Mutation of the murine Bloom's syndrome gene produces global genome destabilization

Bloom's syndrome (BS) is a genetic disorder characterized cellularly by increases in sister chromatid exchanges (SCEs) and numbers of micronuclei. BS is caused by mutation in the BLM DNA helicase gene and involves a greatly enhanced risk of developing the range of malignancies seen in the general population. With a mouse model for the disease, we set out to determine the relationship between genomic instability and neoplasia. We used a novel two-step analysis to investigate a panel of eight cell lines developed from mammary tumors that appeared in Blm conditional knockout mice. First, the panel of cell lines was examined for instability. High numbers of SCEs were uniformly seen in members of the panel, and several lines produced chromosomal instability (CIN) manifested by high numbers of chromosomal structural aberrations (CAs) and chromosome missegregation events. Second, to see if Blm mutation was responsible for the CIN, time-dependent analysis was conducted on a tumor line harboring a functional floxed Blm allele. The floxed allele was deleted in vitro, and mutant as well as control subclones were cultured for 100 passages. By passage 100, six of nine mutant subclones had acquired high CIN. Nine mutant subclones produced 50-fold more CAs than did nine control subclones. Finally, chromosome loss preceded the appearance of CIN, suggesting that this loss provides a potential mechanism for the induction of instability in mutant subclones. Such aneuploidy or CIN is a universal feature of neoplasia but has an uncertain function in oncogenesis. Our results show that Blm gene mutation produces this instability, strengthening a role for CIN in the development of human cancer.     

Role of Bim in regulating CD8+ T-cell responses during chronic viral infection

Apoptosis is critical for the development and maintenance of the immune system. The proapoptotic Bcl-2 family member Bim is important for normal immune system homeostasis. Although previous experiments have shown that Bim is critical for the apoptosis of antigen-specific CD8(+) T cells during acute viral infection, the role of Bim during chronic viral infection is unclear. Using lymphocytic choriomeningitis virus clone 13 infection of mice, we demonstrate a role for Bim in CD8(+) T-cell apoptosis during chronic viral infection. Enumeration of antigen-specific CD8(+) T cells by major histocompatibility complex class I tetramer staining revealed that CD8(+) D(b)NP396-404(+) T cells, which undergo extensive deletion in wild-type mice, exhibited almost no decrease in Bim mutant mice. This contrasts with CD8(+) D(b)GP33-41(+) and CD8(+) D(b)GP276-286(+) T cells that underwent similar decreases in numbers in both Bim mutant and wild-type mice. Increased numbers of CD8(+) D(b)NP396-404(+) T cells in Bim mutant mice were due to lack of apoptosis and could not be explained by altered proliferation, differential homing to tissues, or increased help from CD4(+) T cells. When viral titers were examined, high levels were initially observed in both groups, but in Bim mutant mice, clearance from the spleen and sera was slightly accelerated. These experiments demonstrate the critical role of Bim during chronic viral infection to down-regulate CD8(+) T-cell responses and have implications for designing strategies for optimizing immunotherapies during situations where antigen persists, such as chronic infection, autoimmune syndromes, and cancer.     

High-potency human immunodeficiency virus vaccination leads to delayed and reduced CD8+ T-cell expansion but improved virus control

CD8(+) T lymphocytes are thought to play an important role in the control of acute and chronic human immunodeficiency virus infections. However, there is a significant delay between infection and the first observed increase in virus-specific CD8(+) T-cell numbers. Prior to this time, viral kinetics are not significantly different between controls and vaccinees. Surprisingly, higher initial virus-specific CD8(+) T-cell numbers lead to a longer delay prior to initial CD8(+) T-cell expansion, and slower CD8(+) T-cell increases. Nevertheless, higher initial CD8(+) T-cell numbers were associated with reduced peak and chronic viral loads and reduced CD4(+) T-cell depletion.     

Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.     

Impairment of T-cell functions with the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin

It has been observed that the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin, designated Dalton's lymphoma (DL), in a murine host induces inhibition of various immune responses and is associated with an involution of thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes caused by tumour serum-dependent induction of apoptosis with a decrease of CD4(+)CD8(+), CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. Here, we report that thymocytes of DL-bearing mice are defective in their proliferative ability and in their response to non-specific mitogenic stimulus in vitro. Also, antigen-specific T-cell proliferative ability representing the fundamental T(H) function declines under DL-bearing conditions and upon treatment with serum of DL-bearing mice. Moreover, a significant inhibition of T-cell cytolytic activity with a decreased ability to produce interferon gamma is shown by the T cells of DL-bearing mice and by the T cells treated with DL-ascitic fluid, DL-conditioned medium or serum of DL-bearing mice. Further, addition of interleukin-2 and anti-interleukin-10 to the cultures of thymocytes treated with serum of DL-bearing mice is found to inhibit the induction of apoptosis in thymocytes, a phenomenon associated with the progression of DL growth. Analysis of the results indicates an immune deviation with the predominance of a T(H2)-type response with the progression of tumour. We further discuss the possible mechanisms that may explain the observed tumour-induced diminution of T-cell immunity.     

The early kinetics of cytomegalovirus-specific CD8+ T-cell responses are not affected by antigen load or the absence of perforin or gamma interferon

Both innate and adaptive immune responses participate in the control of murine cytomegalovirus (mCMV) infection. In some mouse strains, like BALB/c, the control of infection relies on the activities of CD8(+) T cells. mCMV-specific CD8(+) T-cell responses are unusual in that, even after mCMV has been controlled in the periphery, the numbers of circulating virus-specific CD8(+) T cells remain high compared to those observed in other viral infections. To better understand the generation and maintenance of mCMV-specific CD8(+) T-cell responses, we evaluated how antigen load and effector molecules, such as perforin (Prf) and gamma interferon (IFN-gamma), influence these responses during acute infection in vivo. Viral burden affected the magnitude, but not the early kinetics, of antigen-specific CD8(+) T-cell responses. Similarly, the magnitude of virus-specific CD8(+) T-cell expansion was affected by Prf and IFN-gamma, but contraction of antigen-specific responses occurred normally in both Prf- and IFN-gamma-deficient mice. These data indicate that control of mCMV-specific CD8(+) T-cell expansion and contraction is more complex than anticipated and, despite the role of Prf or IFN-gamma in controlling viral replication, a full program of T-cell expansion and contraction can occur in their absence.     

Experimental Trypanosoma cruzi infection alters the shaping of the central and peripheral T-cell repertoire

We investigated the thymic and peripheral T-lymphocyte subsets in BALB/c mice undergoing acute or chronic Trypanosoma cruzi infection, in terms of expression of particular Vbeta rearrangements of the T-cell receptor. We first confirmed the severe depletion of CD4(+)CD8(+) thymocytes following acute T. cruzi infection. By contrast, the numbers of CD4(+)CD8(+) cells in subcutaneous lymph nodes increased up to 16 times. In subcutaneous lymph nodes, we found CD4(+)CD8(+) cells that expressed prohibited segments TCRVbeta5 and TCRVbeta12 (which are physiologically deleted in the thymus of BALB/c mice), as did some mature single-positive cells (CD4(+)CD8(-) and CD4(-)CD8(+)). In the thymus of infected animals, although higher numbers of immature cells bearing such Vbeta segments were seen, they were no longer detected in the mature single-positive stage, suggesting that negative selection occurs normally. We also found increased numbers of cells bearing the potentially autoreactive phenotype TCRVbeta5(+) and TCRVbeta12(+) in T-lymphocyte subsets from subcutaneous lymph nodes of T. cruzi chronically infected mice. In conclusion, our data indicate that immature T lymphocytes bearing prohibited TCRVbeta segments leave the thymus and gain the lymph nodes, where they further differentiate into mature CD4(+) or CD8(+) cells. Conjointly, these findings show changes in the shaping of the central and peripheral T-cell repertoire in both acute and chronic phases of murine T. cruzi infection. The release of potentially autoreactive T cells in the periphery of the immune system may contribute to the autoimmune process found in both murine and human Chagas' disease.     

Oligomeric ring structure of the Bloom's syndrome helicase.

Bloom's syndrome is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The Bloom's syndrome gene product, BLM, belongs to the RecQ subfamily of DNA helicases and is required for the maintenance of genomic stability in human cells - in particular, the suppression of reciprocal exchanges between sister chromatids. We have investigated the quaternary structure of BLM using a combination of size-exclusion chromatography and electron microscopy with reference-free image processing. We found that BLM forms hexameric ring structures with an overall diameter of approximately 13 nm surrounding a central hole of approximately 3.5 nm diameter. A fourfold symmetric square form with approximately 11 nm sides and a hole of approximately 4 nm diameter was also detected, which might represent a distinct oligomeric species or a side view of the hexameric form. Chromatography studies indicated that the majority of enzymatically active BLM has an apparent molecular mass of > 700 kDa, which is consistent with an oligomeric structure for BLM. This provides the first structural analysis of an oligomeric ring helicase of eukaryotic cellular origin. These results have implications for the mechanism of action of BLM and suggest that other RecQ family helicases, including the WRN protein associated with Werner's syndrome, might also adopt ring structures.     

B7-1 overexpression by thymic epithelial cells results in transient and long-lasting effects on thymocytes and peripheral T helper cells but does not result in immunodeficiency.

The B7-1 (CD80) molecule provides costimulatory function for the activation of T helper lymphocytes upon encounter with antigen. To investigate the role of this molecule in thymocyte maturation, we have generated transgenic (Tg) mice in which CD80 expression is driven by the keratin 14 promoter (K14). This overexpression of CD80 resulted in the loss of detectable cell surface CD28 expression on thymocytes and a significant reduction in both the surface T cell receptor expression and the ratio of CD4(+) to CD8(+) single-positive thymocytes in Tg animals compared to nontransgenic (non-Tg) controls. While many of these defects were transient, the significant decrease in CD4(+) versus CD8(+) T cell ratio persisted peripherally. Peripheral T cells from these Tg mice were found to be significantly hyporesponsive to T cell mitogens and in mixed leukocyte reaction, effects that our data indicate are due to reduced IL-2 production by Tg T cells upon activation. Despite these functional defects, immunization with both complex and simple protein antigens produced no differences in the proliferative or humoral responses to these antigens between Tg and non-Tg groups. These data indicate that thymic CD80 signaling results in the deletion of significant numbers of CD4(+) T cells but does not culminate in antigen-specific immunodeficiency.     

Selective reduction of post-selection CD8 thymocyte proliferation in IL-15Rα deficient mice

Peripheral CD8(+) T cells are defective in both IL-15 and IL-15Rα knock-out (KO) mice; however, whether IL-15/IL-15Rα deficiency has a similar effect on CD8 single-positive (SP) thymocytes remains unclear. In this study, we investigated whether the absence of IL-15 transpresentation in IL-15Rα KO mice results in a defect in thymic CD8 single positive (SP) TCR(hi) thymocytes. Comparison of CD8SP TCR(hi) thymocytes from IL-15Rα KO mice with their wild type (WT) counterparts by flow cytometry showed a significant reduction in the percentage of CD69(-) CD8SP TCR(hi) thymocytes, which represent thymic premigrants. In addition, analysis of in vivo 5-bromo-2-deoxyuridine (BrdU) incorporation demonstrated that premigrant expansion of CD8SP TCR(hi) thymocytes was reduced in IL-15Rα KO mice. The presence of IL-15 transpresentation-dependent expansion in CD8SP TCR(hi) thymocytes was assessed by culturing total thymocytes in IL-15Rα-Fc fusion protein-pre-bound plates that were pre-incubated with IL-15 to mimic IL-15 transpresentation in vitro. The results demonstrated that CD8SP thymocytes selectively outgrew other thymic subsets. The contribution of the newly divided CD8SP thymocytes to the peripheral CD8(+) T cell pool was examined using double labeling with intrathymically injected FITC and intravenously injected BrdU. A marked decrease in FITC(+) BrdU(+) CD8(+) T cells was observed in the IL-15Rα KO lymph nodes. Through these experiments, we identified an IL-15 transpresentation-dependent proliferation process selective for the mature CD8SP premigrant subpopulation. Importantly, this process may contribute to the maintenance of the normal peripheral CD8(+) T cell pool.     

Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4(+) T-cell depletion and the effect of HAART on the restoration of CD4(+) T cells in GALT. Severe depletion of intestinal CD4(+) T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4(+) T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4(+) T cells, despite the delay in comparison to peripheral blood CD4(+) T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4(+) T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4(+) T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4(+) T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.     

MMP19 is essential for T cell development and T cell-mediated cutaneous immune responses

Matrix metalloproteinase-19 (MMP19) affects cell proliferation, adhesion, and migration in vitro but its physiological role in vivo is poorly understood. To determine the function of MMP19, we generated mice deficient for MMP19 by disrupting the catalytic domain of mmp19 gene. Although MMP19-deficient mice do not show overt developmental and morphological abnormalities they display a distinct physiological phenotype. In a model of contact hypersensitivity (CHS) MMP19-deficient mice showed impaired T cell-mediated immune reaction that was characterized by limited influx of inflammatory cells, low proliferation of keratinocytes, and reduced number of activated CD8(+) T cells in draining lymph nodes. In the inflamed tissue, the low number of CD8(+) T cells in MMP19-deficient mice correlated with low amounts of proinflammatory cytokines, especially lymphotactin and interferon-inducible T cell alpha chemoattractant (I-TAC). Further analyses showed that T cell populations in the blood of immature, unsensitized mice were diminished and that this alteration originated from an altered maturation of thymocytes. In the thymus, thymocytes exhibited low proliferation rates and the number of CD4(+)CD8(+) double-positive cells was remarkably augmented. Based on the phenotype of MMP19-deficient mice we propose that MMP19 is an important factor in cutaneous immune responses and influences the development of T cells.     

Down-regulation of p27Kip1 expression is required for development and function of T cells

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant (p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.     

A cell autonomous role for the Notch ligand Delta-like 3 in αβ T-cell development

Notch signalling is critical to help direct T-cell lineage commitment in early T-cell progenitors and in the development of αβ T-cells. Epithelial and stromal cell populations in the thymus express the Notch DSL (Delta, Serrate and Lag2)ligands Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged 1 and Jagged 2, and induce Notch signalling in thymocytes that express the Notch receptor. At present there is nothing known about the role of the Delta-like 3 (Dll3) ligand in the immune system. Here we describe a novel cell autonomous role for Dll3 in αβ T-cell development. We show that Dll3 cannot activate Notch when expressed in trans but like other Notch ligands it can inhibit Notch signalling when expressed in cis with the receptor. The loss of Dll3 leads to an increase in Hes5 expression in double positive thymocytes and their increased production of mature CD4(+) and CD8(+) T cells. Studies using competitive irradiation chimeras proved that Dll3 acts in a cell autonomous manner to regulate positive selection but not negative selection of autoreactive T cells. Our results indicate that Dll3 has a unique function during T-cell development that is distinct from the role played by the other DSL ligands of Notch and is in keeping with other recent studies indicating that Dll1 and Dll3 ligands have non-overlapping roles during embryonic development.     

The Bloom's syndrome gene product interacts with topoisomerase III

Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age. The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae. This family shows strong evolutionary conservation of protein structure and function. Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III. Here, we have investigated whether this interaction has been conserved in human cells. We show that BLM and hTOPO IIIalpha, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts. Moreover, the purified BLM and hTOPO IIIalpha proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct. We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIalpha. Furthermore, through characterizing a genetic interaction between BLM and TOP3 in S. cerevisiae, we have identified a functional role for the hTOPO IIIalpha interaction domains in BLM.     

CD4 T cells contribute to virus control and pathology following central nervous system infection with neurotropic mouse hepatitis virus

Replication of the neurotropic mouse hepatitis virus strain JHM (JHMV) is controlled primarily by CD8(+) T-cell effectors utilizing gamma interferon (IFN-gamma) and perforin-mediated cytotoxicity. CD4(+) T cells provide an auxiliary function(s) for CD8(+) T-cell survival; however, their direct contribution to control of virus replication and pathology is unclear. To examine a direct role of CD4(+) T cells in viral clearance and pathology, pathogenesis was compared in mice deficient in both perforin and IFN-gamma that were selectively reconstituted for these functions via transfer of virus-specific memory CD4(+) T cells. CD4(+) T cells from immunized wild-type, perforin-deficient, and IFN-gamma-deficient donors all initially reduced virus replication. However, prolonged viral control by IFN-gamma-competent donors suggested that IFN-gamma is important for sustained virus control. Local release of IFN-gamma was evident by up-regulation of class II molecules on microglia in recipients of IFN-gamma producing CD4(+) T cells. CD4(+) T-cell-mediated antiviral activity correlated with diminished clinical symptoms, pathology, and demyelination. Both wild-type donor CD90.1 and recipient CD90.2 CD4(+) T cells trafficked into the central nervous system (CNS) parenchyma and localized to infected white matter, correlating with decreased numbers of virus-infected oligodendrocytes in the CNS. These data support a direct, if limited, antiviral role for CD4(+) T cells early during acute JHMV encephalomyelitis. Although the antiviral effector mechanism is initially independent of IFN-gamma secretion, sustained control of CNS virus replication by CD4(+) T cells requires IFN-gamma.     

TCR gamma delta+ and CD161+ thymocytes express HIV-1 in the SCID-hu mouse,potentially contributing to immune dysfunction in HIV infection

The vast diversity of the T cell repertoire renders the adaptive immune response capable of recognizing a broad spectrum of potential antigenic peptides. However, certain T cell rearrangements are conserved for recognition of specific pathogens, as is the case for TCRgammadelta cells. In addition, an immunoregulatory class of T cells expressing the NK receptor protein 1A (CD161) responds to nonpeptide Ags presented on the MHC-like CD1d molecule. The effect of HIV-1 infection on these specialized T cells in the thymus was studied using the SCID-hu mouse model. We were able to identify CD161-expressing CD3(+) cells but not the CD1d-restricted invariant Valpha24/Vbeta11/CD161(+) NK T cells in the thymus. A subset of TCRgammadelta cells and CD161-expressing thymocytes express CD4, CXCR4, and CCR5 during development in the thymus and are susceptible to HIV-1 infection. TCRgammadelta thymocytes were productively infectable by both X4 and R5 virus, and thymic HIV-1 infection induced depletion of CD4(+) TCRgammadelta cells. Similarly, CD4(+)CD161(+) thymocytes were depleted by thymic HIV-1 infection, leading to enrichment of CD4(-)CD161(+) thymocytes. Furthermore, compared with the general CD4-negative thymocyte population, CD4(-)CD161(+) NK T thymocytes exhibited as much as a 27-fold lower frequency of virus-expressing cells. We conclude that HIV-1 infection and/or disruption of cells important in both innate and acquired immunity may contribute to the overall immune dysfunction seen in HIV-1 disease.