T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma
Authors: Chen Dong
Stephanie S Watowich
Qiang Tian
Bhanu P Pappu
Anton M Jetten
Li Ma
Askar Akimzhanov
Xuexian O Yang
Roza Nurieva
Hong Soon Kang
Kimberly S Schluns
Athanasia D Panopoulos
Yeonseok Chung
Bhavin Shah
Journal: Immunity (2008)
Discipline: IMMUNOLOGY
Keywords: T cell; functional differentiation; lineage-specific transcription factors; T helper 17; Th17
Affiliations: Department of Immunology, M.D. Anderson Cancer Center, Houston, TX 77030, USA.

Abstract:  T cell functional differentiation is mediated by lineage-specific transcription factors. T helper 17 (Th17) has been recently identified as a distinct Th lineage mediating tissue inflammation. Retinoic acid receptor-related orphan receptor gamma (ROR gamma) was shown to regulate Th17 differentiation; ROR gamma deficiency, however, did not completely abolish Th17 cytokine expression. Here, we report Th17 cells highly expressed another related nuclear receptor, ROR alpha, induced by transforming growth factor-beta and interleukin-6 (IL-6), which is dependent on signal transducer and activator of transcription 3. Overexpression of ROR alpha promoted Th17 differentiation, possibly through the conserved noncoding sequence 2 in Il17-Il17f locus. ROR alpha deficiency resulted in reduced IL-17 expression in vitro and in vivo. Furthermore, ROR alpha and ROR gamma coexpression synergistically led to greater Th17 differentiation. Double deficiencies in ROR alpha and ROR gamma globally impaired Th17 generation and completely protected mice against experimental autoimmune encephalomyelitis. Therefore, Th17 differentiation is directed by two lineage-specific nuclear receptors, ROR alpha and ROR gamma. 

Retracted: No

PubPeer comments:
 Fig 1A and Fig 6A showed different ways of drawing gates. Since cell proportions are being compared, could the authors please explain why the gates were drawn differently?   , 
 #1 Is there any reason why gating criteria should be *the same* for different combinations of surface proteins, indicating different aspects of the conditions of the cells? , 
 The correct gating should be made based on the isotype staining, instead of using the same gating for all samples with different treatments. So in other words, the gating in each sample could be different to each other in panel A. Sometimes, it is acceptable to apply the same gating to all the samples if it does not change the overall conclusion. Isocoma Rusbyi pointed out that in the top panel, one gating looks different from all other 7 gatings. It is not the best gating. But let me ask you, do you think there would be a huge change if that gating (in red box) were applied to all others? For the bottom panel, the best cut-off line gating should be based on isotype staining of IL-4 on a duplicate sample. But the gating does not necessarily to be the same as in other samples. Using contour or zebra plot to determine the cut-off line is acceptable to me. , 
 First of all, prior to addressing this specific question, we would like to clarify that there are no guidelines within the immunology research field that FACS gating should be kept exactly the SAME for various samples, especially for samples collected from different tissues, at different time points and or stained with different combinations of intracellular or surface markers. In fact, it is the well-accepted consensus by the research community that gating could be and should be adjusted according to factors including but not limited to controls used and intensities of isotype staining. The scientific principles underlying such adjustments include varying levels of auto-fluorescence and varying degrees of laser compensation. With the above being said, we still spend time to compose comprehensive answers to each specific question on FACS gating with the hope that the user community would witness our good will and support of the Pubpeeru2019s principles of fostering open and honest discussions on scientifically related issues of high values. In Fig. 1A (should be Fig. 2A), we determined IL-17- and IFNgamma-expressing cells after transduction with RORa-expressing or a control vector during CD4+ T cell differentiation . Background fluorescence signals are not identical across the board in all samples receiving various treatment conditions, likely due to the different cytokine milieu in culture that impacted on cell status. To accurately quantitate cytokine-producing cells, it is ideal to define cytokine-expressing cells relative to the negative population in the same sample and the upper boundary of the IL-17 gate relative to the population contour center served this purpose. In Fig. 7A (mistakenly referred to as Fig. 6A by the anonymous commenter), the background staining in this sample was higher and thus the bottom boundary of the IL-17 gate was set relative to the negative population contour center. Overall, we felt that the gating strategy employed in these figures are appropriate and that the concerns raised are of minimal scientific merits. -Xuexian Yang and Chen Dong ,