kappaB activation, and selective down-regulation of cIAP2 causes cells blocked in mitosis with nocodazole to become susceptible to apoptosis, indicating that the G2/M-phase-specific expression of cIAP2 contributes to the survival of mitotically arrested cells. Our studies describing the NF-kappaB-independent G2/M-phase-specific expression of cIAP2 will help in further understanding the molecular basis of cIAP2 over-expression in a variety of human cancers.

The ubiquitin system in immune regulation.

The ubiquitin system plays a pivotal role in the regulation of immune responses. This system includes a large family of E3 ubiquitin ligases of over 700 proteins and about 100 deubiquitinating enzymes, with the majority of their biological functions remaining unknown. Over the last decade, through a combination of genetic, biochemical, and molecular approaches, tremendous progress has been made in our understanding of how the process of protein ubiquitination and its reversal deubiquitination controls the basic aspect of the immune system including lymphocyte development, differentiation, activation, and tolerance induction and regulates the pathophysiological abnormalities such as autoimmunity, allergy, and malignant formation. In this review, we selected some of the published literature to discuss the roles of protein-ubiquitin conjugation and deubiquitination in T-cell activation and anergy, regulatory T-cell and T-helper cell differentiation, regulation of NF-κB signaling, and hematopoiesis in both normal and dysregulated conditions. A comprehensive understanding of the relationship between the ubiquitin system and immunity will provide insight into the molecular mechanisms of immune regulation and at the same time will advance new therapeutic intervention for human immunological diseases.