Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma.

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)-ß-mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single-cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo.

Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24-48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.

A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q.

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

The effects of secondary structure and O2 on the formation of direct strand breaks upon UV irradiation of 5-bromodeoxyuridine-containing oligonucleotides.

BACKGROUND: 5-Bromodeoxyuridine is a radiosensitizing agent that is currently being evaluated in clinical trials as an adjuvant in the treatment of a variety of cancers. gamma-Radiolysis and UV irradiation of oligonucleotides containing 5-bromodeoxyuridine result in the formation of direct strand breaks at the 5'-adjacent nucleotide by oxidation of the respective deoxyribose. We investigated the effects of DNA secondary structure and O2 on the induction of direct strand breaks in 5-bromodeoxyuridine-containing oligonucleotides. RESULTS: The efficiency of direct strand break formation in duplex DNA is dependent upon O2 and results in fragments containing 3'-phosphate and the labile 3'-ketodeoxyadenosine termini. The ratio of the 3'-termini is also dependent upon O2 and structure. Deuterium product isotope effects and tritium-transfer studies indicate that hydrogen-atom abstraction from the C1'- and C2'-positions occurs in an O2- and structure-dependent manner. CONCLUSIONS: The reaction mechanisms by which DNA containing 5-bromodeoxyuridine is sensitized to damage by UV irradiation are dependent upon whether the substrate is hybridized and upon the presence or absence of O2. Oxygen reduces the efficiency of direct strand break formation in duplex DNA, but does not affect the overall strand damage. It is proposed that the sigma radical abstracts hydrogen atoms from the C1'- and C2'-positions of the 5'-adjacent deoxyribose moiety, whereas the nucleobase peroxyl radical selectively abstracts the C1'-hydrogen atom from this site. This is the second example of DNA damage amplification by a nucleobase peroxyl radical, and might be indicative of a general reaction pattern for this family of reactive intermediates.

Discriminative stimulus properties of the putative dopamine D3 receptor agonist, (+)-PD 128907: role of presynaptic dopamine D2 autoreceptors.

The putative D3 receptor agonist, (+)-PD 128907, is widely used to study the functional relevance of D3 receptors in vivo. Given that non-selective D2/3/4 receptor agonists serve as effective discriminative stimuli in rats we have trained animals to discriminate (+)-PD 128907 (30 microg kg(-1), s.c.) from saline and examined the pharmacological specificity of the response. Consistent with a D3 receptor mediated response, the non-selective D2/3 receptor agonist apomorphine and the D3 preferring agonists 7-OH-DPAT and (-) quinpirole generalised to the cue whilst the D2/3 receptor antagonists haloperidol, raclopride, spiperone and (+)-butaclamol antagonised drug lever responding. In contrast, the D1 selective agonist (+/-)-SKF 81297 and D1/5 selective antagonist, R-(+)-SCH 23390 had no effect. Results also suggest that presynaptic dopamine receptors are involved. Thus the dopamine depleting agent alpha-methyl-p-tyrosine potentiated the effects of a submaximal dose of (+)-PD 128907 whereas amphetamine failed to generalise per se and blocked (+)-PD 128907 lever selection. However, studies using subtype selective antagonists argue against a role for the D3 receptor. Thus the 10-fold selective D2 receptor antagonist L-741,626 blocked the (+)-PD 128907 discriminative stimulus whereas L-745,829 and GR 103,691, antagonists > 40 and > 100-fold selective for D3 receptors, failed to modify the response. These results suggest that presynaptic D2 receptors mediate the discriminative stimulus properties of (+)-PD 128907 and highlight the lack of selectivity of (+)-PD 128907 for D3 receptors in vivo.

The behavioural and neurochemical profile of the putative dopamine D3 receptor agonist, (+)-PD 128907, in the rat.

The functional relevance of the dopamine D3 receptor is still unresolved, largely because of the absence of selective D3 receptor ligands. In the present study we have examined the in vivo profile of (+)-PD 128907, a potent and functionally selective D3 receptor agonist. Low doses of (+)-PD 128907 reduced spontaneous locomotor activity in the rat (ED50 = 13 +/- 3 micrograms/kg, s.c.) a response which was comparable with the non-selective D2,3 receptor agonist apomorphine (ED50 = 13 +/- 1.6 micrograms/kg, s.c.). In addition (+)-PD 128907 impaired prepulse inhibition of the acoustic startle response, with significant effects observed at doses of 30 micrograms/kg when appropriate prepulse intensities were used. Higher doses reversed gamma-butyrolactone-induced catecholamine synthesis (ED50 = 95 +/- 22 and 207 +/- 37 micrograms/kg in accumbens and striatum respectively) and induced yawning (100-300 micrograms/kg), penile grooming (30-1000 micrograms/kg) and sniffing (> or = 300 micrograms/kg) although doses 3- to 10-fold greater than apomorphine were required to produce maximal effects. In contrast to apomorphine, however, (+)-PD 128907 failed to induce intense stereotyped licking and biting in the rat. In view of the potency and selectivity of (+)-PD 128907 for the D3 receptor, a role in the control of locomotor activity is suggested. In addition, the observation that (+)-PD 128907 disrupts prepulse inhibition, a phenomenon which is also impaired in schizophrenic subjects, may indicate the pathological importance of this receptor subtype.

The hypothermic effect of 5-CT in mice is mediated through the 5-HT7 receptor.

The 5-HT(7) receptor is a recent addition to the 5-HT receptor family and to date there is no clear idea as to its potential role in the CNS. The receptor has been mapped by in situ hybridization and 5-HT(7)-like immunoreactivity and has been detected in discrete areas of the brain including the hypothalamus (Oliver et al., 1999). This suggests the receptor may be involved in temperature regulation and have shown that a selective 5-HT(7) receptor antagonist reverses the hypothermic effect of 5-CT in guinea-pigs. The current study confirmed that the 5-HT(7) receptor antagonists, SB-269970 (1-30 mg/kg, i.p.) and SB-258719 (5-20 mg/kg, i.p.), but not the 5-HT(1A) receptor antagonist, WAY 100635(0.1-1 mg/kg, s.c.), or the 5-HT(1B/D) antagonist, GR127935 (1.25-5 mg/kg, i.p.), reversed the hypothermic effect of 5-CT in mice. In addition the effect of 5-CT on body temperature was examined on 5-HT(7) receptor null mutant mice. 5-CT (0.1-1 mg/kg, i.p.) significantly reduced rectal temperature in wildtype but not 5-HT(7) receptor knockout mice. This suggests that the hypothermic effects of 5-CT are mediated through the 5-HT(7) receptor. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986).

Salmonella typhimurium (TA100) mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and its open- and closed-ring analogs.

The mutagenicities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, compound 1), 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, compound 6), and 2-(dichloromethyl)-3,3-dichloropropenal (TCB, compound 7) were determined in the same assay and in repetitive determinations using Salmonella typhimurium (TA 100) without microsomal fraction activation. In addition, the mutagenicity of 2-methyl-3,3-dichloropropenal (compound 8) was assayed in the same manner although not simultaneously with MX, RMX, and TCB. This study was undertaken to ascertain the role of open- and closed-ring forms of MX in the mutagenicity of MX. MX proved to be roughly 100 times more mutagenic than the open-ring analogue TCB and 10 times more mutagenic than the closed-ring analogue RMX. Compound 8 was inactive. Assay stability of the three active compounds in Vogel-Bonner medium at 38 degrees C was estimated as the chemical half-life values by following the change in UV absorbance at selected wave lengths. Half-life values were 10.7, 2.6, and 2.8 hr, respectively, for MX, RMX, and TCB. The enhanced mutagenicity of MX relative to RMX and TCB is attributed to the intrinsic mutagenicity of MX and its greater stability is judged to play only a minor role. Moreover, the greater mutagenicity of the closed-ring analogue RMX relative to the open-ring analogue TCB points to the ring form of MX as the active species even though the open form of MX is predominant under assay conditions.

Effects of ibuprofen on the in vitro invasiveness of a human transitional cell carcinoma.

Non-steroidal anti-inflammatory drugs, such as ibuprofen, have demonstrated significant anti-cancer activity in both animals and humans. We examined the anti-invasive effects of ibuprofen on the human UM-UC urinary bladder carcinoma cell line using a rapid in vitro tumor cell invasion assay. The inhibitory effects of ibuprofen on the invasiveness and motility of the human UM-UC transitional cell carcinoma (TCC) cell line were evaluated using Matrigel coated polycarbonate filters (8 microns pore size) from Transwell cluster plates. In addition, the potential role of prostaglandin E2 in this process was examined. Ibuprofen exposure at non-cytotoxic concentrations resulted in a significant (p < 0.05) dose-dependent reduction of invasion when compared to vehicle exposed controls. Even at the highest concentration, ibuprofen had no effect on the rate of tumor cell division. Similarly, the highest concentration of ibuprofen did not alter tumor cell motility through uncoated 8 microns-pore polycarbonate filters. Addition of both prostaglandin E2 and ibuprofen to the culture medium restored tumor cell invasiveness through Matrigel-coated membranes to levels nearly identical to vehicle exposed controls (DMSO-no ibuprofen). The results indicate that ibuprofen is effective in preventing tumor cell invasion in this in vitro model. Prostaglandin E2 reverses the anti-invasive effects of ibuprofen. The anti-invasive effect of ibuprofen warrants further study alone or in combination with other therapies used in the treatment of early stage transitional cell carcinoma of the urinary bladder.

Creation of mice expressing human antibody light chains by introduction of a yeast artificial chromosome containing the core region of the human immunoglobulin kappa locus.

We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.

High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells.

High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigen-presentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication.

The human immunoglobulin VH repertoire.

A complete map of the human immunoglobulin VH locus on chromosome 14 has recently been constructed. The locus is 1100kb in length and contains 51 functional VH segments interspersed amongst a similar number of pseudogenes. Here, Graham Cook and Ian Tomlinson review the organization of the locus, its polymorphism and the repertoire it encodes.

Lymphoid-specific transcriptional activation by components of the IgH enhancer: studies on the E2/E3 and octanucleotide elements.

The IgH enhancer is a strong lymphoid-specific activator and is composed of multiple factor-binding motifs. One of these, the octamer, is common to enhancer and promoter, binds ubiquitous and lymphoid-specific factors and is able to act as a lymphoid-specific transcriptional activator. However, it is also found as an essential component of promoters active in non-lymphoid cells. From analysis of the activities of synthetic promoters, we suggest that recruitment of the lymphoid-specific octamer-binding protein next to the TATA is sufficient to create a functional lymphoid-specific promoter whereas the ubiquitous octamer binding protein is not active in single copy but can act in concert with other promoter binding factors. However, the activity of the IgH enhancer is not dependent on the octamer and we identify the E2/E3 elements as also being sufficient to confer lymphoid-specificity on a linked gene. Activity of the E2/E3 region results from the synergistic activity of the two motifs, E2 alone being able to confer a low level of activity which is dramatically increased by the adjacent E3. Thus, in the case of both the E2/E3 and the octamer motifs, interactions between adjacent elements can play a critical role in determining the tissue specificity of activity.

Contextual fear conditioning and baseline startle responses in the rat fear-potentiated startle test: a comparison of benzodiazepine/gamma-aminobutyric acid-A receptor agonists.

In the rat, fear-potentiated startle (FPS) test animals are first trained to associate brief light presentations with a mild electric footshock and then tested for startle responses to acoustic stimuli, delivered either in darkness (i.e. baseline startle) or after the conditioning stimulus. Following light presentation the magnitude of the startle response is markedly increased, and the test is commonly used to distinguish anxiolytic drug effects (i.e. a reduction in FPS) from non-specific effects such as sedation/muscle relaxation. However, recent studies suggest that the environment in which the animal is trained may also contribute towards the acquisition of a conditioned fear response (i.e. contextual fear conditioning) and that this may elevate startle responses recorded in the dark. In the present study, therefore, we have compared the benzodiazepine/gamma-aminobutyric acid-A receptor agonist chlordiazepoxide with the partial agonists FG 8205 and bretazenil, which are known to have a reduced propensity to produce sedation/myorelaxation, using two different FPS procedures: (i) conditioning and testing in stabilimeter chambers, and (ii) conditioning and testing in different environments. The results show that FPS can be demonstrated in both procedures and that treatment with chlordiazepoxide, FG 8205 or bretazenil dose-dependently attenuates the response. However, animals conditioned and tested in stabilimeter chambers also showed a significant increase in dark-startle amplitudes compared with non-shocked rats, suggesting that this response was elevated by contextual fear conditioning. Furthermore, despite clear differences in side-effect liabilities, FG 8205 and bretazenil significantly reduced dark-startle responses, suggesting that this measure is also sensitive to the anxiolytic effects of benzodiazepines. In contrast, when animals were conditioned and tested in different environments, dark-startle responses were not significantly different from those recorded in non-shocked rats and treatment with FG 8205 or bretazenil had no effect. Thus, conditioning and testing animals in different environments may provide a more effective means of distinguishing anxiolytic from non-specific drug effects in the rat FPS test.

MAFA-L, an ITIM-containing receptor encoded by the human NK cell gene complex and expressed by basophils and NK cells.

The natural killer cell gene complex on human chromosome 12p12-13 encodes several C-type lectin receptor genes expressed by NK cells and other hematopoietic cells. We have identified a novel receptor gene in this region encoding a putative type II transmembrane glycoprotein. The product is 54% identical to the rat mast cell function-associated antigen (MAFA), which inhibits mast cell activation by IgE. The human MAFA-like receptor (MAFA-L) and the rat MAFA protein are expressed by basophils and both have an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail, consistent with an inhibitory role in basophil activation. Unlike rat MAFA, expression of the MAFA-L gene is not limited to mast cells and basophils. In common with other genes in the NK cell gene complex MAFA-L is also expressed by natural killer cells as well as the monocyte-like cell-line U937. Expression in NK cells is restricted to peripheral blood NK cells, decidual NK cells do not express MAFA-L. While MAFA-L and rat MAFA might have a similar role in basophils, the expression of MAFA-L in other cell types implies additional functions for this molecule. The presence of the MAFA-L gene in the human NK cell complex indicates that this locus encodes C-type lectin receptors expressed by a variety of cells important in host defense.

Effect on mutagenicity of the stepwise removal of hydroxyl group and chlorine atoms from 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: 13C NMR chemical shifts as determinants of mutagenicity.

The response of mutagenicity to the stepwise replacement of chlorine atoms and the hydroxyl group by hydrogen in 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, 1) was determined in several assays by using Salmonella typhimurium tester strain (TA100). In all, eight MX derivatives were assayed. Several were studied together in at least one assay. In addition to MX, the seven included 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, 2), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (3), 3-chloro-4-(chloromethyl)-2(5H)-furanone (4), 4-(chloromethyl)-5-hydroxy-2(5H)-furanone (5), 4-chloromethyl-2(5H)-furanone (6), and 4-(dichloromethyl)-2(5H)-furanone (8). Compounds 1-6 were mutagenic. Compound 8 gave erratic results. 4-(Acetoxymethyl)-2(5H)-furanone (11) was nonmutagenic. The largest drop in mutagenicity amounted to a factor of about 10(2) for the replacement of the hydroxyl group or a C-3 chlorine atom from 3. Other replacements of the hydroxyl group or a C-3 or C-6 chlorine atom amounted to mutagenicity diminished by a factor of only 10. On the basis of the rates of UV spectral changes under assay conditions, chemical half-life values (ct 1/2) for 1-6 and 8 were estimated as indicators of compound stability. However, mutagenicity differences were shown to result principally from the intrinsic mutagenicities of the six compounds 1-6 rather than from differences in stability.(ABSTRACT TRUNCATED AT 250 WORDS)