Tissue-resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer.

Tissue-resident memory T (TRM ) cells are CD8+ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in TRM cells and correlate it with their functional potential. Fifty-three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in TRM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in-vitro stimulation were used to evaluate TRM cell phenotypes. We discovered that tumour TRM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin-expressing TRM cells. Surprisingly, programmed cell death 1 (PD-1) expression is high in tumour TRM cells, suggesting exhaustion. Following interleukin-15 and T cell receptor stimulation, perforin and T-bet expressions are enhanced, indicating that TRM cells from tumours are not terminally exhausted. Moreover, a high number of TRM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, TRM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies TRM cells as potential new targets for cancer immunotherapy.

Long-term responses of the green-algal lichen Parmelia caperata to natural CO2 enrichment.

Acclimation to elevated CO2 was investigated in Parmelia caperata originating from the vicinity of a natural CO2 spring, where the average daytime CO2 concentration was 729 ± 39 µmol mol-1 dry air. Thalli showed no evidence of a down-regulation in photosynthetic capacity following long-term exposure to CO2 enrichment in the field; carboxylation efficiency, total Ribulose bisphosphate carboxylase/oxygenase (Rubisco) content, apparent quantum yield of CO2 assimilation, and the light-saturated rate of CO2 assimilation (measured under ambient and saturating CO2 concentrations) were similar in thalli from the naturally CO2 enriched site and an adjacent control site where the average long-term CO2 concentration was about 355 µmol mol-1. Thalli from both CO2 environments exhibited low CO2 compensation points and early saturation of CO2 uptake kinetics in response to increasing external CO2 concentrations, suggesting the presence of an active carbon-concentrating mechanism. Consistent with the lack of significant effects on photosynthetic metabolism, no changes were found in the nitrogen content of thalli following prolonged exposure to elevated CO2. Detailed intrathalline analysis revealed a decreased investment of nitrogen in Rubisco in the pyrenoid of algae located in the elongation zone of thalli originating from elevated CO2, an effect associated with a reduction in the percentage of the cell volume occupied by lipid bodies and starch grains. Although these differences did not affect the photosynthetic capacity of thalli, there was evidence of enhanced limitations to CO2 assimilation in lichens originating from the CO2-enriched site. The light-saturated rate of CO2 assimilation measured at the average growth CO2 concentration was found to be significantly lower in thalli originating from a CO2-enriched atmosphere compared with that of thalli originating and measured at ambient CO2. At lower photosynthetic photon flux densities, the light compensation point of net CO2 assimilation was significantly higher in thalli originating from elevated CO2, and this effect was associated with higher usnic acid content.

Growth and vitality of epiphytic lichens : II. Modelling of carbon gain using field and laboratory data.

Photosynthetic and respiratory CO2 gas exchange was measured under controlled climate conditions in the laboratory in two epiphytic lichens, Lobaria pulmonaria and Platismatia glauca, with the aim of modelling their net productivity using field microclimate data. For both, the thallus water content (WC) and the light intensity had the greatest impact on photosynthesis. L. pulmonaria had optimum net photosynthesis (NP) at WCs between 75-175% of the thallus dry weight (DW), while P. glauca required a WC of c. 85% for maximal NP without depression at higher WCs. Both species reached light compensation of NP at 5-10 µmol photons m-2 s-1 and were saturated at 100-150 µmol photons m-2 s-1. Respiratory CO2 loss corresponded to 35-40% of gross photosynthesis at 85-100% WC and 15° C, in both species. Growth of the two species were followed in transplanted thalli during a 16-month period at two contrasting sites, a forest edge adjacent to a 15 year old clear-cut and within the interior of a mature Picea abies forest. At these sites, the microclimate parameters; light, temperature, relative humidity (RH) and thallus WC were also monitored. Judged from the microclimate data, the lichens were active for 13-19% of the time with thallus WC monitoring, where >60% of the active time occurred in darkness. When photosynthetically active, the edge transplants received a 2-3 times higher light dose and were active for a longer accumulated time compared to the interior transplants. The field microclimate data in conjunction with the laboratory data predicted a 4 times higher DW yield of the edge transplants compared to the interior transplants. However, the DW yield of L. pulmonaria was overestimated at the edge and underestimated for P. glauca in the interior by our model. Possible reasons for these discrepancies and the validity of using laboratory data and microclimate monitoring to predict growth rates of lichens under varying field conditions are discussed.

Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa.

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO2-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +/- 5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 microM azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa.(ABSTRACT TRUNCATED AT 250 WORDS)

Photosynthetic Light Utilization Efficiency, Photosystem II Heterogeneity, and Fluorescence Quenching in Chlamydomonas reinhardtii during the Induction of the CO(2)-Concentrating Mechanism.

The photosynthetic light-response curve, the relative amounts of the different photosystem II (PSII) units, and fluorescence quenching were altered in an adaptive manner when CO(2)-enriched wild-type Chlamydomonas reinhardtii cells were transferred to low levels of CO(2). This treatment is known to result in the induction of an energy-dependent CO(2)-concentrating mechanism (CCM) that increases the internal inorganic carbon concentration and thus the photosynthetic CO(2) utilization efficiency. After 3 to 6 h of low inorganic carbon treatment, several changes in the photosynthetic energy-transducing reactions appeared and proceeded for about 12 h. After this time, the fluorescence parameter variable/maximal fluorescence yield and the amounts of both PSIIalpha and PSIIbeta (secondary quinone electron acceptor of PSII-reducing) centers had decreased, whereas the amount of PSIIbeta (secondary quinone electron acceptor of PSII-nonreducing) centers had increased. The yield of noncyclic electron transport also decreased during the induction of the CCM, whereas both photochemical and nonphotochemical quenching of PSII fluorescence increased. Concurrent with these changes, the photosynthetic light-utilization efficiency also decreased significantly, largely attributed to a decline in the curvature parameter theta, the convexity of the photosynthetic light-response curve. Thus, it is concluded that the increased CO(2) utilization efficiency in algal cells possessing the CCM is maintained at the cost of a reduced light utilization efficiency, most probably due to the reduced energy flow through PSII.

Acclimation of Photosynthetic Light Reactions during Induction of Inorganic Carbon Accumulation in the Green Alga Chlamydomonas reinhardtii.

Cells of the unicellular green algae Chlamydomonas reinhardtii were grown in high dissolved inorganic carbon (DIC) concentrations (supplied with 50 milliliters per liter CO(2)[g]) and transferred to low DIC concentrations (supplied with

Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii.

The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.

A correlation between changes in luminescence decay kinetics and the appearance of a CO2-accumulating mechanism in Scenedesmus obliquus.

In experiments with the unicellular green algae Scenedesmus obliquus a correlation was found between the presence of the CO2-accumulating mechanism and the appearance of polyphasic luminescence decay kinetics. A potentiometric titration method was used to measure and calculate photosynthetic carbon uptake.Polyphasic luminescence decay kinetics was found when the algae showed photosynthetic characteristics typical of algae adapted to low-CO2 conditions. When high-CO2 grown algae were transferred to low-CO2 conditions they gradually developed polyphasic decay kinetics during the first 25-30 minutes. When low-CO2 grown algae were transferred to high-CO2 conditions the polyphasic decay kinetics disappeared. To account for these results a working hypothesis is presented on the basis of the energy requirement for a CO2-accumulating mechanism.