B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

In vivo responses to vaccination with Mage-b, GM-CSF and thioglycollate in a highly metastatic mouse breast tumor model, 4T1.

Metastatic breast cancer is an important contributor to morbidity and mortality. Hence, new therapies are needed that target breast cancer metastases. Here, we focus on Mage-b as a possible vaccine target to prevent the development of breast cancer metastases, through activation of Mage-b-specific cytotoxic T lymphocytes (CTL). The syngeneic cell line 4T1, highly expressing Mage-b, was used as a pre-clinical metastatic mouse breast tumor model. BALB/c mice received three preventive intraperitoneal immunizations with Mage-b DNA vaccine mixed with plasmid DNA, secreting granulocyte-macrophage colony stimulating factor (GM-CSF). In addition, antigen-presenting cells were more efficiently recruited to the peritoneal cavity by the injection of thioglycollate broth (TGB), prior to each immunization. Immunization with Mage-b/GM-CSF/TGB significantly reduced the number of metastases by 67% compared to the saline/GM-CSF/TGB and by 69% compared to the vector control/GM-CSF/TGB. Also, tumor growth was significantly reduced by 45% in mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/ GM-CSF/TGB and by 47% compared to the control vector/ GM-CSF/TGB group. In vivo, the number of CD8 T cells significantly increased in the primary tumors and metastases of mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/GM-CSF/TGB and the control vector/ GM-CSF/TGB group, while the number of CD4 T cells significantly decreased. The combination of Mage-b, GM-CSF and TGB did not only induce significantly higher levels of IFNgamma in the lymph nodes of vaccinated compared to control mice, but also induced significantly higher expression levels of Fas-ligand (FasL) in the primary tumors (expressing Fas protein constitutively), compared to the control mice. Whether the interaction between Fas and FasL may have contributed to the smaller tumors needs to be further analyzed.

Increased cell-to-cell variation in gene expression in ageing mouse heart.

The accumulation of somatic DNA damage has been implicated as a cause of ageing in metazoa. One possible mechanism by which increased DNA damage could lead to cellular degeneration and death is by stochastic deregulation of gene expression. Here we directly test for increased transcriptional noise in aged tissue by dissociating single cardiomyocytes from fresh heart samples of both young and old mice, followed by global mRNA amplification and quantification of mRNA levels in a panel of housekeeping and heart-specific genes. Although gene expression levels already varied among cardiomyocytes from young heart, this heterogeneity was significantly elevated at old age. We had demonstrated previously an increased load of genome rearrangements and other mutations in the heart of aged mice. To confirm that increased stochasticity of gene expression could be a result of increased genome damage, we treated mouse embryonic fibroblasts in culture with hydrogen peroxide. Such treatment resulted in a significant increase in cell-to-cell variation in gene expression, which was found to parallel the induction and persistence of genome rearrangement mutations at a lacZ reporter locus. These results underscore the stochastic nature of the ageing process, and could provide a mechanism for age-related cellular degeneration and death in tissues of multicellular organisms.

Aging and genome maintenance.

Genomic instability in somatic cells has been implicated as a major stochastic mechanism of aging. Using a transgenic mouse model with chromosomally integrated lacZ mutational target genes, we found mutations to accumulate with age at an organ- and tissue-specific rate. Also the spectrum of age-accumulated mutations was found to differ greatly from organ to organ; while initially similar, mutation spectra of different tissues diverged significantly over the lifetime. To explain how genomic instability, which is inherently stochastic, can be a causal factor in aging, it is proposed that randomly induced mutations may adversely affect normal patterns of gene regulation, resulting in a mosaic of cells at various stages on a trajectory of degeneration, eventually resulting in cell death or neoplastic transformation. To directly address this question we demonstrate that it is now possible to analyze single cells, isolated from old and young tissues, for specific alterations in gene expression.

DNA polymerase theta is preferentially expressed in lymphoid tissues and upregulated in human cancers.

DNA polymerase theta (Pol theta) is a recently identified family A polymerase that contains an intrinsic helicase domain. Drosophila Pol theta mutants are hypersensitive to bifunctional DNA crosslinking agents and exhibit an elevated frequency of spontaneous chromosomal aberrations, suggesting a role for Pol theta in repair of DNA interstrand crosslinks and in the general maintenance of genome stability. To investigate a possible involvement of Pol theta in tumorigenesis, we have examined its expression in various normal and malignant tissues. Paired tumor and adjacent nontumorous tissues from patients with lung (n = 27), stomach (n = 28) and colon (n = 26) cancer, as well as 26 normal human tissues, were examined for Pol theta expression by RT-PCR, Northern or Western blot analysis. Pol theta was predominantly expressed in primary lymphoid organs including the fetal liver, thymus and bone marrow where lymphocyte progenitors undergo V(D)J rearrangements of their antigen receptor genes. In addition, Pol theta expression was upregulated in germinal center B cells, in which class switch recombination of the immunoglobulin genes occurs. Examination of Pol theta expression in matched cancer specimens revealed that Pol theta was barely detectable in the nontumorous tissues but was upregulated in 17 of 27 (63%) lung, 11 of 28 (39%) stomach and 20 of 26 (77%) colon cancers. Moreover, patients with high levels of Pol theta expression had a significantly poorer clinical outcome compared with those expressing low levels of Pol theta. These results implicate that Pol theta may have a specialized function in lymphocytes and that its overexpression may contribute to tumor progression.

Stage-specific expression of Clast6/E3/LAPTM5 during B cell differentiation: elevated expression in human B lymphomas.

We have identified a CD40-regulated gene, Clast6/E3/LAPTM5, by subtraction of cDNAs derived from resting and CD40 ligand-treated B cells. Clast6/E3/LAPTM5 is abundantly expressed in resting mature B cells but is rapidly and transiently repressed by treatment with CD40 ligand, a T helper signal that induces B cell activation. Using a fluorescence activated cell sorter, we have purified B-lineage cells into distinct populations based on their differential expression of cell surface markers. Clast6/E3/LAPTM5 was found to be highly expressed in progenitor and precursor B cells, downregulated in late pre-B and immature B cells, and upregulated again in mature B and the germinal center B cells. Interestingly, Clast6/E3/LAPTM5 was expressed at high levels in malignant B lymphomas. These results reveal stage-specific expression of Clast6/E3/LAPTM5 during B cell differentiation and implicates its possible involvement in B cell malignancies.

Growth retardation, polyploidy, and multinucleation induced by Clast3, a novel cell cycle-regulated protein.

We have identified a novel gene, Clast3, by subtraction of cDNAs derived from activated and naive B lymphocytes. Clast3 expression is elevated in cycling cells and down-regulated in cells undergoing growth arrest, indicating that its expression is controlled in a cell cycle-dependent manner. The deduced amino acid sequence of Clast3 cDNA exhibits no significant homology to the known proteins in mammalian and other species. Immunofluorescence staining revealed that Clast3 localizes into discrete nuclear foci. Forced expression of Clast3 results in growth retardation, polyploidy, and generation of multinucleated cells. Treatment of Clast3 transfectants with nocodazole, a spindle-damaging agent, greatly enhances the incidence of the multinucleated cells, suggesting that Clast3 overexpression impairs the same checkpoint activated by nocodazole. Down-regulation of Clast3 expression by antisense oligonucleotides results in a decrease of cells at G(2)-M phase and a concomitant increase of apoptotic cells. These findings indicate that Clast3 is a novel cell cycle-regulated protein and that its constitutive overexpression induces polyploidy and multinucleation by interfering with the mitotic spindle checkpoint.

A promoter region of midkine gene can activate transcription of an exogenous suicide gene in human pancreatic cancer.

We examined a possible application of regulatory regions of the midkine (MK) gene for suicide gene therapy of pancreatic cancer. The expression of MK has been demonstrated in human pancreatic cancer tissues but scarcely in normal adult tissues. Northern blot analysis confirmed that human pancreatic cancer cell lines expressed the MK gene. A 609-bp genomic fragment in the 5'-regulatory region of the MK gene, when transfected into human pancreatic cancer cells, activated the transcription of a fused reporter gene to an extent greater than the SV40 promoter. In contrast, the 609-bp fragment-mediated promoter activity tested in fibroblast cells was significantly weak. Human pancreatic cancer cells (AsPC-1) that were transduced with the herpes simplex virus-thymidine kinase gene linked with the 609-bp promoter markedly increased their sensitivity to a prodrug, ganciclovir, compared with untransduced cells. The present study suggests that preferential cytotoxic effects for pancreatic tumors can be achieved by using the MK promoter.

Clast5/Stra13 is a negative regulator of B lymphocyte activation.

CD40 is a member of the tumor necrosis factor receptor family and mediates a variety of functions of B cells, including B cell survival, proliferation, immunoglobulin gene class switching, memory B cell formation, and regulation of Fas-mediated apoptosis. To begin to elucidate the molecular mechanism governing such diverse functions of CD40, we have isolated a gene from mouse splenic B cells, termed Clast5, whose expression is strongly repressed during B cell activation. Clast5 is identical with Stra13, a recently identified member of the basic helix-loop-helix family of transcription factors. Clast5/Stra13 is highly expressed in unstimulated, resting B cells and is rapidly downregulated by a variety of stimuli that activate B cells, including CD40 ligand, anti-IgM antibodies, lipopolysaccharides and interleukin-4. Forced expression of Clast5/Stra13 in B cells delayed the cell cycle progression into S phase and strongly suppressed Fas-mediated apoptosis. Moreover, Clast5/Stra13 inhibited the colony formation in fibroblasts. Our results suggest that Clast5/Stra13 functions as a negative regulator of B cell activation by inhibiting cell cycle progression and cell growth.

Secretion of interleukin-10 from murine colon carcinoma cells suppresses systemic antitumor immunity and impairs protective immunity induced against the tumors.

Interleukin-10 (IL-10) is a T helper type 2 (Th2) cytokine that suppresses Th1-mediated, cell-mediated immune responses and reciprocally enhances antibody-mediated responses. Previous studies, however, demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. We then examined whether tumor-derived IL-10 could modulate systemic immune responses. Murine colon carcinoma (Colon 26) cells that were retrovirally transduced with the murine IL-10 gene (Colon 26/IL-10) were inoculated in syngeneic immunocompetent or T cell-defective nude mice. Growth of Colon 26/IL-10 tumors was augmented in immunocompetent and, to less extent, in nude mice compared with that of wild-type tumors developed in respective mice. Growth of wild-type tumors was accelerated to the same level as that of Colon 26/IL-10 tumors when wild type and Colon 26/IL-10 cells were respectively inoculated in different flanks of the same immunocompetent mice. This enhanced growth of wild-type tumors was not observed in nude mice. Immunocompetent mice that had rejected IL-2- or IL-12-secreting Colon 26 cells developed protective immunity and became completely resistant to wild-type Colon 26 cells subsequently challenged. However, some of the mice that had rejected IL-2 or IL-12 producers developed Colon 26/IL-10 tumors inoculated thereafter. The present study showed that production of IL-10 from tumor cells impaired T cell- and non-T cell-mediated systemic antitumor immunity in hosts.