Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance

Experimental cancer immunology and therap

AMPA, kainate and NMDA receptor densities in the hippocampus of untreated male rats and females in estrus and diestrus

Steroid hormones systematically affect numerous neuronal targets, thus influencing, in a permanent or a transitory manner, the way the brain reacts to external and internal stimuli. The hippocampus is an important brain region for learning and memory and the glutamatergic intrahippocampal pathway plays a major role in performing such functions. We applied quantitative in vitro receptor autoradiography to examine how the in vivo hormone milieu affects the densities of AMPA, kainate, and NMDA receptors in the hippocampus of adult male rats and females in estrus and diestrus. All three examined receptor types presented significant gender-specific differences in their densities. The hippocampus of male rats contains significantly more AMPA, kainate, and NMDA receptors than that of female rats. Female rats in diestrus have significantly higher AMPA receptor densities than female rats in estrus. AMPA changes occurred to the same extent in CA1-3 and in the dentate gyrus. Significant differences in the densities of NMDA receptors were observed in the CA1-3 regions, whereas kainate receptor differences were restricted to the CA1 region. These results further support that steroid hormones, through their modulation of AMPA and NMDA receptors, may be involved in the control of synaptic efficacy and, therefore, influence learning and memory

Aligned Schwann cells within 3D tissue-like gels provide guidance to regenerating neurites

There is a clinical demand to shorten the delay of reinnervation and improve functional recovery after peripheral nerve injury. A peripheral nerve repair device with the ability to direct and promote cellular growth across a lesion would be a promising alternative to nerve autograft repair, which is the current gold standard treatment. The growth of axons across a lesion is most effective when supported by columns of aligned Schwann cells, as found in an autograft. Here we report a technique to generate aligned Schwann cells within a stable and robust 3D collagen matrix, providing a cellular biomaterial that confers alignment on regenerating neurons. Collagen gels containing F7 Schwann cells were tethered for 24 h to permit cellular self-alignment and then plastic compressed by the rapid removal of the interstitial fluid from fully hydrated gels. This process generates stable tissue-like gels with cells situated within a dense, strong, three-dimensional matrix. Cell alignment was monitored before and after plastic compression using CellTracker dye and confocal image analysis. Dissociated dorsal root ganglia (DRG) cells were cultured on the surface of the material for 3 days and neurite growth was quantified using immunostaining and confocal microscopy. Chains of aligned Schwann cells were formed within the collagen matrix and persisted following plastic compression. This robust, aligned cellular biomaterial promotes and guides neuronal growth in a manner that mimics a nerve autograft. The next stage of this work is to integrate this cellular material into a repair device. Plastic compressed gels containing aligned Schwann cells have been rolled into columns which can then be packed together. In vitro testing of this engineered endoneurium, within a silicone outer tube, demonstrates the potential of such a device to function as an implantable conduit for peripheral nerve repair

AMPA, kainate, and NMDA receptor densities in the hippocampus of untreated male rats and females in estrus and diestrus

Steroid hormones systematically affect numerous neuronal targets, thus influencing, in a permanent or a transitory manner, the way the brain reacts to external and internal stimuli. The hippocampus is an important brain region for learning and memory and the glutamatergic intrahippocampal pathway plays a major role in performing such functions. We applied quantitative in vitro receptor autoradiography to examine how the in vivo hormone milieu affects the densities of AMPA, kainate, and NMDA receptors in the hippocampus of adult male rats and females in estrus and diestrus. All three examined receptor types presented significant gender-specific differences in their densities. The hippocampus of male rats contains significantly more AMPA, kainate, and NMDA receptors than that of female rats. Female rats in diestrus have significantly higher AMPA receptor densities than female rats in estrus. AMPA changes occurred to the same extent in CA1-3 and in the dentate gyrus. Significant differences in the densities of NMDA receptors were observed in the CA1-3 regions, whereas kainate receptor differences were restricted to the CA1 region. These results further support that steroid hormones, through their modulation of AMPA and NMDA receptors, may be involved in the control of synaptic efficacy and, therefore, influence learning and memory