LncRNA XLOC_003810 promotes T cell activation and inhibits PD-1/PD-L1 expression in patients with myasthenia gravis-related thymoma
Authors: Li Niu
Shenglin Xu
Yong Hu
Kun Cao
Bo Hu
Zheng Jiang
Journal: Scandinavian journal of immunology (2020)
Discipline: IMMUNOLOGY
Keywords: PD-1; PD-L1; T cell; XLOC_003810; myasthenia gravis
Affiliations: Department of Orthopedics, The First Affiliated Hospital of Anhui Medical University, Hefei, China., Department of Radiotherapy, The First Affiliated Hospital of Anhui Medical University, Hefei, China.

Abstract:  This study aimed to investigate the effect of long non-coding RNA XLOC_003810 on the activation of CD4+ T cells and expression of PD-1/PD-L1 in patients with myasthenia gravis-related thymoma (MG-T). Thymus specimens and thymic mononuclear cells were obtained from MG and MG-T patients or cardiac surgery patients undergoing thoracotomy who were selected as negative controls (NC). XLOC_003810 expression was examined using quantitative real-time PCR (qRT-PCR). Frequency of CD4+ T cells and proportion of CD4+ PD-1+ T cells and CD14+ PD-L1+ monocytes were quantified by flow cytometry. The release of inflammatory cytokines was measured by qRT-PCR and enzyme-linked immunosorbent assay. Compared with the NC group, expression of XLOC_003810, frequency of CD4+ T cells and the production of inflammatory cytokines were increased in patients with MG and MG-T. XLOC_003810 overexpression significantly increased the frequency of CD4+ T cells, facilitated the production of inflammatory cytokines and decreased the proportion of CD4+ PD-1+ T cells and CD14+ PD-L1+ monocytes in the thymic mononuclear cells. In contrast, XLOC_003810 knockdown exerted the opposite effect. Together, XLOC_003810 promotes T cell activation and inhibits PD-1/PD-L1 pathway in patients with MG-T. 

Retracted: No

PubPeer comments:
 Earlier this year, PLOS ONE retracted two articles on ethical grounds due to figure images of mice bearing hideous cancer xenografts. **Too large:** https: pubpeer.com publications 40B3BF202DC5C813EAB8F4AAEC4BBA **Even larger and necrotic too:** https: pubpeer.com publications 6FCE06A14C3AC48194A0FFCEF0ACCB PLOS ONE will be operating under US animal husbandry ethics guidelines. At least in the UK, tumour xenografts should not be allowed to grow large, should not be necrotic and must not inhibit locomotion. The xenografted animal must be sacrificed before it will suffer distress. As can be seen in the embedded image from a 2019 paper published by Frontiersin, for five mice, the grafted cancers (not xenografts as it is a mouse cancer cell line) have grown into whoppers, three are severely necrotic, and they are all to varying extents limiting the free movement of the front left leg - with the G1 mouse being barely able to place its paw on the ground. ![image] However it is not just five mice that had to suffer but 25 since there were five per condition according to the figure legend and the Fig. 6F plot. All the PBS mice and many mice in other groups were directly killed by their tumours (Fig. 6E). Oddly, the PBS survival curve is incompatible with five dead mice but would fit three. The largest mouse-killing tumours approached eight grams (Fig. 6F). Yet body weight did not change (Fig. 6D), indicating that eight grams worth of non-cancerous meecy tissue was lost in compensation. To recap, the mice that you see here, dear reader, constitute a mere fifth of the suffering inflicted on the 25 cancer-injected mice. Tongji University ethics committee happily signed off on this. But what about the publisher? Frontiersin, being domiciled in Switzerland, are not subject to EU animal ethics regulations. I have no idea what the Swiss regulations are like and have not looked into them at this point. Of course it is well known that Frontiersin journal editors that reject too many papers are likely to get fired. https: forbetterscience.com 2018 03 06 editor-sacked-over-rejection-rate-not-inline-with-frontiers-core-principles https: www.sciencemag.org news 2015 05 open-access-publisher-sacks-31-editors-amid-fierce-row-over-independence And it is well known that Frontiersin reviewers that reject papers are often secretly delisted and replaced by more compliant ones. https: twitter.com c_hertweck status 1293483645777453062 Therefore the question arises: Did a reviewer of this manuscript spot the ethics violation, reject the paper, but then was delisted and replaced? Enquiring minds want to know. , 
 The Frontiers in Chemistry editorial office has now been invited to contemplate the ethics of the above images. , 
 #1 Thanks for your comments about animal ethics in this article. We are willing to discuss and clarify the issues you have raised. First of all, our animal experiments followed the laboratory ethical requirement that mice should be sacrificed when the tumor size over 2cm. In other words, the tumor of a mouse over 2 cm is equivalent to death (endpoint of the observation). So, we did not allow the mouse tumor to continue to grow too larger and view the mice to be killed by the tumor painfully. Secondly, the mice were photographed under anesthesia (Figure 6b). The tumor size did not limit the mice s movement as your description. In fact, every time we treated mice with laser, the mice were also under anesthesia. Thirdly, the tumors of mice in the laser-treated group exhibited necrosis, which is owing to the need for treatment efficacy. Of course, the questions you raised are also worthy of our attention. It is critical to alleviating the pain of treatment and amply anti-tumor effect in both, so we will pay more attention to this issue in the future. Besides, there were also some point-to-point answers: Thanks for your comments, we found that we made a data processing error of PBS survival curve in Figure 6E. We will contact the editorial office for errata as soon as possible. In Figure 6F, the observation endpoint of body weight is Day19, while the largest PBS tumor was observed in Day34. Regrettably, my data was shown in a way that is easily misunderstood. Finally, thank you again for pointing out the problems. Your comments can make more people pay attention to the ethics of experimental animals, which is very meaningful. We will contact the Frontiers in Chemistry editorial office to apply for errata and add more detailed explanations and clarifications on these issues. , 
 I thank the author for commenting and providing Frontiers with an erratum. Regarding the erratum and above comments, however, the ethical problem with these images cannot be said to be solved. The only change is the survival plot for the control which is now consistent with five mice (Fig. 6E). I do recommend that the authors read these guidelines in order to understand, for example, why onset of necrosis dictates an endpoint and that large tumours inhibit the free locomotion of host mice: https: www.ncbi.nlm.nih.gov pmc articles PMC2883160 How could the control mouse at left (Fig. 6A) be able to run in its exercise wheel? A statement made above is not really true: *First of all, our animal experiments followed the laboratory ethical requirement that mice should be sacrificed when the tumor size over 2cm.* Google, who is our friend, will reveal the formula for the volume of the sphere. V = (4 3)Pi.r^3 Even better, our friend will do the calculation for us. As we have weights, not volumes, we need to consider the density of tumours, which is typically quoted as values such as 1.01 or 1.05 grams cubic centimeter. Itu2019s very close to the density of water. A 2cm diameter means a radius of 1 cm. Given a radius of 1 cm, Google says that the volume would be 4.19 cm^3 which is approximately 4 grams for tumour weight. In Fig. 6F, the largest tumour has a weight a smidgeon under 8 grams. The multitalented Google can even tell us the radius for a perfect 8 cm^3 sphere. It is 1.24 cm, meaning that the diameter is ~2.5 cm. And that is for a perfect sphere. If the tumour is approximately oval, as is likely, the long edge will be longer than 2.5 cm. A difference between 2.0 and 2.5 cm is not much - but it doubles the volume and weight of the cancer. We also know that the mouse with the largest tumour died on day 34 (Fig. 6E). Unfortunately we know that it was not sacrificed due to exceeding the ethical tumour diameter, but that it died a natural death. And we know this because the figure legend tells us that no mice were sacrificed until day 40. *F) Excised tumor weight of mice in above treatment groups when natural death or on Day 40 (endpoint).* ![image] , 
 Thank you for providing useful information and calculation of an idealized model. We have carefully studied the information about animal ethics you provided and we think that we should be more careful about experimental animal ethics. Mice with tumors exceeding ethical requirements (uff1e2 cm) were euthanized (equivalent to the endpoint of observation) in this work. And we have mentioned it in the Erratum. The difference in theoretical calculations makes us realize that it should consider animal welfare as much as possible, though various factors including detection accuracy and growth rate affect the implementation. In the future experimental process, more stringent criteria and more comprehensive assessment should be adopted to protect animal welfare, ensure the timely termination of the experiment. ,