CD8(+) T-cell responses against latent viruses can cover considerable portions of the CD8(+) T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in ...humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8(+) T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8(+) T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8(+) T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Resident memory T cells (T
) reside in the lung epithelium and mediate protective immunity against respiratory pathogens. Although lung CD8
T
have been extensively characterized, the properties of ...CD4
T
remain unclear. Here we determined the transcriptional signature of CD4
T
, identified by the expression of CD103, retrieved from human lung resection material. Various tissue homing molecules were specifically upregulated on CD4
T
, whereas expression of tissue egress and lymph node homing molecules were low. CD103
T
expressed low levels of T-bet, only a small portion expressed Eomesodermin (Eomes), and although the mRNA levels for Hobit were increased, protein expression was absent. On the other hand, the CD103
T
showed a Notch signature. CD4
CD103
T
constitutively expressed high transcript levels of numerous cytotoxic mediators that was functionally reflected by a fast recall response, magnitude of cytokine production, and a high degree of polyfunctionality. Interestingly, the superior cytokine production appears to be because of an accessible interferon-γ (IFNγ) locus and was partially because of rapid translation of preformed mRNA. Our studies provide a molecular understanding of the maintenance and potential function of CD4
T
in the human lung. Understanding the specific properties of CD4
T
is required to rationally improve vaccine design.
An organized layer of connective tissue coursing from aorta to esophagus was recently discovered in the mediastinum. The relations with other peri‐esophageal fascias have not been described and it is ...unclear whether this layer can be visualized by non‐invasive imaging. This study aimed to provide a comprehensive description of the peri‐esophageal fascias and determine whether the connective tissue layer between aorta and esophagus can be visualized by magnetic resonance imaging (MRI). First, T2‐weighted MRI scanning of the thoracic region of a human cadaver was performed, followed by histological examination of transverse sections of the peri‐esophageal tissue between the thyroid gland and the diaphragm. Secondly, pretreatment motion‐triggered MRI scans were prospectively obtained from 34 patients with esophageal cancer and independently assessed by two radiologists for the presence and location of the connective tissue layer coursing from aorta to esophagus. A layer of connective tissue coursing from the anterior aspect of the descending aorta to the left lateral aspect of the esophagus, with a thin extension coursing to the right pleural reflection, was visualized ex vivo in the cadaver on MR images, macroscopic tissue sections, and after histologic staining, as well as on in vivo MR images. The layer connecting esophagus and aorta was named ‘aorto‐esophageal ligament’ and the layer connecting aorta to the right pleural reflection ‘aorto‐pleural ligament’. These connective tissue layers divides the posterior mediastinum in an anterior compartment containing the esophagus, (carinal) lymph nodes and vagus nerve, and a posterior compartment, containing the azygos vein, thoracic duct and occasionally lymph nodes. The anterior compartment was named ‘peri‐esophageal compartment’ and the posterior compartment ‘para‐aortic compartment’. The connective tissue layers superior to the aortic arch and at the diaphragm corresponded with the currently available anatomic descriptions. This study confirms the existence of the previously described connective tissue layer coursing from aorta to esophagus, challenging the long‐standing paradigm that no such structure exists. A comprehensive, detailed description of the peri‐esophageal fascias is provided and, furthermore, it is shown that the connective tissue layer coursing from aorta to esophagus can be visualized in vivo by MRI.
In vitro CD40-stimulated chronic lymphocytic leukemia (CLL) cells are resistant to cytotoxic drugs. In sharp contrast, we here show that CD40 stimulation sensitizes CLL cells to rituximab-mediated ...cell death. This increased sensitivity is specific for anti-CD20 treatment. Rituximab-mediated death in CD40-stimulated CLL cells shows rapid kinetics (within hours), and is caspase and p53 independent, but depends on extracellular Ca(2+) and reactive oxygen species (ROS) production. By increasing basal ROS production, CD40 stimulation sensitizes CLL cells to rituximab-mediated death. Our findings provide a rationale for combination treatment of CLL with cytotoxic drugs and anti-CD20 monoclonal antibodies.
•Different image post-processing introduces ADC variation between centres.•Aligning ADC calculation methods between centres improves reproducibility.•Calculation-related ADC variation is larger than ...delineation-related ADC variation.
The apparent diffusion coefficient (ADC), a potential imaging biomarker for radiotherapy response, needs to be reproducible before translation into clinical use. The aim of this study was to evaluate the multi-centre delineation- and calculation-related ADC variation and give recommendations to minimize it.
Nine centres received identical diffusion-weighted and anatomical magnetic resonance images of different cancerous tumours (adrenal gland, pelvic oligo metastasis, pancreas, and prostate). All centres delineated the gross tumour volume (GTV), clinical target volume (CTV), and viable tumour volume (VTV), and calculated ADCs using both their local calculation methods and each of the following calculation conditions: b-values 0–500 vs. 150–500 s/mm2, region-of-interest (ROI)-based vs. voxel-based calculation, and mean vs. median. ADC variation was assessed using the mean coefficient of variation across delineations (CVD) and calculation methods (CVC). Absolute ADC differences between calculation conditions were evaluated using Friedman’s test. Recommendations for ADC calculation were formulated based on observations and discussions within the Elekta MRI-linac consortium image analysis working group.
The median (range) CVD and CVC were 0.06 (0.02–0.32) and 0.17 (0.08–0.26), respectively. The ADC estimates differed 18% between b-value sets and 4% between ROI/voxel-based calculation (p-values < 0.01). No significant difference was observed between mean and median (p = 0.64). Aligning calculation conditions between centres reduced CVC to 0.04 (0.01–0.16). CVD was comparable between ROI types.
Overall, calculation methods had a larger impact on ADC reproducibility compared to delineation. Based on the results, significant sources of variation were identified, which should be considered when initiating new studies, in particular multi-centre investigations.
Because biomass is a widely available, renewable resource, its utilisation for the production of energy has great potential for reducing CO(2) emissions and thereby preventing global warming. In this ...mini-review the 'state of the art' of several fermentation processes is discussed, starting with the most advanced process of ethanol production. This is followed by methane production, an established process for waste water purification which is gaining more attention because of the inherent energy production. Subsequently ABE fermentation is discussed and finally the biological production of hydrogen. The last section proposes a new way to assess and compare the different processes by relating their merit to 'work content' values and 'lost work' instead of the combustion values of their products. It is argued that, especially when dealing with energy from biomass, the application of this methodology will provide a uniform valuation for different processes and products. The described fermentation processes enable the supply of pure energy carriers, either gaseous or liquid, from biomass, yet the introduction of these processes is hampered by two major problems. The first is related to technological shortcomings in the mobilisation of fermentable components from the biomass. The second, having a much greater impact, is linked with socio-economics: until full externality costs are attributed to fossil fuels, accounting for their role in pollution and global warming, the competitiveness of the processes described here will hardly stand a chance.
Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 ...stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavy-chain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-κB (NF-κB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-X.sub.L levels, respectively. A dichotomy in NF-κB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-X.sub.L levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-X.sub.L and remained chemoresistant. The pivotal contribution of Bcl-X.sub.L to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-X.sub.L levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-κB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-X.sub.L levels. Oncogene (2010) 29, 5071-5082; doi: 10.1038/onc.2010.248; published online 28 June 2010 Keywords: CLL; CD40; TLR9; NF-κB; Bcl-X.sub.L; IgVH mutation status
The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced ...apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Deltapsi(m)), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Deltapsi(m), and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling ...immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.
Summary
Rabbit anti‐thymocyte globulin (rATG) induces a long‐lasting lymphocytopenia. CD4+ T cells remain depleted for up to 2 years, whereas the CD8+ T cell compartment is refilled rapidly by highly ...differentiated CD27‐CD45RA+CD57+effector‐type cells. Because the presence of these highly differentiated CD8+ T cells has been associated with cytomegalovirus (CMV) infection, we questioned to what extent restoration of CMV T cell immunity contributes to the re‐emergence of T cells following rATG treatment. We compared T cell repopulation in six CMV‐seropositive patients with CMV reactivation (reactivating CMV+) to that in three CMV+ patients without reactivation (non‐reactivating CMV+), and to that in three CMV‐seronegative recipients receiving a kidney from a CMV‐seronegative donor (CMV−/−). All patients received rATG because of acute allograft rejection. Total CD4 and CD8 counts, frequency and phenotype of virus‐specific CD8+ T cells were determined. In reactivating CMV+ patients, total CD8+ T cells reappeared rapidly, whereas in non‐reactivating CMV+ patients they lagged behind. In CMV−/− patients, CD8+ T cell counts had not yet reached pretransplant levels after 2 years. CMV reactivation was indeed followed by a progressive accumulation of CMV‐specific CD8+ T cells. During lymphocytopenia following rATG treatment, serum interleukin (IL)‐7 levels were elevated. Although this was most prominent in the CMV‐seronegative patients, it did not result in an advantage in T cell repopulation in these patients. Repopulated CD8+ T cells showed increased skewing in their Vβ repertoire in both CMV−/− and reactivating CMV‐seropositive patients. We conclude that rapid T cell repopulation following rATG treatment is driven mainly by CMV.
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