Transcription‐Related Dynamics from Immune Disability into Endogenous Innovation

Abstract So far, thymus involution in adults is believed to be irreversible, and endogenous innovation for thymus‐related immunodeficiency remains to be an intractable puzzle. With the expectation of addressing this dilemma, human ovarian surface epithelium (OSE) has been reengineered as epithelial‐mesenchymal transition (EMT)‐tridimensional‐spheroid biologics (ETSB) using a dynamic EMT‐3D‐floating system along with 160 Gy X‐ray‐amelioration, which inoculates subcutaneously into aging rhesus and athymic Balb/cnu/nu mice. Herein, it is bioinformatically validated that ETSB can reset Clock/Arntl‐Per3/Tim molecule rhythm dynamics to re‐prime thymus residual (parathyroid or fatty‐like invalid vesicles yet no thymic architecture) to evolutionary transcription with overall cortex‐medulla endogenized by TECs undergoing MET/EMT reversion. Rhythm dynamics immediately resettles the bHLH‐LTβR‐NFκB‐RelA/B loop as a cascade to provoke the core immune microenvironment for multifunctional innovation of dynamic TCR orchestration, with harmonious naïve T‐subsets and TRECs renewals (P < 0.005). Subsequently, peripheral biological burden and tumor metastasis dynamics are addressed by innovative TCR‐defense/attack dynamics quickly (P < 0.005 vs Control), yet without autoimmune indication to hosts. Moreover, a functional blockade of core‐rhythm dynamics deeply impedes the endogenous innovation of invalid thymus residual. Thus this study may help pioneer a prospective strategy to innovate panoramic central‐peripheral immune microenvironments and defense dynamics for immune‐deficient/aging victims.


Fig.S2. Endogenous innovation for invalid residues with deserted microenvironments. Related to
A, Study strategy for the experimental layout in rhesus. The hosts (5-6-year old rhesus equivalent to 25-36year old man) were subjected to five times of (h)ETSB inoculations or other corresponding regimens at indicated months during two years (Month 0-1-6-12-24 protocol), with MRI or other detections two year later. B, Photographs show representative appearances among different groups two year after the last experimental inoculation termination. C, As in (B), MRI was adopted to scan rhesus thorax for different groups two year after experimental procedure termination, with single arrow indicating the sight of atrophied/aging or vanished thymus in front of trachea over heart and multiple-arrows revealing in situ evolved thymus(n=6). D, Graphs represent the thymic size based on tridimensional scan and weight among different groups. E, Lung and spleen weights keep similar among different groups (P>0.05).
F, Schematic depiction for experimental layout in Balb/c nu/nu nude mice.
G, MRI was used as non-invasive detection to scan pre-and post-therapy thorax, with arrows indicating the athymic sign in front of trachea over heart and white circles revealing in situ evolved thymus(n=6).
H, In situ inspection manifests thoracic cavity in nude mice, with tridimensional-innovation of left/right thymic lobes indicated by four arrows and superficial hyperemia above heart. Panoramic histomorphometry was used in different groups to monitor how parathyroid-like residual in voided microenvironment (v-m) was endogenized into cortex (C) and medulla (M) innovation with tridimensional core microenvironment (t-m) revival.
B, Graphs represent the thymic size of aging hosts before ETSB administration at month-16. C, Graphs represent the thymic size based on aging hosts after ETSB administration at month-18. n=6. D, FACS assay for thymocytes illustrates the central-phased in situ/naïve-αβ/γδTCR index of aging hosts before ETSB administration. E, FACS assay for thymocytes further illustrates the central-phased in situ/naïve-αβ/γδTCR index week 1~2 after 4th ETSB administration so as to compare TCR defense dynamics of aging hosts among different groups. n=5. A, Immunofluorescence molecule scan was utilized to detect timeless expression in homeostatic ETSB and Arntl/Tim-low/negative ETSB (3D-ETSB Tim-).
B, Since it is hard for Arntl/Tim-null OSEs (OSEs Tim-) to regenerate EMT-3D-spheroids, liposomal quercetin (Lipo-Que) modification for dynamic blockade of Clock/Arntl-Tim feedback regulatory loop was adopted for the 3D-ETSB Timpreparation. About 250 3D-floating ETSB spheroids per ml were exposed to 15µg of Lipo-Que for 12 hours.
C, Lipo-Que modification at different time-points was adopted and selected for the blockade of Clock/Arntl-Tim regulatory loop and meanwhile without hindering 3D-EMT spheroid transformation from OSEs. D, Immunofluorescence assay was used to see if the expression of Nanog (FITC) and Oct-4 (AMCA-labelled) in 3D-ETSB (same field) would be impeded synchronously by Lipo-Que modification at day 14.
E, Weighted gene co-expression network analysis (WGCNA) was performed for different gene modules based on whole transcriptome RNAseq with R-Package/Cytoscape Series and indicated that homeostatic ETSB (upper modules) would resettle Clock/Arntl-Per3/Tim-LTβR-NF-κB-RelA/B feedback loops as cascades to re-prime secondary central/thymus rhythm-peripheral/defense networks (lower modules) into innovative dynamic collaboration yet could be impeded functionally by relevant blockade.  E, Consequently, ETSB could re-prime molecule clock to evoke withered thymic rudiment into endogenous innovation, as Clock-Arntl/Per3-Tim axis resets TECs by undergoing dynamic MET/EMT reversion in central microenvironment for thymic progenitors remodeled and subjected to multifunctional revival of dynamic TCR orchestration to innovate innate-adaptive immunocompetence against evolving biologic burdens/tumor challenges. Where αβTCR-dominated evolving-repertoire could eliminate non-stem terminal cancer-cell subsets; Vγ4TCR-dominated evolving-subsets can accurately attack peripheral EMT/CSC-evolving pools, and are thereby able to fully address therapy-resistance/relapse-metastasis.

In vitro EMT-3D-floating system establishment and ETSB preparation
Human ovary tissues were obtained after informed consent and ovarian surface epithelium (OSE) was digested by collagenase II for 30 min at 37°C to separate surface epithelial cells. Mononuclear cells were collected and filtered through a 70-mum nylon mesh (BD Biosciences) to weed out unwanted cells. Then the selected cells were suspended in DMEM supplemented with 15% FBS, 10U bFGF, 2 mM L-glutamine and 0.1mg Amikacin/ml at an initial seeding density of 5×10 5 cells/ml. Two days later, the nonadhesive cells were removed by washing with serum-free DMEM. Next, media were replaced twice per week for a consecutive 14-day anchorage-independent screening period in serum-free medium [31,32] to generate floating EMT-3D spheroids via ameliorative dynamic shaking suspension model. Immunofluorescence with flow cytometry was used to detect characteristic markers of EMT-3D-spheroids for CD44, CD73, CD133, CD200, Nanog, Oct4, Sox2, PDL1, Arntl-Clock/Per3-Tim(E-15, H-276, G4). Purified EMT-3D-spheroids with positive markers were propagated in dynamic suspension with ameliorative DMEM/F12/1640-integrated medium until generating more than 250 floating 3D-spheroids/ml with 195±25µm/each D ( Fig.S1 and Video.1-2). These spheroids were collected and X-ray ameliorated using RS-2000 biological irradiator (www.radsource.com) at 160 Gy so as to keep the cells metabolically alive yet unable to proliferate and address renewal, frozen in one batch and resuscitated as EMT-3D-spheroid biologics (ETSB). Via the same procedure, mOSE-derived ETSB (mETSB) was prepared from C57BL/6 mice ovary by the same formula. Common OSEs in adherent culture were X-ray-ameliorated as common 2D-biologics (CB). High-throughput transcriptome sequencing and quantitative RT-PCR were collectively used to detect dynamic transcriptional characteristics of EMT-3Dspheroid establishment.

qRT-PCR analysis
RNA was isolated using an RNeasy Mini Kit (Qiagen) and subjected to on-column DNase digestion. Less than 5 mg total RNA was reverse transcribed to cDNA with 1st strand cDNA synthsis (PrimeScript TM RT reagent Kit with gDNA Eraser, Takara); qPCR amplification for certain primers were performed with SYBR Premix Ex Taq TM II (Takara) and CFX96 Real-Time PCR Detection System (Bio-Rad). The relative expression of mRNA was normalized to β-actin expression and calculated by using the 2 −ΔΔCT method. All primers were designed with NCBI Primer Blast, primer sequences for amplification were listed in Supplemental Table S1-S4. Data from qRT-PCR were analyzed with GraphPad Prism Version 6.0, differences between groups were statistically evaluated by sample one-tailed Student's t-test with p<0.05 considered as significant. Genomic DNA was extracted and purified from the rhesus PBMCs using TIANamp Genomic DNA kit (TIANGEN). The DNA concentration was then determined using NanoDrop2000. qRT-PCR assays for rhesus TREC quantification [33,34] were performed in quadruplicate and TREC number per genomic DNA (100 ng) was calculated using the software provided with the Bio-Rad CFX96 Real-Time System and LightCycler 480 System (Roche Diagnostics).

Inoculation regimen in immune deficiency and senescence hosts
Research protocol involving animals was reviewed and approved by institute's Animal Care and Use Committee. Athymic Balb/c nu/nu nude mice 6 weeks of age, C57BL/6 and Balb/c mice with the same weeks were purchased from Huafukang Bioscience Company and maintained in air-filtered laminar flow cabinets under aseptic conditions with a 12-h light/dark cycle. Arntl/Bmal1 -/knockout model was constructed by Cyagen through the deletion of Exon 6,7,8 and 9 in arntl gene locus of chromosome 7 on C57BL/6 backgrounds using CRISPR/Cas9 techniques. Mice were fed with AIN-93M rodent diet and autoclaved reverse-osmosis treated water to ensure proper health and acclimate to their living environments before study initiation. One week later the mice were randomly assigned to Control, CB and mETSB and hETSB groups with 24 hosts in each group including 12 females and 12 males. ETSB group hosts were subjected to four times of ETSB of subcutaneous inoculation into left flank (5×10 3 of biologics spheroids per time). Primary inoculation was followed by the second inoculation 2 weeks later, then by 1 week apart. Hosts after receiving four times of subcutaneous ETSB-inoculation would be used as Post-Balb/c nu/nu for further study. Control group and CB groups were subjected to corresponding inoculation regimens. Targeted irradiation for normal mice at week 6~7 was performed in a specialized chamber exposing only the thymic area to a targeted dose (9.5Gy) of X-ray radiation. Adult rhesus macaques 5-6-year old, have lived their entire lives at the Primate Research Center of Scientific Park, and have known birthdates, pedigrees, and complete medical histories.
Prior to the start of this study, no animals had any clinical or experimental history that would be expected to affect disease susceptibility or immunity. The animals have been fed a semi-purified, nutritionally fortified, low fat diet containing 15% protein and 10% fat and lived under the circadian model condition (Natural light regime, 16 h-light: 8 h-dark cycle, LD 16:8, with ZT0 defined as lights on) with drinking water and daily activities freely. As part of the study design, animals were evenly matched and randomized to Control, CB or (h)ETSB groups with 6 hosts in each group including 3 females and 3 males and treated for presenting conditions. The hosts received five times of (h)ETSB inoculations (5×10 5 of biologics spheroids per time) subcutaneously into both upper arms, or other corresponding regimens during two years (Month 0-1-6-12-24 protocol), with subsequent relevant detections covering thorax MRI two year more later. Every subject was sampled for the peripheral blood 5ml every 4 hours at each circadian time point (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22) in a circadian day. Lymphocytes were separated from blood and total RNA was extracted from each sample. RT-PCR was used to determine the temporal changes in mRNA levels of Clock/Arntl and other core genes during different zeitgeber times. The circadian parameters were obtained and analyzed by both cosine function/Cosinor analyses and amplitude F test to reveal the rhythmic transcriptions of core clock genes in LD (16:8) condition.

High-throughput transcriptome-sequencing and ELISpot assay with thymic rudiment nursing
Whole transcriptome RNAseq library was prepared for dynamic transcriptional characteristics detection of thymocytes and splenocytes. Total RNA was isolated from tissues using a Trizol reagent (ambion, life technology). The RNA quality was assessed using a BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA), and the samples were stored at −80°C until use. RNA integrity numbers (RIN) of these RNA samples have succeeded 8.0 and were appropriate for cDNA library construction using TruSeqTM RNA Sample Preparation Kit. The libraries were established by NEBNext Ultra RNA Library Prep Kit for Illumina, purified by Beckman AMPure XP beads, and quantified by ABI 7500 real time PCR system using KAPA SYBR green fast universal (2x) qPCR master mix. Samples were clustered on cBot cluster generation system and sequenced on HiSeqTM 4000 platform according to manufacturer's instructions. The Dual-Color IFN-γ/IL-4/17 ELISpot Kits (R&D Systems# ELD5217) was adopted for relevant cells monitoring. Briefly, thymocytes were harvested from thymus of Post-Balb/c nu/nu hosts. 5×10 4 recipient thymocytes as responder cells and 3×10 3 ETSB cells as biological burden/stimulators were respectively performed according to manufacturer's protocol. 72 hours later, spots were automatically scanned using an ELISpot plate reader (Cellular Technology Ltd., Cleveland, OH) for scanning. Vesicular thymic rudiments were harvested from Balb/c nu/nu nude mice 7 weeks of age and nursed in RPMI 1640 supplemented with 20% FBS at 37°C with 5% CO 2 for one week; and then nursed in ameliorative DMEM/F12/1640 integrated medium with 3D-ETSB at 120 spheroids/ml for another four weeks under floating separation condition via 6.5-μm filter screen for avoiding direct attachment between thymic rudiments and ETSB.

Side effect evaluation
Health status of all inoculated hosts was observed successively for relevant clinical indexes such as weight loss, ruffled fur, diarrhea, anorexia, cachexia, skin ulceration or toxic deaths. The tissues of heart, liver, spleen, lung, kidney, brain, and so on were fixed in 4% neutral buffered formalin solution and embedded in paraffin.
Slices of 4 microns were stained with hematoxylin and eosin (HE) and observed by two pathologists in a blinded manner. Tumor growth and metastasis, covering detection of the inguinal sentinel LNs, were monitored once per three days consecutively by measuring the largest diameter and respective perpendicular diameters using a caliper and plotted at three-day intervals throughout the whole experiment. Tumor volume (mm 3 ) was determined by the formula: 0.52×length (mm)×width 2 (mm) 2 . As for TCR pathway blockade experiments, additional post-Balb/c nu/nu hosts were established as described above and performed by i.v. injection of anti-Vγ1TCR (clone 2.11, 100 mg/mouse), anti-Vγ4TCR (clone UC3, 100 mg/mouse), anti-CD28 (27.51 mAb) or anti-αβTCR (clone H57-597, 100 mg/mouse) mAb on 7 days and 1 day before and 4 days after tumor-challenge, with normal rat IgG injected according to the same protocol as control. Depletion was confirmed by FACS analysis, and then hosts were allowed for a 6-week observation period for tumor growth and metastasis.
Briefly, the mixture of lecithin, cholesterol, PEG, and Que was dissolved in chloroform/methanol (3:1, v/v), and evaporated to dryness using a rotary evaporator under reduced pressure. The lipid films were dissolved in 5% glucose solution under ultrasonication, and subsequently concentrated and lyophilized. About 250 3Dfloating ETSB spheroids per ml at day-14 were exposed to 15µg of Lipo-Que for 12 hours [37,38] . Control cultures as homeostatic ETSB were left untreated. Clock/Per3-Tim blockades were detected by immunofluorescence for the ETSB and then evaluated using qRT-PCR analysis.

Magnetic bead microarray and western blotting
Non-necrotic fresh tumors or tumor-free local inoculum and thymus were cut into small pieces, homogenized in liquid nitrogen, emulsified by ultrasonication, extracted with ice-cold RIPA lysis buffer at a ratio of 100mg:1ml, cracked 30min onto ice, passed through a fine mesh sieve (Bellco Glass) and then centrifuged (12000rpm, 20min, 4°C). The extracted total protein was concentrated to 12mg/ml and then detected for peripheral molecule microenvironment of immunoregulatory network according to MILLIPLEX ® MAP magnetic bead panel kit (Luminex, USA). Nanog and IL17 levels were determined by the blotting using 50g total protein from each sample.

Statistical analysis
SPSS software package system was used for most statistical analyses. Data were subjected to one-way ANOVA plus Tukey post-hoc test or two-way ANOVA and repeated measures when comparing more than two groups. Log-rank test and Kaplan-Meier method were used to analyze tumor-free survival differences.
Numerical values are reported as means ± one standard deviation (SD). Statistical significance was assumed for P<0.05. Weighted gene co-expression network analysis (WGCNA) in bioinformatics was performed for different gene modules with R Package (version 3.2) as well as Cytoscape (version 3.6.0) based on whole transcriptome RNAseq. Heatmaps were generated by R package with hierarchical clustering algorithm .   Whole transcriptomes were subjected to MDS on expressed genes to assess sample diversity and relatedness among 3D-ETSB (green), 2D-CB (red) and Control/wild OSEs (black). ETSB symbols clustered more together than CB, meaning closer relatedness within ETSB samples.

Antibodies used for IF and Flow cytometry
Meanwhile, ETSB samples are very distinct from CB and Control samples, indicating diverse transcriptional characteristics among them and dynamic variation of Control/wild OSEs until transcription terminating.