This study also aims to find out angiographic findings of cerebral collateral circulation to predict ICA BTO results simultaneously.Endoscopic nasopharyngectomy (ENPG) is a promising way in treating recurrent nasopharyngeal carcinoma (rNPC), but sometimes may require therapeutic internal carotid artery (ICA) occlusion beforehand. Balloon test occlusion (BTO) is performed to evaluate cerebral ischemic tolerance for ICA sacrifice. However, absence of neurological deficits during BTO does not preclude occur of delayed cerebral ischemia after permanent ICA occlusion. In this study, we evaluate the utility of near-infrared spectroscopy (NIRS) regional cerebral oxygen saturation . NIRS rSO2 monitoring wasn’t able to detect BTO false‐negative results (p = 0.310). The anterior Circle was functionally much more important than the posterior Circle among the primary collateral pathways. The presence of secondary collateral pathways was considered as a sign of deteriorated cerebral hemodynamic condition during ICA BTO. In Types 5 and 6, reverse blood flow to the ICA during BTO protected patients from delayed cerebral ischemia after therapeutic ICA occlusion (p = 0.0357). In Subgroup IV, absence of the posterior Circle was significantly associated with BTO-positive results (p = 0.0426).The most suitable cut-off △rSO2 outcomes of ICA sacrifice. However, in order to ensure a safe carotid artery occlusion, more quantitative adjunctive blood flow measurements are recommended when angiography of cerebral collateral circulation doesn’t fully support rSO2 outcome among clinically ICA BTO-negative cases.Angiography of cerebral collateral circulation during ICA BTO is significantly correlated with ICA BTO results. Angiographic ICA BTO can be performed in conjunction with NIRS cerebral oximeter for its advantage of being noninvasive, real-time, cost-effective, simple for operation and most importantly for its correct prediction of most rSO Nasopharyngeal carcinoma (NPC) features itself by its distinct geographical distribution, particularly prevails in east and southeast Asia . RadiothHowever, preoperative safety management of internal carotid artery (ICA) is of vital importance before surgery, because ICA bleeding can be catastrophic during the operation, especially for tumors invading the ICA. In fact, patients harboring head and neck tumors may require therapeutic occlusion of ICA before tumor resection as preoperative preparation . PermaneMeanwhile, blood flow obstructed during BTO of ICA can be recruited from other places, depending on the development of collateral pathways including the Circle of Willis (CoW) . Hence, 2) and angiographic crossflow assessment. In this study, we evaluated the utility of near-infrared spectroscopy (NIRS) cerebral oximeter, which could calculate and monitor rSO2 in real time during ICA BTO, to quantify ischemic tolerance and to identify the valid cut-off values for safe carotid artery occlusion. This study also tried to find out some angiographic findings of cerebral collateral circulation to predict ICA BTO results, which might help to choose a more suitable and individualized adjunctive measurement during BTO  who underwent BTO for ICA on the lesion side were enrolled in the study. Apart from 81 patients, another six patients didn’t manage to perform ICA BTO, because they were identified with chronic internal carotid artery occlusion (CICAO) on the lesion side after DSA confirmation before BTO  were placed on the forehead of both sides, and rSOAll procedures were performed with single plane DSA equipment, Innova 3100-IQ  and Artis Q Zeego . After local anesthesia induction, a 6-French femoral sheath was inserted into the right femoral artery. After that, control anteroposterior and lateral angiography of bilateral ICAs and unilateral vertebral artery (VA) were performed at six frames per second before BTO with a diagnostic catheter. Heparin was administered intravenously after a puncture to prevent procedure-related thromboembolic complications. A nondetachable balloon catheter was then introduced and placed in the distal cervical segment of ICA on the lesion side through the balloon guiding catheter. The balloon positioned in the lower segment of the ICA might lead to bradycardia and a transient drop of blood pressure (BP) caused by carotid sinus reflex, which might affect the result of BTO.2 abnormality was detected during the procedure, the balloon was deflated immediately regardless of the collateral angiography. Under such circumstance, the BTO was considered positive. Once the patient clinically tolerated the 30-min occlusion, the BTO was judged to be negative. Patient developing delayed cerebral ischemia after therapeutic ICA occlusion was judged to be false-negative.After performing the initial neurological evaluation, the balloon  was then inflated under fluoroscopic visualization with angiographic confirmation of complete flow arrest of the vessel. The tested ICA was occluded for a total duration of 30 min and a neurological assessment was repeated immediately after the occlusion and every 5 min during the test. If the patient didn’t show with sign of the symptoms caused by hemodynamic cerebral ischemia 15 min after the temporary occlusion of ICA, the operator would puncture the left femoral artery and performed angiography of the contralateral ICA and the unilateral VA to evaluate the collateral circulation with the balloon inflated. As soon as any neurological deficit with rSOHemostasis at the femoral punctures were treated by electronic compressor. After permanent ICA occlusion by coiling, patients were transferred to the intensive care unit (ICU) where fluid balance, neurologic status, and blood pressure were carefully monitored. Subcutaneous heparin at therapeutic dosages was continued for 48 h. Routine head CT scanning was performed 24 h after carotid occlusion. Attention was focused on detecting symptoms of cerebral ischemia that might have been caused by the occlusion of a carotid artery. BP monitoring was another concern after the operation. BP should be kept pharmacologically elevated for 48 h to avoid possible episode of hypotension if necessary. Early stage of permanent blockage of the blood flow to the ICA might lead to an increased pressure within the carotid sinus, hence resulting in a temporary drop in BP and subsequent craniocerebral hypoperfusion. If no delayed cerebral hemodynamic ischemia was detected, the patients were discharged 5 days after the treatment and then transferred to EYE & ENT hospital of Fudan University for salvage ENPG.It is widely acknowledged that the CoW exhibits considerable anatomical variations –21. A coUnder this theory, for two vessels of similar length and pressure, a two times of difference in vessel diameter leads to a sixteen times of difference in blood flow. The Pouseille equation shows that vessel diameter influences the blood supply significantly.Thus, we speculate that relative position of tested side ICA and dominant A1 will have a great impact on collateral blood flow recruitment and subsequently affect results of BTO. In the presence of AcomA, the A1 to A2 flow dominance was classified on a 3-point scale: symmetric, no clear dominance of the inflow contribution of one A1 segment over the other; dominant, one A1 segment clearly contributes more inflow to an A2 than the other; and complete, no detectable inflow contribution from the contralateral segment. As for the posterior Circle of CoW, it is composed of the posterior communicating arteries (PcomA) and the P1 segments of the posterior cerebral arteries (PCA) . SimilarIn our study, the CoW anatomical variations were categorized into several Subgroups Figure 1Continuous variables were reported as mean with standard deviations (SD) or median with interquartile ranges (IQR). Categorical variables were compared by using Fisher exact tests and Mann–Whitney U tests. Receiver operating characteristic (ROC) curve analysis was used to determine the most suitable cut-off value based on the shortest distance from the curve to the upper-left corner. The analysis was performed using SPSS Version 25.0 and Medcalc Version 19.0. The level of significance was established at a 0.05 level (two-sided).87 BTO of ICA were performed, as six patients underwent BTO of bilateral ICAs . 17 cases were judged to be BTO-positive, three cases were judged to be BTO false-negative and the remaining 67 cases were judged to be BTO-negative. Clinical features of all 87 cases were given in 2, among which 12 cases were BTO-positive, 47 cases were BTO-negative and three cases were BTO false-negative. One BTO-positive case (Case No. 77) was excluded in ROC curve analysis because he was found significantly increase in rSO2, as the patient complained of unbearable headache during the procedure. Decrease in muscle strength of the right limb (Grade 3/5) was also detected by our clinician. The increase in rSO2 was considered to be attributed to increased blood pressure caused by the pain, and the subsequent angiography of the collateral circulation showed insufficient perfusion of the tested side MCA area. The remaining 11 BTO-positive demonstrated significant drop in tested side rSO2. As for the decrease in rSO2, there was no significant difference between BTO false-negative group  and BTO negative group  (p = 0.310). In our study, the most suitable cut-off △rSO2 value for detecting the BTO-positive group obtained through ROC curve analysis was 5%   Figure 279 of 87 cases performed complete collateral angiography of contralateral ICA and unilateral VA. Case No. 58 performed complete angiography of collateral circulation, but was excluded from all Types for his unique collateral flow configuration. 79 of 87 cases were included in Subgroups and Types based on their anatomic configurations and collateral flow configurations Figure 1via the anterior Circle alone (Type 1) and three cases of tested side cerebral hemisphere blood supply were completely recruited via the posterior Circle alone (Type 2). No immediate or delayed hemodynamic cerebral ischemia complications were found in cases in Type 1, while two cases among Type 2 were BTO-positive, but with no statistical difference of ischemic complication rates between two types (p = 0.143).A total of seven cases were classified in Subgroup I, among which four cases of tested side cerebral hemisphere blood supply were completely recruited via the anterior Circle. Type 3 represented cross flow via the anterior Circle alone to the tested side MCA area, whereas Type 4 represented cross flow via both anterior Circle and posterior Circle to the tested side MCA area. Type 6 in Subgroup III and Type 5 had similar cerebral hemodynamic features during ICA BTO as they both represented cross flow via the posterior Circle alone to the tested side MCA area. The summary of tested side MCA area blood supply categorization and its BTO results for BTO alone group and therapeutic ICA occlusion group are given in via the posterior Circle alone (Type 5 +Type 6) and those with collateral flow via the anterior Circle alone (Type 3)/via both anterior Circle and posterior Circle (Type 4), a higher BTO-positive rate  and BTO false-negative rate  in (Type 5 + Type 6) were observed, compared with (Type 3 + Type 4). Cross flow via the posterior Circle alone to the tested side MCA area was significantly associated with cerebral ischemic complications .All types had a complete anterior Circle in this subgroup and A1 to A2 blood flow showed no clear dominance of the inflow contribution of one A1 segment over the other. The classification in Subgroup II was based on the different blood supply to the tested side MCA area. Tested side ACA area of all cases in Subgroup II has already gained enough blood perfusion (ASITN/SIR Grade 4)  via the p = 0.0357).In cases among Types 5 and 6, we found that no patient experienced cerebral ischemia complications during and after BTO when the sign of reverse blood flow to the ICA was detected Table 3.2 abnormality detected during the BTO procedure simultaneously. So the case was considered to be BTO-negative, although angiographic findings showed cross flow not only via the posterior Circle to the affected hemisphere but also pial collaterals from contralateral MCA to contralateral ACA.Subgroup III showed no evidence of A1 segment on one side, with total supply to both A2s from the single A1. In Subgroup III, Type 6 was classified into p = 0.0426; OR:29.4000; 95%CI:1.1190–772.4193) (p = 0.470)  with severe and immediate neurological deficits was excluded, because angiography of collateral blood flow failed to complete in time), the absence of the posterior Circle was significantly associated with BTO-positive results (72.4193) Table 4.Case Nos. 76 and 80: A 63-year old male (Subgroup IV) was admitted to our neurosurgery department for bilateral ICA BTO and intended to have his left ICA occluded permanently. Control angiography of bilateral ICA and unilateral VA was shown in 2 did decrease by 8%, as soon as the balloon was inflated (via the anterior Circle to right MCA area during right ICA BTO (ASITN/SIR Grade1)  was admitted to our neurosurgery department for left ICA BTO and intended to have his left ICA occluded permanently. Control angiography of bilateral ICA and unilateral VA was shown in via the posterior Circle and no reverse blood flow to the left ICA was found  not long before and was wearing a tracheostomy tube, he wasn’t able to communicate with us verbally. Control angiography of bilateral ICA and unilateral VA was similar to Case No. 50  or via the anterior Circle alone (Type 3) demonstrated a significant reduction in BTO-positive rate and BTO false-negative rate in comparison with patients whose tested side MCA area was supplied via the posterior Circle alone (Type 5 + Type 6). In accordance with It has been widely accepted that the CoW is the major and most effective collateral pathway in the brain, which can respond quickly to low perfusion areas by recruiting blood from the contralateral hemisphere misphere . In the It is true that appearance on collateral flow can be evaluated by using the American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology (ASITN/SIR) Collateral Flow Grading System for angiography . HoweverHowever, reverse blood flow to tested side ICA from the posterior Circle during BTO can be easily detected in the DSA angiography. For cases among Types 5 and 6,cases without reverse blood flow to ICA suffered more from immediate or delayed cerebral ischemia by contrast with cases with reverse blood flow to ICA. So we suggest that more attention should be paid to clinical surveillance during BTO for patients with similar hemodynamic change like Types 5 and 6.We also recommend that should the sigh of reverse blood flow to tested side ICA from the posterior Circle, which indicated more than enough collateral blood perfusion, was detected among cases in Types 5 and 6, therapeutic ICA occlusion could be performed with no need to worry about delayed cerebral ischemic complicationsp = 1)  via the anterior Circle, originally collateral blood flow remains unchanged, but the cross flow has two more ACA areas to supply compared with the other situation. One situation shows smaller blood flow to a small low perfusion area, whereas the other situation shows normal blood flow to a larger low perfusion area. Cross flow via the anterior Circle might not be sufficient under these two circumstances, so that’s the reason why collateral flow from the posterior Circle is indispensable for BTO-negative results in Subgroup IV. It is also crucial to point out not only BTO-positive cases but also BTO-negative cases presented themselves in Type 12. This situation could be explained by the fact that tested side MCA area could recruit enough blood perfusion via the anterior Circle, when there is rather little difference between A1 diameters on both sides, thus making subtle impact on cross flow via the anterior Circle. So, as a matter of fact, although statistical analysis shows that configurations of blood supply to tested side MCA area in BTO-negative group in Subgroup IV don’t differ between group, whose tested side ICA is contralateral to dominant A1, and group, whose tested side ICA is ipsilateral to dominant A1, we speculate that difference will occur with the rise of sample size  and ICA (tested side) was not associated with BTO-positive rate  of four cases presented herself with symptoms of occasional limb weakness and dizziness to our division 7 days after permanent ICA occlusion. Head CT scanning showed newly-developed lacunar infraction in the left cerebral hemisphere in contrast to previous scanning. Contralateral ICA stenosis was detected last time during BTO and we once hesitated to perform the permanent ICA occlusion, but both rSOstenosis . Combinivia leptomengningeal branches from PCA to the tested side MCA area without the presence of PcomA, was diagnosed with acute cerebral hemodynamic ischemia owing to drowsiness, decrease in muscle strength and simultaneous significant reduction in tested side rSO2 during the procedure. Furthermore, Case No. 62 in Type 7 not only gained its collateral flow via the posterior Circle on the tested side, but also via leptomengningeal arteries contralateral to the tested side. The rSO2 monitoring of this case also showed no abnormal fluctuation on tested side. Evidence above demonstrates the fact that secondary collateral pathways, which include but are not limited to extracranial-intracranial anastomoses via the meningeal or ophthalmic arteries and pial collaterals, need more time to function  and Allcock maneuver  before ICA BTO. What also needed to mention was that another seven BTO-positive cases(Case Nos. 81–87) in our study were excluded from all the Subgroups,as they failed to perform angiography of collateral flow in time. For patients those who quickly fail the BTO, earlier awareness of their CoW development might spare them from unnecessary BTO. Anyway, though BTO is a simple procedure, it was reported to increase the rate of complications for neuroangiography from 1.3%  to 3 to 2 change during BTO. rSO2 monitoring is a quantitative method that also carries the merits of being noninvasive and cost-effective. The baseline values of rSO2 in our study ranged from 64 to 80% (mean 72.3 ± 5.1%) and was similar to previous reports , single-photon emission CT(SPECT) and xenon-enhanced CT(XeCT), might be essential for cases with certain kind of collateral flow configurations during BTO. According to our study, clinically BTO-negative cases, who recruit collateral flow via both primary and secondary pathways (e.g. Case No. 62), or who have similar hemodynamic changes like Types 5 and 6 (e.g. Case No. 50), or who have contralateral ICA stenosis (e.g. Case No. 69) are proper candidates for BTO in conjunction with these more quantitative blood flow measurements before permanent ICA occlusion. In fact, cases, whose angiography of collateral circulation doesn’t fully support clinically BTO-negative results, are recommended to use more quantitative blood flow measurements to avoid delayed cerebral ischemia after permanent ICA occlusion. Tomoyoshi Kuribara et al. reported that patients whose MTT obtained through CT perfusion at less than 16.4% increase compared with that on the contralateral side might be treated with abrupt ICA occlusion , National Natural Science Foundation of China (81874215), and Natural Science Research Project of Minhang District (Shanghai) (2019MHZ090).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer BL declared a shared affiliation with the authors to the handling editor at time of review.


The impact of prematurity on human development and neonatal diseases, such as bronchopulmonary dysplasia, has been widely reported. However, little is known about the effects of prematurity on the programs of stem cell self-renewal and differentiation of the upper respiratory epithelium, which is key for adaptation to neonatal life. We developed a minimally invasive methodology for isolation of neonatal basal cells from nasopharyngeal (NP) aspirates and performed functional analysis in organotypic cultures to address this issue. We show that preterm NP progenitors have a markedly distinct molecular signature of abnormal proliferation and mitochondria quality control compared to term progenitors. Preterm progenitors had lower oxygen consumption at baseline and were unable to ramp up consumption to the levels of term cells when challenged. Although they formed a mucociliary epithelium, ciliary function tended to decline in premature cells as they differentiated, compared to term cells. Together, these differences suggested increased sensitivity of preterm progenitors to environmental stressors under non-homeostatic conditions. The conducting airways are crucial for cleaning the air from particulates and biological agents and are responsible for metabolizing inhaled toxic compounds and environmental pollutants. These functions are exerted by a variety of differentiated airway cell types, including distinct secretory , multiciliated and other minor populations  regionally balanced from the nasal passages to the terminal bronchioles2. By turning on specific programs of cell fate, these multipotent progenitor cells acquire molecular and morphological features to generate the various luminal cell types. Studies in mouse genetic models show that basal cells form during prenatal development and together with undifferentiated embryonic precursors, contribute to the pool of differentiated luminal cells5. The transition from prenatal to postnatal life imposes major challenges to the epithelium that must respond to the markedly different hyperoxic environment triggered by the first breath, as well as have an innate immune mechanism able to handle exposure to biological agents not present during intrauterine life. Given that these depend on differentiation/maturation events that are completed largely by the end of the third trimester, premature birth can be associated with respiratory diseases of the neonate with increased morbidity and mortality.The various populations of differentiated luminal airway cells arise and are maintained by a pool of undifferentiated stem/progenitor cells (stem cell compartment) largely comprised of basal cells, multipotent progenitors basally located along the basement membrane12. Are the intrinsic programs of self-renewal and cell fate of basal cells affected by premature birth in infants? If so, how and to which extent this impairs the ability to form specific cell types? If exposed to the same physiologic normal conditions can premature and mature stem cell develop similarly? These compelling questions are of high significance but difficult to systematically address. Assessment of the intrinsic properties of basal stem/progenitor cells from neonates would require invasive procedures, such as intubation, trachea/bronchial brushings or even biopsy. Clinically, these opportunities would not occur in healthy term or preterm newborns in neonatal intensive care unit (NICU) settings.While the impact of prematurity on the differentiated luminal cells of the airway epithelium has been reported in diseases such as bronchopulmonary dysplasia, little is known about how it affects the stem cell compartment of the airwaysHere we have developed a minimally invasive methodology for systematic isolation and functional analysis of the stem cell properties of human basal cells from nasopharyngeal aspirates. We show that these cells can be expanded and passaged without losing their original identity or behavior. We used this system as a platform to investigate how prematurity influences the stem cell behavior and the differentiation capacity of airway basal cells in preterm compared to term neonates. RNAseq showed remarkably distinct molecular signatures between basal cells from these groups, with preterm cells showing features of mitochondrial dysfunction and functional evidence of decreased oxygen consumption. Prematurity did not prevent epithelial differentiation but still maintained altered gene signature and showed a decline in ciliary beating surface area, suggesting that the epithelium may be more susceptible to environmental challenges and injuries.13. This minimally invasive procedure is performed within the first minutes of neonatal life and consists of suctioning the remaining fluid in the upper airspaces  using a soft canula. NP aspirates are routinely discarded and known to be comprised of a loosely defined mixture of airway epithelial, inflammatory and amniotic cells in abundant mucous secretion. We reasoned that these aspirates included a stem cell population that, even if small, could provide an opportunity to investigate the program of self-renewal and differentiation of the airway epithelium of these neonates.Nasopharyngeal (NP) aspiration is a routine procedure in newborn care widely used to help clear the airways from residual amniotic fluid and secretions4 cells from each sample onto collagen-coated Transwell inserts. Cells were cultured in BEGM medium under submerged conditions until confluency (7–10 days), as similarly reported for primary cultures of tracheobronchial progenitor cells14. Immunofluorescence (IF) and confocal analysis of confluent NP cells showed the extensive P63+, KRT5+ labeling characteristic of basal cells  and seeded approximately 3.5 × 1016. Analysis of these cultures over the following three weeks post ALI conditions showed a pattern of differentiation similar to that reported for tracheobronchial cells with abundant secretory (MUC5AC+) and multiciliated (a-TUB+) cells  using conditions previously established for primary human tracheobronchial epithelial cells19. The understanding of these programs has been hindered by the paucity of systems to properly assess the intrinsic properties of the epithelial barrier and their response to the extra-uterine environment. We reasoned that the analysis of the NP aspirates (above) could provide an opportunity to study the impact of prematurity in the epithelial barrier in neonates. Thus, we collected NP aspirates from preterm neonates  and healthy term neonates  within the first minutes of life, as part of our delivery room resuscitation procedures at CUIMC, and cultured as described above.In neonatal settings prematurity represents the single most important factor influencing mortality and long-term morbidity, including lung conditions associated with aberrant developmental programsFirst, we asked whether we could identify changes in the transcriptomics of the stem cell compartment of the NP epithelium, making preterm and term progenitors intrinsically different when expanded under similar culture conditions. Preterm and term cells (4 subjects per group) reached confluency within 7–10 days and showed the cobblestone morphology with strong nuclear P63 labeling typical of basal cells Fig. a. Three Our organotypic cultures Fig.  provided22. Then, we looked for expression of these genes in our NP progenitor RNAseq dataset. Analysis of transcript abundance showed that, in spite of their neonatal origin, the NP progenitors expressed all twenty canonical basal cell markers . Notably, expression of these genes decreased dramatically as they differentiated in both preterm and term cells, supporting their enrichment at the progenitor cell stage  of preterm and term cultures to have an idea of the overall effect of prematurity on the transcriptome of NP progenitors prior to and after differentiation DEseq2 package). Intriguingly, while PCA showed no clear separation between the preterm and term samples in differentiated cultures (ALI day 21), a distinct pattern of distribution was identified in NP progenitors prior to differentiation , E2F Transcription Factor 3 (E2F3) , and Cell Division Cycle 25A (CDC25A)  , and p21 Activated Kinase 6 (PAK6) , further supporting the idea of higher proliferation activity compared to term cells25 , PRODH , TGM3 , NOXO1 , and ZNF365  as the most downregulated genes in preterm progenitors , an inducer of P53 activity was differentially downregulated in preterm   triggering cell damage and death. In stem cells, high ROS can alter cell metabolism, damaging lipids, proteins, DNA, and ultimately impair self-renewal31. Somewhat surprisingly, we could not find a gene signature consistent with oxidative stress in preterm cells. Gene markers, such as NEFL2 (NRF2), DDIT4, ATF3, HMOX, ENC1, SCO2, or those encoding antioxidant enzymes SODs, GPXs, PRDXs were not significantly altered32.Environmental stressors are known to elicit reactive mechanisms in the stem cell pool that can lead to aberrant cell behavior programs–17, 1.7 fold) and the E3 Ubiquitin Protein Ligase PRKN , two key mediators of autophagy responsible for mitochondria quality control and maintenance of cellular homeostasis and survival34 35  can be used to characterize the cellular bioenergetics at different states of mitochondrial respiration  and define the capacity of mitochondrial respiratory chain to phosphorylate ADP to ATP.Our transcriptome analysis of preterm and term NP progenitors revealed significant differences in expression of genes associated with mitochondrial integrity and function. To investigate whether these changes reflected in differences in the cellular bioenergetics we analyzed the cellular mitochondrial respiration profile of these two populations (Seahorse XFe24 Agilent) Fig. a. The ceNP progenitors from preterm (n = 5) and term (n = 4) newborns were expanded to confluency in XF cell culture microplates and incubated with Agilent Seahorse XF Base Medium for one hour prior to the assay. OCR was measured in three independent experiments , under unchallenged baseline conditions (step 1), after adding the ATPase inhibitor oligomycin to determine the resting/residual OCR once proton-motive force is no longer required for ATP synthesis (step 2), then after adding the mitochondrial uncoupler FCCP to dissipate the proton-motive force and stimulate high oxygen consumption (step 3), and lastly, inhibiting respiratory chain complex I and III , to abolish mitochondrial oxygen consumption and dissect out mitochondria non-specific cellular OCR (step 4). To control for mitochondrial mass, the mitochondria content of preterm and term NP progenitors was assessed using MitoTracker Green FM (Invitrogen).We found significantly decreased baseline OCR in preterm compared to term cells, suggesting that in an aerobic environment and unchallenged homeostatic conditions, premature NP progenitors are metabolically less active. This was further supported by the significantly lower oxygen consumption in preterm cells after treatment with oligomycin and after FCCP. The most remarkable difference was detected under FCCP treatment, which indicated that the maximal ability of the mitochondrial respiratory chain to work under increased energy demand to accelerate OCR in response to mitochondrial challenge (uncoupling) was markedly decreased in preterm cells compared to term cells. Interestingly, the cellular respiratory control ratio (RCR), the metrics of mitochondrial coupling calculated as the ratio of uncoupled to oligomycin-inhibited respiration, did not differ between groups Fig. a. This i37. Also, OCR showed significant negative correlation with mitochondria quality control genes, such as PINK1 . These observations suggest that the overall decreased mitochondrial activity and response to FCCP in preterm cells may be at least in part linked to an effect of prematurity on specific genes affecting mitochondrial integrity and function.The significantly poorer response to FCCP in premature NP cells suggests decreased ability of the mitochondrial respiratory chain to meet metabolic challenge compared to term cells. Notably, Pearson’s correlation analysis showed that maximal OCR correlated negatively with the expression of selective mitochondria-associated genes encoding complex I of the electron transport chain, such as NDUFA3 and NDUFA6 but not with other complex I genes, such as NDUFV1-3  cells as well as smaller population of basal cells that remained undifferentiated in a pattern of distribution indistinguishable between preterm and term cultures Fig. a. We per40. Thus, we investigated whether multiciliated cells arising from differentiation of preterm progenitors were functionally different from those arising from term. Ciliary beating frequency (CBF) is a hallmark parameter widely used to assess cilia function and integrity of the mucociliary defense mechanism41. We used high speed video microscopy (Zeiss Live-Cell Imaging) to measure CBF in organotypic cultures derived from preterm and term at two time points: ALI culture day 15 (n = 5 each) and day 21 (n = 4 each).We reasoned that the prematurity-associated decrease in mitochondria activity in NP progenitors, although not preventing differentiation, could have a negative impact in the function of specific cell types derived from preterm cells. Multiciliated cells require high energy for ciliary beating and cilia maintenance. Moreover, altered mitochondria function with decreased ATP production and impaired mitophagy have been associated with impaired ciliary function in cigarette smoke exposure of the airway epithelium44. The lower CBF in adult cultures was interesting and could be ascribed to age-related  or site-specific  differences in ciliary activity45.Analysis of ten random fields per well, and 3 wells per donor culture, showed an average CBF between 16–18 Hz in both preterm and term NP cultures either at ALI day 15 or day 21. No significant difference in CBF was found between preterm and term cells at both stages . For this, ciliary movement was recorded by high-speed video microscopy in 10 random fields/well in organotypic cultures at ALI day 15 and then day 21. Data were analyzed using a customized script based on a Fast Fourier Transform algorithm  and expressed as percentage of moving (“beating”) pixels per field. The analysis revealed that, although with great variability, the ciliary beating surface tended to increase from ALI day 15 to 21 in term cultures but not in preterm  and after  differentiation. NDUFA6 has been implicated in viral-induced apoptosis in several cell lines. In HIV-infected cells NDUFA6 gene silencing selectively induces apoptosis, an effect that is rescued by NDUFA6 overexpression46. Intriguingly, this gene is upregulated in human subjects exposed to air pollution, which is thought to represent a compensatory response for the mitochondrial damage induced by environmental agents47. How the decreased NDUFA6 expression contributes to differences in mitochondria activity between preterm and term and inability to ramp up expression later during differentiation is currently unclear. Our survey also showed that among the few genes differentially upregulated in day 21 preterm, two are associated with stress conditions. IL1RN , a known negative regulator of IL-1 signaling facilitates infection and is associated with frequent cycles of mitochondrial damage-repair in sepsis48. RHCG, a Rh family C glycoprotein is a negative regulator of NFKb expressed in airway secretory cells and upregulated in a model of squamous metaplasia in adult human bronchial epithelial cultures49.We did a survey in our transcriptome analysis of preterm and term ALI day 21 cultures searching for differentially expressed genes that could be associated with the observed difference in behavior. In contrast to our findings in the undifferentiated progenitors, a comparison of the signatures at ALI day 21 revealed only a small number of genes (8) differentially expressed between preterm and term cultures. NDUFA6 (NADH:ubiquinone oxidoreductase subunit A6) encodes a component of the mitochondria complex I oxidative phosphorylation chain whose mutations are associated with mitochondrial disease36 Fig. b. NDUFA6Although we could not directly implicate the genes above in the preterm phenotype, they point to prematurity having a potential impact in the mechanisms that maintain energy homeostasis and integrity of the NP epithelium.50, who tend to be the sickest patients. In our experience, parents are agreeable to have their neonate participate in studies on clinically discarded material. This opportunistic source of proximal airway basal cells from NP aspirates is advantageous compared to nasal brushings53, particularly in the extremely premature and clinically labile neonates.Here we established a minimally invasive methodology for systematic isolation and functional analysis of the stem cell behavior and differentiation program of human basal cells from nasopharyngeal aspirates. We show that this approach can be used routinely in NICU settings as an invaluable platform to investigate the impact of prematurity in the stem cell pool and barrier function of the upper respiratory tract epithelium. Due to its minimally invasive nature, it can be performed on healthy term neonates as well as extremely premature neonates, and is not limited to intubated neonatesTo our knowledge, this is the first report studying the program of basal cells collected from preterm and term neonates within the initial minutes of life. The uniqueness of this approach is that isolated cells have minimal exposure to the postnatal environment before culture. NP aspirates at birth reflect the innate cell population prior to prolonged exposure of supplemental oxygen, barotrauma, dynamic microbiome, infection, or medications, all of which have the potential to change the cells’ characteristics. This allows comparing the cellular behavior and the developmental program of progenitors from distinct gestational ages. The platform is versatile and allows assessment of a broad range of parameters from gene expression signatures and morphological features to functional parameters, such as bioenergetics and ciliary dynamics.54. Here we found that premature NP progenitors carry a program of gene expression that favors cell expansion while inhibiting functions, such as mitochondria quality control and autophagic cell survival, distinctive from their term counterpart.Cell-intrinsic differences between airway epithelial cultures derived from children and adults have been reported. Basal cells isolated from pediatric tracheobronchial biopsies were shown to have higher colony-forming ability and outcompeted the adult cells in proliferation assays55. This is consistent with our findings of differentially expressed mitochondrial complex I genes and decreased maximal respiratory capacity in preterm cells compared to term. Complex I is a crucial component of the electron transport chain and is considered the rate-limiting step of mitochondria respiration in the control of energy production. Notably, complex I dysfunction is the most common cause of mitochondrial disease in infancy and childhood and has been also implicated in age-related diseases36. In our study we cannot precisely identify the nature of the primary defect, but the strong correlation of decreased maximal OCR with the downregulation of PINK1 in preterm cells suggests that mechanisms that regulate mitochondria function and cellular integrity are less active. The key role of PINK1 in mitochondria quality control is widely recognized across species from Drosophila to mammals58.The activity of pulmonary mitochondrial respiratory chain complexes is developmentally regulated and in mice, increases during postnatal lung development59.Based on these observations, we speculate that preterm progenitors may be less fit to withstand continued physiologic demands over time and may have an overall maladaptive cellular response to stress compared to term cells. Indeed, these cells were unable to ramp up oxygen consumption under FCCP challenge in our assays. Moreover, the organotypic cultures differentiated from preterm NP progenitors could still maintain the ciliary beating surface area comparable to term cultures at an early stage, but not at a later stage. The accompanying differences in expression in only a small number of genes, including NDUFA6 suggest that, although not necessarily abnormal, the preterm-derived epithelium may be less fit to withstand continued or additional demands under challenging conditions. Analysis of cellular bioenergetics in the NP-derived differentiated epithelium and studying the effect of environmental exposures will provide valuable insightWe propose that NP-derived organotypic cultures has the potential to elucidate mechanisms controlling epithelial homeostasis and injury-repair of the upper respiratory tract and help understanding human neonatal diseases.60. Demographics of the NP aspirate donors used for the present study are listed in Supplementary Table All subjects were born at Columbia University Irving Medical Center (CUIMC) between April and August 2018. A total of 24 neonates were enrolled under this protocol, from which 11 were used in this study, after conditions for sample collection and NP cell culture were optimized. Study inclusion criteria were preterm neonates 23 0/7–30 6/7 weeks gestational age (n = 16) or healthy term neonates 37 0/7–40 6/7 weeks gestational age (n = 8), with maternal prenatal labs of rubella immune and negative hepatitis B surface antigen, Human Immunodeficiency Virus, Rapid Plasma Reagin, gonorrhea, and chlamydia tests. Exclusion criteria were neonates with suspected prenatal genetic anomaly. Neonates who clinically required suctioning to clear their upper respiratory tract airspaces in the delivery room within the first 15 min of life were considered for this study. Term infants were born via caesarian section and were admitted to the well-baby nursery. Preterm infants did not require intubation at delivery, and all received nasal bubble continuous positive airway pressure for maximum respiratory support. None were born in the setting of clinical chorioamnionitis. Prenatal care reflects contemporary medicine practices, including mothers of preterm neonates receiving at least 1 course of antenatal betamethasone for lung maturity, as clinically able for threatened preterm birth. Although oligohydramnios was detected by prenatal ultrasound in three of the preterm neonates, the respiratory support required during the NICU hospitalization was not clinically concerning for hypoplastic lungs2, as previously reported for HBE or tracheal epithelial cultures14. Media was changed 24 h after and subsequently every 48 h until cells reached approximately ~ 90% confluence (7–10 days). Cells were expanded to passage 2 prior to seeding 3.5 × 104 cells on 24 well collagen-coated Transwell plates and cultured under submerged conditions in BEGM at 37 °C, 5% CO2. Once cells reached 100% confluence (approximately 5–7 days), the upper chamber medium was aspirated to expose cells to air–liquid-interface (ALI) and the medium in the lower chamber was replaced with Human Air Liquid Interface (hALI) media14 and allowed to differentiate for up to 21 days, with media changed every 48 h. Cultures were harvested for analysis at ALI day 0  or at days 15 and 21. In most experiments NP cultures were performed side-by side with ALI cultures of adult human tracheal progenitors  under the same culture conditions for comparisons. These were isolated from healthy adult lung organ donors not used for transplant. Patient demographics were anonymous.The NP aspirates were resuspended in 5 mL sterile normal saline to dilute mucus, centrifuged  and the supernatant (including mucus) was discarded. The pellet was resuspended in 2 mL Bronchial Epithelium Growth Media (BEGM) containing penicillin–streptomycin, gentamicin, tobramycin, ceftazidime, vancomycin, and amphotericin B. Cells were seeded on 24-well collagen-coated plates (13.2 μL/mL PBS) and cultured under submerged conditions in BEGM at 37 °C, 5% CO61. Briefly, Transwell membranes were fixed with 4% (PFA) for 10 min, room temperature. For the immunofluorescence assays, membranes were washed with phosphate buffered saline (PBS) then permeabilized by 0.2% TritonX-100 in PBS for 10 min twice and blocked in 1% bovine serum albumin (BSA) for 1.5 h prior to incubation with primary and secondary antibodies. Membranes were incubated with primary antibodies in 1% BSA overnight at 4 °C: Mouse monoclonal P63 (D-9) antibody , rabbit monoclonal P63-alpha (D2K8X) XP antibody , rabbit polyclonal keratin-5 antibody , chicken polyclonal keratin-5 antibody , rabbit monoclonal Nkx2.1 antibody , rabbit polyclonal cleaved caspase-3 (Asp175) antibody , mouse monoclonal Ki67 antibody , rabbit monoclonal acetyl-alpha-tubulin antibody , mouse monoclonal mucin 5AC antibody . F-actin was stained by Alexa Fluor™ 647 Phalloidin . Membranes were washed with PBS prior to incubation with the appropriate secondary antibodies and 2,4-diamindino-2-phenylindole (DAPI) selected from the following and added at the corresponding dilutions and incubated in 1% BAS for 1 h at room temperature: Donkey anti-chicken IgY (IgG) (H + L), Alexa Fluor 488 nm , Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 nm , Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 nm , Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 568 , Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 568 , Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 , Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 , DAPI NucBlue™ Live ReadyProbes™ Reagent . Afterwards membranes were washed, mounted on microscope slides with ProLong Gold Antifade Mountant . Membranes were then imaged using a Zeiss confocal 710 microscope.Indirect immunofluorescence was performed in NP confluent (undifferentiated progenitors) cultures and differentiated ALI organotypic cultures as described in Mori et al.62, After removing the adapter and low quality reads, the clean reads were mapped to the human genomes (HG38) using the TopHat (v 2.0.13). The read count was calculated using HTSeq (v.0.6). All the analysis was performed in R-3.6.3. Differential expression and principal component analysis were performed by DESeq2. Differentially expressed gene transcripts were selected based on an adjusted p-value (p-adjusted) less than 0.05. Heatmap analyses were performed using ComplexHeatmap. The heatmap depicted in Fig. goseq. For pathway analysis, a list of all genes detected in the samples was ranked on the t-statistic generated by DESeq2. This list was analyzed with the GSEA package in R using the complete Molecular Signatures Database MSigDB v7.1 gene set. Volcano plot was labeled using MASS and calibrate. CIBERSORTx38 was used to estimate the cell type abundances from our bulk ALI day 21 transcriptome with published human lung tissue single-cell RNA-sequencing data39 as signature matrix. Categories in differentiating ALI cultures included ciliated (multiciliated), secretory goblet and club cells, differentiating basal cells, and others (undetermined)39.Transcriptome analysis was performed in NP progenitor cultures from four term and four preterm donors selected based on their similarity in gestational age, to minimize the effect of potential differences in lung development  software. Ciliary beating frequency (CBF) was analyzed across each video using the fast Fourier transform (FFT) within Matlab  as previously described44. Only pixels (termed beating pixels) with magnitude greater than 1, as well as CBF between 2 and 60 Hz were included in our analysis. The percentage of ciliary beating surface (CBS) in each field was calculated by dividing the number of moving pixels by the total number of pixels in that video frame . Statistical analysis was performed using Two-tailed Student’s t test (Graphpad or Excel).Ciliary beat frequency (CBF) was measured by high speed video microscopy and analyzed using methodologies reported in Oltean et al.63. Cells were seeded in collagen-coated XF 24-well cell culture microplates (Agilent) at a density of 4 × 104 cells/well in 100 µL of growth medium and incubated for 2 h at 37 °C in 5% CO2 to attach and then another 200 µL of growth medium were added. On the next experimental day, the growth medium was replaced with 500 µL of Agilent Seahorse XF Base Medium  supplemented with 2 mM glutamine , 1 mM pyruvate , and 10 mM glucose . Cells were incubated for one hour prior to the assay in a non-CO2 incubator. Oxygen consumption rate (OCR) and extracellular acidification rate were recorded at baseline followed by the additions of 0.5 µg/mL oligomycin, 1.2 µM FCCP, and 0.5 µM rotenone plus 1 mM antimycin A. Non-mitochondrial oxygen consumption was subtracted from all OCR values, and technical replicates identified as outliers were omitted from further calculations. Values were normalized by protein content in the well. For NP-derived basal cells from each newborn donor (Supplementary Table 4 cells/coverslip in the growth media. On the next experimental day, the growth media was replaced with the staining media and incubated for 20 min at 37 °C.Cellular mitochondrial respiration was assessed on NP-derived basal cells from all newborns listed in Supplementary Table Supplementary Information.


STAT3 gene, and also enrichment to the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. Known activating STAT3 mutations were found both in MS patients and controls and overall 1/5 of the mutations were previously described cancer mutations. The detected clustering suggests a selection advantage of the mutated CD8+ clones and calls for further research on possible phenotypic effects.Somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. Previously we and others have demonstrated that especially CD8+ T cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (MS) and rheumatoid arthritis. Here we concentrated on CD8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between MS patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? We separated peripheral blood CD8+ cells from newly diagnosed relapsing MS patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the CD8+ cells’ DNA, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. We discovered nonsynonymous somatic mutations in all MS patients’ and controls’ CD8+ cell DNA samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2–8.6%). The mutations showed statistically significant clustering especially to the  The mutation rates differ between tissues, ranging from 3.5 x 10ase-pair , 3.KRAS mutations have been observed in arteriovenous malformations in the brain [FAS have been discovered, and these mutations induced a defect in T-cell apoptosis [KRAS gene, which also impair T-cell apoptosis [UPA1 gene were reported in men with an adult-onset inflammatory syndrome called VEXAS  [Somatic mutations have an established role in cancer; their role in non-malignant diseases is also gaining attention. Single cell sequence analyses have raised the hypothesis that somatic mutations may modulate brain aging and neurodegeneration . Activathe brain . There apoptosis . Similarpoptosis . Recentlsomatic) .JAK1 and STAT3 mutations have been discovered in the gut intraepithelial lymphocytes that underwent malignant transformation in refractory celiac disease [STAT3 mutations in CD8+ cells of patients with large granular lymphocyte (LGL) leukemia and rheumatoid arthritis [STAT3 mutations were not found, but other somatic mutations were discovered in the expanded CD8+ effector-memory subset in 20% of patients vs. 5% of controls [The role of somatic mutations in more common autoimmune disorders is not clear. Activating  disease . Anotherrthritis , 11. In controls .Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and among the most common causes of neurological disability in young adults. In relapsing MS, which constitute about 90% of cases, there is evidence based on genetics, environmental risk factors and treatment paradigms that peripheral leukocyte dysfunction plays a major role in the disease –15. InflIndirect evidence of somatic mutations in MS patients’ cultured T-lymphocytes was reported in 1990s by using the hypoxanthine guanine phosphoribosyltransferase (HPRT) assay. This assay measures 6-thioguanine resistance of cultured cells, caused by inactivating somatic mutations of the X-chromosomal HPRT gene or other mechanisms. MS patients’ HPRT-deficient T-lymphocyte clones, but not wild-type clones, were potentially autoreactive, i.e. proliferated in response to myelin autoantigen  and a hiHere we extend these findings by using an improved methodology for mutation discovery and analyze a cohort of 21 newly-diagnosed MS patients and 21 matched controls. The comparison between groups allows us to form an initial picture of whether the previously reported findings in MS are disease specific or not. Another major goal enabled by the sample size  is an assessment of non-random accumulation of somatic mutations in certain genes, which we carry out in addition to the case-control comparison. Because the detection of somatic variants in small cell clones is very dependent on sequencing depth, we opted to limit our search to a subset of the exome containing key immunological and cancer-related genes, which allowed us to reach high sequencing depths of over 2000x and obtain a more detailed view over small allelic fraction events.Patients (n = 21) were recruited at the Helsinki University Hospital Department of Neurology outpatient clinic during their diagnostic examinations and were selected from a larger collection of patients to fulfill the inclusion criteria and age- and sex-matching. Inclusion criteria for MS patients were: 1. McDonald 2010 criteria for relapsing MS, 2. CSF oligoclonal bands and 3. Barkhof’s magnetic resonance imaging criteria (20 patients fulfilled 4/4 criteria one patient fulfilled ¾) for anatomical dissemination of demyelination . The locPeripheral blood mononuclear cells (PBMCs) were extracted from 120–140 ml venous EDTA blood using Ficoll-Paque PLUS . First, 13 ml of Ficoll-Paque was added to a centrifuge tube. Then, 9 ml of blood diluted with 28 ml of PBS was layered on top of it. The tube was centrifuged at 800 x g for 30 minutes after which the PBMC layer was transferred to a new tube with a pipette. The cells were washed twice, using PBS and centrifugation at 500 x g for 15 minutes and at 500 x g for 10 minutes. From the PBMCs, positive separation with MACS CD8 antibody MicroBeads  was performed using an OctoMACS magnetic separator (Miltenyi Biotec) following the manufacturers protocol. We simultaneously separated also other cells, which will be analyzed later. From the separated cell populations, DNA and RNA were extracted using the InviTrap Spin Universal RNA Mini Kit  according to manufacturer’s instructions. The purities of the separated CD8+ cells were tested in 31 (74%) of the 42 of samples by flow cytometric analysis, in which T cells (CD3+) represented 89–99% of the cells and the observed purities for CD8 were all ≥ 87%. T-cell receptor Vβ repertoire was not included to the analyses, because in our previous analysis the presence of a large Vβ clone did not significantly predict the detection of a somatic mutations with small allelic fractions .A gene panel that consists of 2524 genes related to immunity and cancer (Immunopanel-2524) was designed for mutation screening [A “reference DNA” pool was collected from 8 whole blood DNAs of healthy donors, to act as technical control material. These DNA samples were sequenced in an identical manner to a combined median depth of 4343x. The samples for the reference DNA pool were obtained via Meilahti Biobank from individuals aged 20–35 years without hematological or autoimmune conditions.The sequencing reads were trimmed for adapters and base quality using Trimmomatic , after w−5 in the ExAC or gnomAD germline population databases were also filtered to remove remaining possible germline contamination and to exclude common mapping errors and other artefacts. To remove common erroneous variant calls, the ExAC and gnomAD database variant lists that were used were purposefully of the unfiltered type i.e., they included even the population variant calls that don’t pass the quality control filters of ExAC or gnomAD, and likely represent repeating artefacts.The somatic variant calls were filtered using several methods. TNScope produced VCF filter statistics were checked, and suspect artefact or possible germline calls were removed from each sample’s data. Germline variant calls were removed within-sample based on a call’s allelic fraction, and germline variants detected in any of the other samples were also removed to avoid possible cross-contamination. Furthermore, somatic variants were only called at high depth (>100x) locations in order to facilitate accurate calculation of the allelic fraction and to avoid germline mix-up. Strand bias filtering was used to discard systematic sequencing errors that preferably show up in one sequencing direction only. Known segmental duplication areas were excluded from the analysis to avoid errors from mismapping of pseudogenes at these loci. Errors from mismapping of reads were mitigated by discarding highly clustered variants and by requiring a similarly high mapping quality from variant reads as from the reference allele containing reads. Variants with a frequency of >10In order to automatically calibrate the variant calling sensitivity level against several classes of library preparation related base substitution errors, an empirical trinucleotide-context aware noise frequency was calculated for each possible base substitution, separately for each sample. The noise frequencies were calculated from the data by counting the number of base mismatches against the reference genome, for all sequenced coordinates, excluding germline variant sites, tabulated separately for each possible 192 trinucleotide context and base substitution combinations. Using the empirical noise frequencies calculated this way, a binomial test that takes into account the number of variant observations, the sequencing depth, and the noise frequency was used to discard likely library preparation related artefacts, by requiring more evidence at noisier locations.We paid especial attention to base changes of type C>T (G>A) because previous studies have indicated a risk of erroneous C>T (G>A) substitutions arising during library preparation . These aAfter filtering for quality, all synonymous variants were excluded (n = 160) from detailed analysis. The variant list was also filtered by excluding genes that are not expressed in CD8+ PBMCs according to RNA-Seq data that we had previously generated , which rAs a quality control measure, targeted PCR amplicons from CD8+ cell DNA were designed to verify some of the discovered variants. The amplicons were sequenced using Illumina MiSeq with 2x300bp reads. Variant loci were inspected using samtools mpileup and base counts were compared to a control DNA amplified in the same manner.We were able to perform unusually high depth sequencing of over 2000x median coverage for 42 CD8+ cell DNA samples. All of the sequenced CD8+ samples were found to contain high-confidence somatic variants. After quality filtering, and including synonymous variants, the number of detected somatic mutations was 652 (median and mean numbers of mutations per subject were 13.5 and 15.5). The total number of mutations was 307 in MS patients  and 345 in controls . After removing synonymous variants and variants in genes not expressed in CD8+ cells a total of 225 nonsynonymous somatic mutations were detected in genes expressed in CD8+ cells.The number of nonsynonymous mutations in genes expressed in CD8+ cells was 104 in MS patients and 121 in controls . A comparison of mutations between cases and controls is shown in The 225 nonsynonymous somatic mutations in genes expressed in CD8+ cells were found in 186 different genes and the mutations mostly did not recur between samples. Three mutations were found in two participants , in one MS patient and in one control. Otherwise, the mutations were singleton observations.Since aging is associated with the number of somatic mutations in most tissues, we tested whether there would be an association between age and the number of mutations . There was an association between participant age and the number of somatic mutations, when normalized by the sequencing depth . As expected, the median sequencing depth had a modest association to the number of somatic mutations detected in that sample .We analyzed enrichment of nonsynonymous mutations to specific genes. Because a large coding region size of a gene increases the likelihood of mutations, we calculated Poisson p-values that take into account the size of each gene, the number of mutations in that gene, the total gene panel area, and the total number of mutations. There were 17 enriched genes that were mutated in two or more participants at p<0.05 .STAT3 gene stood out as the most enriched locus (p = 9 x 10−8) with a total of 6 nonsynonymous somatic mutations  and their mutations in cases and controls are shown in All 225 nonsynonymous somatic mutations in genes expressed in CD8+ cells, their allelic fractions, base counts, presence in databases, discovery p-values and predicted deleteriousness are shown in STAT3 somatic mutations detected in our MS patients (D661Y and S614R variants). For comparison we chose a low-confidence but recurring C>T variant ITPR3*T2584M, which was filtered out in our pipeline. A more relaxed sensitivity level would have called this variant in three MS patients and one control. This variant call represented a potential mutational hotspot, and it has been previously reported in cancer tissue (COSMIC database id COSM1643037). Hence, we chose to test this variant call with amplicon sequencing. By comparing the variant allele fractions in the original panel data and amplicon sequencing data (STAT3 variant (fold change only 0.76–0.85 in amplicon sequencing) but not for the ITPR3 variant . Moreover, the ITPR3 variant was found in amplicon sequencing data of the reference-DNA and in a cell-line control DNA close to the same allele fraction as in the tested CD8+ cell DNAs. These results indicate that the STAT3 variants are true, while the ITPR3 C>T variant is a likely artifact, and support the use of stringent filtering to avoid false positives.To test our somatic variant calling sensitivity and specificity settings , we prepared confirmation PCR amplicons for the two high-confidence ing data , we founWe carried out an additional analysis of synonymous somatic variants in order to include possible effects on RNA structure and other effects not examined by our primary analysis of nonsynonymous mutations. It should be noted that we targeted the coding region of each gene, untranslated regions were not included. After applying the same filters to synonymous variants as we used for the nonsynonymous variants, a total of 72 synonymous somatic mutations were detected in genes expressed in CD8+ cells. Out of these, 43 were found in cases and 29 in controls with no statistical difference in number between the groups (Mann-Whitney U-test p = 0.25). A full list of the detected synonymous mutations is shown in MAP3K12 gene (Poisson test p = 0.00011), with a total of 4 synonymous mutations, 2 in cases and 2 in controls. All four had the same recurring mutation, NM_001193511:c.279A>G, which was novel. The general purpose CADD algorithm did not predict this mutation to be deleterious (CADD PHRED score 9.024), nor did the algorithm TraP, specialized in synonymous variants, classify it as damaging (TraP score 0.038). However, we also utilized the SURF RNA structural predictivity index that combines different secondary structure effects into one summary metric, and this algorithm did predict the mutation to be deleterious. The somatic MAP3K12 synonymous mutation had a SURF PHRED score of 21.46, which is inside the 99th percentile and is higher than the scores obtained by the SURF authors for nine known pathogenic mutations affecting RNA structure [A clustering of somatic synonymous mutations was detected in the tructure . This sutructure . AlthougWe had previously shown that it is possible to detect somatic mutations in about 60% of MS patient blood samples, and that the mutations were mostly (85%) discovered in CD8+ cells in comparison to other, CD4+ or CD19+ cells. In the present work, we thus chose to concentrate on the CD8+ cell fraction and included age- and sex-matched controls to assess whether the finding is MS specific, or generalizes to the population at large. Moreover, we were able to perform especially deep sequencing, reaching coverages greater than 2000x, which enabled a sensitive screen of low allelic fraction events. We found 225 nonsynonymous somatic mutations in genes expressed in CD8+ cells and these were distributed evenly between MS patients and healthy controls, with each sample carrying at least one (median 4 mutations in both groups). Including also synonymous and non CD8+ expressed gene variants in the target area there were a total of 652 high-confidence somatic mutations (median and mean numbers of mutations per subject were 13.5 and 15.5).In order to understand what would be the expected  number of detectable mutations in our experiment, we need to consider some technical issues regarding the number of cells and detectable clones. We used 1000 ng of DNA, which corresponds to 167,000 individual cells (6 pg of DNA per cell), of which ≈90%  were CD8+ cells after the immunomagnetic purification scheme. Our median sequencing depth 2349x, due to the excess of cells, produces essentially haploid sequence from ca. 2100 CD8+ cells . Usually about half of the blood CD8+ cells are naïve cells, with small clone sizes , not detSomatic mutations start to occur and accumulate since the first cell division of the fertilized egg. A recent analysis utilizing individual DNA molecule sequencing of hematopoietic stem cell/progenitor cell colonies and circulating granulocytes from different ages suggest that mutational burden increases linearly with age  Based onSTAT3 gene, most of which (4 out of 6 mutations) have been shown to cause an activating phenotype which affects growth and apoptosis properties of T lymphocytes [STAT3 was the gene containing more nonsynonymous somatic mutations than any other gene, showing a statistically significant enrichment. It is likely that the clones that carry STAT3 mutations have a growth advantage over other clones, increasing their size in the sequenced sample, and thus bring the somatic mutation closer to detection threshold more often than mutations in many other genes. It is of note that the STAT3*D661Y mutation found in one MS patient was also previously discovered in our pilot study in another patient [STAT3 variants in MS patients and controls.Most of the discovered mutations were predictably deleterious  and manyphocytes , 41. In  patient . We are SMARCA2, SOCS1, DNMT3A and PPP3CA. The SMARCA2  gene is involved in chromatin remodeling processes and is considered a tumor suppressor gene that is very often downregulated in various cancers [SMARCA2 have been reported in chronic lymphocytic leukemia [SOCS1 is a gene with known regulatory functions in autoimmunity and a tumor suppressor in cancer [DNMT3A codes a methyltransferase protein that is essential for establishing normal epigenetic DNA methylation patterns. Somatic mutations in DNMT3A have been implicated in age-related clonal hematopoiesis, wherein a hematopoietic stem cell has acquired a growth advantage mutation resulting in an increased clonal size of its peripheral blood cell progeny [DNMT3A loss-of-function somatic mutations are also known for phenotypic effects more specifically tied to T cell behavior. In CD8+ T cells specifically, defects in DNMT3A will cause an accelerated shift towards a long-living memory phenotype [PPP3CA encodes calcineurin-A, which mediates one of the three major signaling pathways of T cell receptor signaling [The other genes that stood out in the number of enriched nonsynonymous mutations were  cancers . On the  cancers . Somaticleukemia  and, mosleukemia . SOCS1 in cancer . STAT3 an cancer . DNMT3A  progeny . On the henotype . PPP3CA ignaling . Ca2+–caignaling .MAP3K12 encoding mitogen-activated protein 3-kinase 12, also known as dual leucine zipper kinase (DLK). It acts upstream of c-Jun N-terminal kinase (JNK) and p38 MAP kinases in the JNK signalling pathway. There are multiple functional outcomes of this pathway in different cell types, one of which is the regulation of STAT3 [MAP3K12 locus has been reported in non-Hodgkin’s lymphomas [One potentially functional and recurring synonymous somatic mutation was found in of STAT3 . Amplifiymphomas .The most commonly mutated genes we found in blood CD8+ cells differ as compared to the top5 mutated genes reported in sun-exposed normal skin  and normal oesophagus  , 55. ThiIt is interesting to consider whether some of the detected somatic mutations might have a role in disease. They can theoretically add some level of cancer risk, as 1/5 of the mutations have been found in cancer cells, even if they likely drive only a limited growth potential in most people, when normal T-cell apoptosis occurs. The mutations were detected in CD8+ cells, and because CD8+ T cells are a potent effector of the immune system, a link to autoimmune disorders is also possible. In our data the somatic mutations were equally distributed between MS patients and healthy controls. However, the antigen specificities and general phenotypic features of the mutated CD8+ clones may be differ in MS patients as compared to controls. An autoreactive clone with an activating somatic mutation would offer a potential mechanistic explanation for a chronic disease like MS. Previously, using the HPRT reporter assay it was shown that MS-patients’ mutated T-lymphocyte clones proliferated in response to myelin antigen . Future STAT3 somatic mutations in our MS patients were actual true positives. Other experiments may witness a different base substitution error profile depending on the specific library preparation conditions they employ [The sequencing depths of over 2000x that we attained in this research call for special care in handling potential false positive errors that may arise in the sample preparation and sequencing processes. Misclassification of low frequency technical artefacts as reportable variants becomes more likely at low allelic fractions. The sequencing artefacts can arise from several different biochemical processes affecting DNA while it is being processed in the laboratory, such as heating induced spontaneous deamination, base oxidization caused by energy from ultrasonic shearing, or damage from free oxygen radicals . To comby employ .As we sequenced a panel of genes chosen because of their association to immunity and cancer, we were not able to run generic enrichment analyses of biological functions using our result variant list, and this is a limitation of the study—the somatic mutations we detected are found in immunity and cancer related genes by design. However, inside the immunity context, STAT3-signaling emerged as one clear pathway possibly affected. Moreover, the repeated hits in genes known to give CD8+ T cells a growth and survival advantage suggest that these mutations may have functional consequences as algorithms predict. As there was an association in our data between the number of mutations detected and the sequencing depth reached by a sample, it is reasonable to expect that even deeper sequencing would increase the number of discovered deleterious somatic mutations and potentially dysregulated clones, even in normal healthy adults such as the control subjects in this study.In conclusion, our results demonstrate that somatic mutations are present both in MS patients’ and controls blood CD8+ T cells and that they cluster on certain genes such as STAT3. Somatic mutations can be important modifiers of the characteristics of CD8+ T cell clones and may result in dysregulated immune responses. The role of somatic mutations in driving autoimmune responses is presently unclear but given the abundance of mutated clones deserves further attention.S1 FigThe per sample depths obtained in sequencing.(PDF)Click here for additional data file.S2 FigAge vs number of exonic somatic mutations for all samples.(PDF)Click here for additional data file.S1 TablePatients’ demographic features.(XLSX)Click here for additional data file.S2 TableControl participants’ demographic features.(XLSX)Click here for additional data file.S3 TableList of genes included in the NGS capture panel.(DOCX)Click here for additional data file.S4 TableMean background noise tabulated by sequence context and base change.(XLSX)Click here for additional data file.S5 TableList of filtering criteria used during somatic variant calling.(XLSX)Click here for additional data file.S6 TableAll nonsynonymous somatic mutations detected in the study.(XLSX)Click here for additional data file.S7 TableTargeted amplicon sequencing results.(XLSX)Click here for additional data file.S8 TableThe synonymous mutations detected in the study.(XLSX)Click here for additional data file.