| 研究課題/領域番号 |
18K14695
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| 研究機関 | 大阪大学 |
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研究代表者 |
パヴィヨン ニコラ 大阪大学, 免疫学フロンティア研究センター, 特任助教(常勤) (80644525)
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| 研究期間 (年度) |
2018-04-01 – 2020-03-31
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| キーワード | immune response / cell-to-cell / label-free microscopy / live cell imaging / macrophage / lymphocyte / Raman spectroscopy / quantitative phase |
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| 研究実績の概要 |
The research has advanced on all the sub-projects devised in the original proposal, where the study of both cytokine-induced and immune synapse activation were to be studied.
For the study of cytokines, our microscope was improved to allow the tracking of multiple cells in the field of view, increasing the throughput and allowing better pairing between the different measured modalities. This made it possible to link our activation indicators based on label-free measurements with markers of specific molecules through immunostaining, showing a strong correlation between the two.
This approach has been employed to then map cells within larger measured regions to study local and cascading effects that can occur during inflammatory responses because of the release of cytokines. The analysis of these preliminary results is currently ongoing. We started to study such effects in the case of co-cultures, where the molecules secreted by T cells are known to promote immune cell migration and activation. Studies with cell lines are currently ongoing.
We also developed the protocols to extract and purify primary T cells populations and macrophages that will be employed to further validate our results on animal models. We could also demonstrate that our detection approach is robust also in the case of highly heterogeneous populations derived from the peritoneal cavity.
We also started to perform preliminary studies with artificial antigen-presenting beads to study the immune synapses and could validate our protocols to activate cells with such beads.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The planned studies on the effects of cytokines on macrophage activation with our label-free system, either through endogenous cascading effects, or through external secretion from lymphocytes in the case of co-cultures, were initiated, but not completed. The delays in this part of the project are due to the necessity of first developing technical solutions to improve the throughput of the microscope system, as well as reliability of the measurements for better validation. Furthermore, it has been necessary to refine the experimental protocols to ensure a more reproducible environment in terms of culturing conditions. The measurements and analysis are nevertheless currently ongoing.
On the other hand, the need to refine and study new experimental protocols allowed us to already prepare the procedures required for the second part of the project, in order to retrieve purified primary cell populations to validate our approach with animal models. Furthermore, we could already study the effect of commercially available coated microbeads to activate these populations.
That advance on the second part of the project implies that overall the research is on track with the original proposal.
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| 今後の研究の推進方策 |
During the second year of the project, we will continue the measurements related to the influence of cytokines, either in terms of feedback signaling (pure macrophage cells cultures), or in the case of extraneous stimulation (co-culture with pre-stimulated T cells), and perform the analysis. This will require the development of new computing tools to efficiently pair the data measured by multiple modalities and the training of new machine learning algorithms for these specific conditions.
In a second stage, these tools will also be applied to measurements of immune synapses. This will be performed through similar approaches (i.e. high throughput measurement with point-based Raman), but also with measurements on smaller populations based on molecular (Raman) imaging to retrieve more specific information about the creation of immune synapses on live cells. In a first stage, artificial antigen-presenting beads will be employed to ensure the immune activation efficiency, as well as to facilitate the localization of the immune synapses. This will enable the creation of a baseline of expected responses, both molecular and morphological, which will allow the study of the more complex responses that can occur in physiological conditions. The measurements will then be performed in co-cultures, where physiological antigen-presenting cells (macrophages, dendritic cells) will be co-cultured with lymphocytes to study the physiological case of immune synapses.
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| 次年度使用額が生じた理由 |
As our measurement equipment is mostly developed at this point in the project, most of the expenses in the next fiscal year are expected to be dedicated to consumables aimed at supporting the biological experiments and analysis.
Additional costs will include travel to present the research work of this project at academic meetings, as well as publication fees to publish the results in peer-reviewed journals.
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