Romidepsin (FK228) regulates the expression of the immune checkpoint ligand PD-L1 and suppresses cellular immune functions in colon cancer.

Romidepsin (FK228), a histone deacetylase inhibitor (HDACi), has anti-tumor effects against several types of solid tumors. Studies have suggested that HDACi could upregulate PD-L1 expression in tumor cells and change the state of anti-tumor immune responses in vivo. However, the influence of enhanced PD-L1 expression in tumor cells induced by romidepsin on anti-tumor immune responses is still under debate. So, the purpose of this study was to explore the anti-tumor effects and influence on immune responses of romidepsin in colon cancer. The results indicated that romidepsin inhibited proliferation, induced G0/G1 cell cycle arrest and increased apoptosis in CT26 and MC38 cells. Romidepsin treatment increased PD-L1 expression in vivo and in vitro via increasing the acetylation levels of histones H3 and H4 and regulating the transcription factor BRD4. In subcutaneous transplant tumor mice and colitis-associated cancer (CAC) mice, romidepsin increased the percentage of FOXP3+ regulatory T cells (Tregs), decreased the ratio of Th1/Th2 cells and the percentage of IFN-γ+ CD8+ T cells in the peripheral blood and the tumor microenvironment. Upon combination with an anti-PD-1 antibody, the anti-tumor effects of romidepsin were enhanced and the influence on CD4+ and CD8+ T cells was partially reversed. Therefore, the combination of romidepsin and anti-PD-1 immunotherapy provides a more potential treatment for colon cancer.

Distinct Neutrophil Populations in the Spleen During PICS.

While mortality after acute sepsis has decreased, the long-term recovery for survivors is still poor, particularly those developing persistent inflammation, immunosuppression, and catabolism syndrome (PICS). While previously thought that activated neutrophils responding to the acute phase of sepsis migrate to the spleen to undergo cell death and contribute to immunosuppression, our data show a significant accumulation of distinct, yet functional, neutrophil populations in the spleen in a murine model of PICS. The exact role and function of neutrophils in this response is still unclear. The objective of our study was to better define the immune function of splenic neutrophils to determine if this could give insight into the pathogenesis of PICS. Using a murine model of cecal ligation and puncture (CLP), which demonstrates all characteristics of PICS by 8 days, spleens were harvested, and neutrophils were identified by Ly6G and CD11b expression via flow cytometry. Nearly all splenic neutrophils expressed CD54, but there were distinct CD54hi and CD54lo cells, with the majority being CD54lo cells during PICS. The CD54hi population showed traditional, proinflammatory properties, but a relatively decreased chemotactic response, while CD54lo cells had significantly higher chemotaxis, yet significantly decreased proinflammatory functions. Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation, we found that the CD54hi population on day 2 after CLP may be participating in emergency myelopoiesis. However, the vast majority of the CD54lo population were paused in the G1 phase at this time point and not proliferating. By day 8 after CLP, most of the CD54hi cells in the spleen were no longer proliferating, while the CD54lo cells were, indicating that CD54lo dominate in extramedullary myelopoiesis at later time points. Almost none of the neutrophils produced arginase or inducible nitric oxide synthase (iNOS), indicating that these are not suppressor cells. Overall, our data demonstrate that neutrophil accumulation in the spleen during PICS is related to extramedullary myelopoiesis, leading to the production of immature neutrophils. While not suppressor cells, the majority have greater chemotactic function but less inflammatory responsiveness, which may contribute to the immunosuppression seen in PICS. Attention to these distinct neutrophil populations after septic or other systemic inflammatory responses is therefore critical to understanding the mechanisms of PICS.

SAMHD1 enhances immunoglobulin hypermutation by promoting transversion mutation.

Activation-induced deaminase (AID) initiates hypermutation of Ig genes in activated B cells by converting C:G into U:G base pairs. G1-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G1 phase to inhibit viral replication. To derestrict G1-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. Samhd1 inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1's restriction of dNTP supply enhances AID's mutagenicity and that the evolution of Ig hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.

Proliferating Helper T Cells Require Rictor/mTORC2 Complex to Integrate Signals from Limiting Environmental Amino Acids.

Antigen-stimulated T cells require elevated importation of essential and non-essential amino acids to generate large numbers of daughter cells necessary for effective immunity to pathogens. When amino acids are limiting, T cells arrest in the G1 phase of the cell cycle, suggesting that they have specific sensing mechanisms to ensure sufficient amino acids are available for multiple rounds of daughter generation. We found that activation of mTORC1, which is regulated by amino acid amounts, was uncoupled from limiting amino acids in the G1 phase of the cell cycle. Instead, we found that Rictor/mTORC2 has an essential role in T cell amino acid sensing. In the absence of Rictor, CD4+ T cells proliferate normally in limiting arginine or leucine. Our data suggest that Rictor/mTORC2 controls an amino acid-sensitive checkpoint that allows T cells to determine whether the microenvironment contains sufficient resources for daughter cell generation.

Immunosuppressive activity of daphnetin, one of coumarin derivatives, is mediated through suppression of NF-κB and NFAT signaling pathways in mouse T cells.

Daphnetin, a plant-derived dihydroxylated derivative of coumarin, is an effective compound extracted from a plant called Daphne Korean Nakai. Coumarin derivates were known for their antithrombotic, anti-inflammatory, and antioxidant activities. The present study was aimed to determine the immunosuppressive effects and the underlying mechanisms of daphnetin on concanavalin A (ConA) induced T lymphocytes in mice. We showed that, in vitro, daphnetin suppressed ConA-induced splenocyte proliferation, influenced production of the cytokines and inhibited cell cycle progression through the G0/G1 transition. The data also revealed that daphnetin could down-regulate activation of ConA induced NF-κB and NFAT signal transduction pathways in mouse T lymphocyte. In vivo, daphnetin treatment significantly inhibited the 2, 4- dinitrofluorobenzene (DNFB) -induced delayed type hypersensitivity (DTH) reactions in mice. Collectively, daphnetin had strong immunosuppressive activity both in vitro and in vivo, suggesting a potential role for daphnetin as an immunosuppressive agent, and established the groundwork for further research on daphnetin.

RP215 single chain fragment variable and single domain recombinant antibodies induce cell cycle arrest at G0/G1 phase in breast cancer.

BACKGROUND: Targeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines. METHODS: The VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function. RESULTS: The novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells. CONCLUSION: Our results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.

[Changes in immune status induced by repeated aggression in male mice].

It has been shown that psychoneurological disorders are accompanied by different disturbances of immunity. Paper aimed to study the effects of repeated experience of aggression in daily agonistic interactions leading to the development of behavioral psychopathology on the parameters of cellular immunity in the thymus and spleen. There were no found the changes in the weight indexes, the number of cells in the thymus, spleen and blood in aggressive mice. In the spleen of aggressive mice percent of B-lymphocytes--CD19+ and CD16/32+, as well as T-lymphocytes CD4+8-, CD4-8+, and CD4+25(hi) decreased and percent of CD4-25+ increased in comparison with the controls. In the thymus percent of CD4-25+ cells are decreased without changes of other types of lymphocytes. Flow cytometric analysis revealed decreased percentage of apoptotic (A(0)) and resting (G0/G1) cells and increased percentage of proliferating cells in phase S+G2/M in the spleen of aggressive male mice in comparison with the control. The percentage of apoptotic thymocytes is increased and the percentage of thymocytes in S+G2/M phase is decreased under the repeated experience of aggression. Data suggest the possible development of an autoimmune procceses in male mice under the influence of repeated experience of aggression.

Inhibitory effects of Trypanosoma cruzi sialoglycoproteins on CD4+ T cells are associated with increased susceptibility to infection.

BACKGROUND: The Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens characterized by decreased IL-2 synthesis. Trypanosoma cruzi mucin (Tc Muc) has been implicated in this phenomenom. These molecules contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the ability of Tc Muc to modulate the activation of CD4(+) T cells. Our data show that cross-linking of CD3 on naïve CD4(+) T cells in the presence of Tc Muc resulted in the inhibition of both cytokine secretion and proliferation. We further show that the sialylated O-Linked Glycan residues from tc mucin potentiate the suppression of T cell response by inducing G1-phase cell cycle arrest associated with upregulation of mitogen inhibitor p27(kip1). These inhibitory effects cannot be reversed by the addition of exogenous IL-2, rendering CD4(+) T cells anergic when activated by TCR triggering. Additionally, in vivo administration of Tc Muc during T. cruzi infection enhanced parasitemia and aggravated heart damage. Analysis of recall responses during infection showed lower frequencies of IFN-γ producing CD4(+) T cells in the spleen of Tc Muc treated mice, compared to untreated controls. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Tc Muc mediates inhibitory efects on CD4(+) T expansion and cytokine production, by blocking cell cycle progression in the G1 phase. We propose that the sialyl motif of Tc Muc is able to interact with sialic acid-binding Ig-like lectins (Siglecs) on CD4(+) T cells, which may allow the parasite to modulate the immune system.

Immunomodulatory properties of colonic mesenchymal stem cells.

AIM: To elucidate the immunomodulatory functions of colonic mesenchymal stem cells (MSCs) in the colonic mucosal immune system. METHODS: The colonic MSCs were isolated, enriched and expanded. The immunosuppressive role of colonic MSCs on activated T cells was evaluated. The cell cycle progression of T cells and the expression of FoxP3+ T cells were assessed by fluorescence-activated cell sorting (FACS). The levels of cytokines and PGE2 were measured by ELISA. RESULT: Mouse colonic MSCs can inhibit the proliferation of activated T cells by arresting cells in G0/G1 phase, induce the expression of CD4+CD25+Foxp3+ T cells (8.05%±0.49% in transwell culture vs 8.45%±0.64% in direct contact culture vs 4.30%±0.28% in control, p<0.05), downregulate the levels of the cytokines TNF-α and IFN-γ, and increase the production of IL-10 (p<0.05). The data obtained from transwell culture and direct contact culture showed no difference (p>0.05). PGE2 level was increased when T cells were cultured with colonic MSCs (385.10±19.45 ng/l in transwell culture vs 387.91±19.85 ng/l in direct contact culture vs 276.21±25.49 ng/l in control, p<0.05). Blocking PGE2 partially reversed the immunosuppression of MSCs on activated T cells proliferation (p<0.05). CONCLUSION: Colonic MSCs have the same immunosuppressive property as other MSCs. They performed their functions partially through secreting soluble factor PGE2. The characterization of these colonic MSCs may be helpful for studying the involvement of stromal cell compartment in colon diseases.

Modeling interleukin-2-based immunotherapy in AIDS pathogenesis.

In this paper, we sought to identify the CD4(+) T-cell dynamics in the course of HIV infection in response to continuous and intermittent intravenous courses of interleukin-2 (IL-2), the principal cytokine responsible for progression of CD4(+) T-lymphocytes from the G1 to the S phase of the cell cycle. Based on multivariate regression models, previous literature has concluded that the increase in survival of CD4(+) T-cell appears to be the critical mechanism leading to sustained CD4(+) T-cell levels in HIV-infected patients receiving intermittent IL-2 therapy. Underscored by comprehensive mathematical modeling, a major finding of the present work is related to the fact that, rather than due to any increase in survival of CD4(+) T-cells, the expressive, selective and sustained CD4(+) T-cell expansions following IL-2 administration may be related to the role of IL-2 in modulating the dynamics of Fas-dependent apoptotic pathways, such as activation-induced cell death (AICD) or HIV-specific apoptotic routes triggered by viral proteins.

Grape antioxidant dietary fiber inhibits intestinal polyposis in ApcMin/+ mice: relation to cell cycle and immune response.

Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.

Analysis of Ig gene hypermutation in Ung(-/-)Polh(-/-) mice suggests that UNG and A:T mutagenesis pathway target different U:G lesions.

The activation-induced cytidine deaminase (AID) initiates Ig gene hypermutation by converting cytosine to uracil (U) and generating a U:G lesion. Genetic and biochemical studies suggest that the AID-triggered U:G lesions are processed by three mutagenic pathways to induce mutations at both C:G and A:T pairs. First, direct replication of the U:G lesion leads to C to T and G to A transitions. Second, U can be excised by the uracil DNA glycosylase (UNG) and the replication/processing of the resulting abasic site leads to transversions and transitions at C:G pairs. Third, the U:G lesion is recognized by an atypical mismatch repair (MMR) pathway which generates mutations at A:T pairs in a DNA polymerase η (POLH)-dependent manner. To further explore whether these three mutagenic pathways function competitively or independently, we have analyzed Ig gene hypermutation in mice deficient in both UNG and POLH. Compared with WT mice, UNG deficiency caused elevated frequency of C:G mutations, suggesting that UNG-mediated U excision led to error-free as well as error-prone repair. In contrast, UNG deficiency did not affect the frequency and patterns of A:T mutations, suggesting that the MMR did not target U:G lesions normally recognized and processed by UNG. In addition, POLH deficiency did not affect the frequency and patterns of C:G mutations and UNG POLH double deficiency showed an additive effect of single deficiency. Based on these observations and previous results, along with the recent finding that UNG excises AID-triggered U predominantly during G1 phase of the cell cycle, it appears that UNG and MMR targets U:G lesions generated during G1 and S phases of the cell cycle, respectively.

Reciprocal control of G1-phase progression is required for Th-POK/Runx3-mediated CD4/8 thymocyte cell fate decision.

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G(1) cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G(1)-phase extension and represses CD8 expression in a G(1) extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G(1) extension and CD8 repression. Importantly, forced G(1) extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G(1) progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G(1)-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

Activation-induced cytidine deaminase-initiated off-target DNA breaks are detected and resolved during S phase.

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G1 phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G1 reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G1 phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G1-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G1. These data suggest that AID-mediated DSBs can evade G1/S checkpoint activation and persist beyond G1, becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.

Polyclonal rabbit antithymocyte globulin induces apoptosis and has cytotoxic effects on human leukemic cells.

UNLABELLED: The possibility of antileukemic activity of antithymocyte globulin (ATG) was investigated in 8 human leukemic cell lines and primary leukemic cells from 15 leukemia patients. The study demonstrated that ATG induced apoptosis and reduced proliferation in both cell lines and primary leukemic cells, particularly in lymphatic origin cells, indicating that ATG has broad-spectrum antileukemic activity, especially for cells of lymphatic origin. BACKGROUND: Polyclonal ATGs are currently used to prevent graft-versus-host disease in allogeneic stem cell transplantation patients and to treat patients with severe aplastic anemia. It contains antibodies against antigens expressed on various hematopoietic cells, we hypothesized that it induces cell death not only in healthy cells but also in malignant hematopoietic cells. MATERIALS AND METHODS: In this study, several human leukemic cell lines and primary leukemic cells from 15 patients with leukemia were used to investigate the ability of polyclonal ATGs to induce apoptosis and proliferation. RESULTS: Polyclonal ATGs induced cell apoptosis in primary leukemic cells and in cell lines in a dose-dependent manner, and induced apoptosis in different populations through a variety of targets. Cell proliferation was significantly reduced in the presence of polyclonal ATGs; it arrested cells in the G0-G1 phase by cell cycle analysis. Treatment with polyclonal ATGs plus complement increased cytolysis of the leukemic cells; complement augments polyclonal ATG-induced leukemic cell death. CONCLUSION: These data show that polyclonal ATG has broad-spectrum antileukemic activity, especially for cells of lymphatic origin, as it induced cell death through a variety of targets. This study provides an experimental basis for the application of polyclonal ATGs in allogeneic hematopoietic stem cell transplantation and in patients with lymphatic leukemia.

Membrane attack by complement: the assembly and biology of terminal complement complexes.

Complement system activation plays an important role in both innate and acquired immunity. Activation of the complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on the cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis.

Bacillus Calmette-Guerin and BCG cell wall skeleton suppressed viability of bladder cancer cells in vitro.

AIM: Bacillus Calmette-Guerin (BCG) is one of therapeutic options for urothelial carcinoma (UC). The objectives of this study were to determine the direct effect of viable or heat-killed BCG and BCG cell wall skeleton (BCG-CWS) on UC cells in vitro. MATERIALS AND METHODS: UC cell lines were co-cultured with viable or heat-killed BCG Immunobladder® (Tokyo 172 strain) and BCG-CWS. Viability of the cells, apoptosis and BrdU incorporation were estimated. RESULTS: BCG induced cell growth retardation in highly malignant UC bearing integrin α5ß1 (VLA5). VLA5-blocking antibody partially abrogated this effect. BCG treatment induced a modest increase in the sub-G(1) fraction of cells and a decrease of BrdU incorporation. Cell growth retardation effect of viable BCG was reproduced by both heat-killed BCG and BCG-CWS. CONCLUSION: The results indicate that VLA5 may be a biomarker of UC with sensitivity to BCG. Moreover, BCG-CWS is a promising substance which might replace BCG, preventing life-threatening complications of viable BCG treatment.

Temporal regulation of Ig gene diversification revealed by single-cell imaging.

Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2.

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I-IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2-dependent yet is gp91phox-independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue-derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum-starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.

The CD8+ memory T-cell state of readiness is actively maintained and reversible.

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.