RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo.The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant  Rhinovirus C has a global distribution and significant clinical impact–especially in those with underlying lung disease. Although RV-C is genetically, structurally, and biologically distinct from RV-A and -B viruses, our understanding of the RV-C life cycle has been largely inferred from these and other related viruses. Here, we performed a detailed analysis of RV-C15 replication in a physiologically-relevant model of human airway epithelium. Our single-cell, microscopy-based approach revealed that–unlike other RVs–the endoplasmic reticulum is the primary site for RV-C15 replication. RV-C15 replication also stimulated STING expression, which was proviral, and triggered dramatic changes in cellular organization, including altered virus receptor distribution, fragmented Golgi stacks, and the induction of incomplete autophagy. Additionally, we observed a loss of epithelial barrier function and a decrease in mucociliary clearance, a major defense mechanism in the lung, during RV-C15 infection. Together, these data reveal novel insight into RV-C15 replication dynamics and resulting cytopathic effects in the primary target cells for infection, thereby furthering our understanding of the pathogenesis of RV-C. Our work highlights similar, as well as unique, aspects of RV-C15 replication compared to related pathogens, which will help guide future studies on the molecular mechanisms of RV-C infection. Rhinoviruses (RVs) are responsible for over 40% of respiratory virus infections in the human population –4. AlthoRV-C was discovered in 2006  and compin vivo mouse model that is naturally susceptible and highly permissive for RV-C replication, previous reports demonstrate that ex vivo tissue and primary airway cultures support infection [in vitro model of human airway epithelium (HAE). Our data identify the endoplasmic reticulum (ER) as the primary site for RV-C15 replication and demonstrate the impact of infection on the structural integrity of ciliated cells and epithelial barrier function, thereby identifying both unique and shared features of RV-C amongst related viruses.Despite these advances, details of the RV-C life cycle and underlying mechanisms that contribute to pathogenesis remain scarce. While such studies remain hampered by the absence of immortalized cell lines or an nfection ,25. None10 RV-C15 RNA copies and quantified viral RNA intracellularly as well as in both the apical and basolateral compartments over time . The dynamics of RV-C15 replication were similar in HAE from two different donors, where cell-associated RV-C15 RNA levels increased during the first 12 hours post-infection (hpi). This correlated with the detection of double-stranded (ds) RNA, a marker of viral replication, by immunofluorescence  and was in line with peak viral release into the apical chamber at 24hpi . The lack of RV-C15 RNA in basolateral supernatants confirmed polarized release of RV-C15 to the airway lumen , consistent with a previous report in nasal cells [Pseudostratified models of HAE at air-liquid interface are permissive for RV-C replication ,26. To dal cells  and the 1D and 1E). Notably, the corrected total cellular fluorescence (CTCF) levels of CDHR3 had a limited relationship with FoxJ1 but were strongly correlated with acetylated alpha-tubulin levels, in line with increased expression of CDHR3 during differentiation . Notably, the loss of apical acetylated alpha-tubulin localization in dsRNA(+) cells  may explain the lack of cilia visualized in these cells by en face IF . The 3D views also suggested CDHR3 was internalized in some dsRNA(+) cells .Given that HAE cultures were permissive for RV-C15 replication, we next sought to investigate cell tropism in our system. Visualization of the RV-C receptor, CDHR3, in non-infected HAE by IF revealed expression at the apical surface in cells that were also positive for Forkhead box protein J1  or acetylated alpha-tubulin . In contrast, CDHR3 protein expression decreased at 12 and 48hpi, coincident with robust detection of the viral capsid protein VP1 . Subsequent single-cell analysis of acetylated alpha-tubulin and FoxJ1 fluorescence levels corroborated our WB data . This was also true for CDHR3, in which fluorescence levels decreased significantly at 12hpi in cells with active viral replication .To better understand the dynamics of FoxJ1, acetylated alpha-tubulin, and CDHR3 during infection, we analyzed global protein expression by Western blot (WB) and quantified fluorescence levels following immunostaining specifically in infected cells. Beyond the intrinsic variation expected across cultures in this model system, neither FoxJ1 nor acetylated alpha-tubulin protein levels were dramatically altered over the course of infection , dsRNA in RV-C15-infected cultures was found in either a perinuclear (S1 video) or ring-like disposition closer to apical and basolateral membranes (S2 video). Neither phenotype was specific for RV-C15, however, as HAE infected with either RV-A16 or RV-A2 revealed similar dsRNA profiles . Additionally, the ring-like distribution pattern was determined to be the minority profile across all dsRNA(+) cells visualized at 12hpi in cultures infected with RV-C15 , RV-A16 , or RV-A2 .Picornaviruses, including RV-A and -B, replicate in association with cellular membranes, leading to the formation of double-membrane vesicles known as replicative complexes –31. ThusFigs S2B and S2D, possibly a result of large intracellular vesicle formation . Subsequent TEM analysis in non-infected HAE revealed a differentiated epithelium with no overt cytopathic effects  and a limited number of vacuoles in the cytoplasm . In contrast, in RV-C15-infected HAE, we identified ciliated cells with many small vesicles clustered near multivesicular bodies, mitochondria, and electron-dense structures similar to β-particles of glycogen . Under higher magnification, we were able to confirm these small vesicles had a double-membrane  similar to replicative complexes previously described for RV-A and RV-B [) [Notably, the nuclei in cells with the ring-like dsRNA pattern were found near the apical cell surface instead of the usual basolateral location in polarized columnar cells (and RV-B . Interes RV-B [) ,33.Fig 3A), giantin signal was spread throughout the cytoplasm in cells with evidence of active viral replication for all RVs tested . The fragmentation of Golgi structures inferred from our IF data was further supported by TEM, where, compared to the non-infected control, Golgi stacks were barely visible in cells from RV-C15-infected HAE cultures .Different cellular membranes can contribute to the formation of replication organelles during viral infection; however, the Golgi is the main source reported for many picornaviruses, including rhinoviruses ,34. ConsFigs S3A and S3B). To further assess the changes in giantin and PI4P levels, we compared the CTCF for these cellular targets in both non-infected and infected cells. Our data revealed both giantin and PI4P levels were elevated and strongly correlated in HAE infected with all RVs, though the change in PI4P was not significant for RV-A2 . A direct relationship was also observed between giantin and dsRNA CTCF in RV-C15- and RV-A16-infected cells while PI4P and viral dsRNA CTCF were only correlated in RV-C15 HAE .In addition to a loss of Golgi integrity, we also observed enhanced detection of PI4P in cells with active RV replication compared to non-infected controls. Similar to giantin, PI4P was dispersed throughout the cytoplasm in infected cells, reflecting the same general distribution (perinuclear or ring-like) as observed for dsRNA , the ratio of colocalization between them was very low at pixel intensity and spatial levels  or PI4P . Spatial colocalization analysis confirmed these observations, with less than 9% of the total dsRNA detected at the same location as giantin or PI4P in RV-C15-infected cells . Thus, despite the fragmentation of the Golgi stacks and increase of PI4P in airway epithelial cells with active RV-C15 replication, our data indicate the Golgi is not the main site for viral genome replication and PI4P is not a marker for sites of RV-C15 replication in HAE as observed for other RVs [The close association between Golgi membranes and picornavirus replication has been shown by the colocalization between viral dsRNA and different Golgi markers  ,31,34. TS4A Fig). However, at the single-cell level, calnexin fluorescence was not only elevated in cells infected with RV-C15, RV-A16, and RV-A2 compared to the negative control at 12hpi  but also directly correlated with fluorescence levels of viral dsRNA . Interestingly, the 3D view of HAE infected with RV-C15 at 12hpi showed calnexin spread throughout the cytoplasm, and there was a strong indication of calnexin and viral dsRNA colocalization , which was further supported by 3D surface plot data . Adding to this, the perinuclear and ring-like distributions of dsRNA were also observed for calnexin in RV-infected cells .Given the low ratio of colocalization between RV-C15 dsRNA and Golgi markers, we next evaluated ER membranes as a potential site for viral genome replication. WB analysis of the ER-associated protein calnexin in RV-C15-infected HAE did not indicate a dramatic change compared to non-infected cultures at 12, 24, or 48hpi . However, this high colocalization ratio was only confirmed for RV-C15-infected cells at the 3D level , suggesting that, unlike RV-A16 and RV-A2, the ER is the main site for RV-C15 genome replication.Pixel intensity-based analysis confirmed the high level of colocalization between viral dsRNA and calnexin in cells infected not only with RV-C15 (56.3%), but also RV-A16 (28%) and RV-A2 (49.1%) at 12hpi . Therefore, we further evaluated levels of both Lamp-1 and LC3b in HAE by IF 12hpi. Lamp-1 and LC3b detection in HAE was significantly stronger in RV-C15- and RV-A2-infected cells while the level of detection in RV-A16-infected HAE was similar to the negative control . Curiously, the 3D view of non-infected cells showed an apical localization of Lamp-1 while this protein was spread through the cytoplasm in RV-infected cells . A high correlation ratio between Lamp-1 fluorescence levels and dsRNA was obtained in RV-infected cells; however, LC3b levels strongly correlated with dsRNA only in cultures infected with RV-C15 and RV-A2 . Together, these data suggest RV-C15 and RV-A2 (but not RV-A16) induce autophagy to significant levels over the baseline in HAE.Previous studies have shown picornaviruses, including several rhinoviruses, manipulate the autophagy pathway to mediate non-lytic release of progeny following replication . Since t). Thus, these data suggest that, similar to RV-A2 but unlike RV-A16, RV-C15 induces incomplete autophagy in HAE.Since the inhibition of autolysosome formation (the last step in the autophagy pathway) is characteristic of picornaviruses that induce this pathway to promote virus release , we furtFig 4) and the induction of incomplete autophagy at 12hpi . Interestingly, STING, which localizes to the ER, was recently shown to induce the autophagy pathway during RV-A16 infection [Fig 6A) as well as robust detection of STING by IF in dsRNA(+) ciliated cells at 12hpi . Notably, 3D view analysis revealed that dsRNA/STING-double positive cells also exhibited dispersed distribution of acetylated alpha-tubulin, suggesting STING levels increase as replication progresses . Indeed, the CTCF of STING not only increased significantly in HAE infected with RV-C15 , but also strongly correlated with dsRNA levels at 12hpi . However, despite the maximum Z-projection, 3D surface plot, and pixel intensity colocalization analysis suggesting high level of dsRNA/STING colocalization in RV-C15-infected HAE (S16 Table), spatial colocalization analysis indicated the median colocalization ratio between both targets was lower than 5% .In this study, we observed RV-C15 genome replication in HAE to be associated with the ER , and infected them with RV-C15, RV-A16, or RV-B14. Immunofluorescence detection of dsRNA at 12hpi confirmed the susceptibility of control cultures expressing a non-targeting guide RNA to RV-C15, RV-A16, and RV-B14 infection . However, the frequency of dsRNA(+) cells was lower in STING-depleted HAE infected with RV-C15 or RV-A16 . While infection with RV-B14 was less efficient overall, a similar frequency of dsRNA(+) cells were observed in both control and knockout HAE . STING-knockout cultures also had significantly lower intracellular viral RNA copy numbers for RV-C15 and RV-A16 but not RV-B14, further validating our results  and suggesting that the increase of STING expression in RV-C15-infected HAE has an advantageous impact on viral genome replication.To better understand the importance of STING to RV-C15 genome replication, we genetically knocked-out STING using a CRISPR/Cas9 approach in immortalized human airway epithelial cells (BCi-NS1.1 cells ), differFigs 5). To extend these observations, we quantified extracellular lactate dehydrogenase (LDH) at the culture-level as an indication of plasma membrane leakage. LDH release was restricted to the apical compartment and did not exceed 20% of the maximum levels obtained from lysed control cultures; however, LDH levels increased over time  in line with RV-C15 replication kinetics . Thus, we sought to further evaluate the global impact of RV-C15 infection on epithelial integrity. Interrogation of HAE permeability during RV-C15 infection revealed a significant, albeit transient, decrease in transepithelial electrical resistance (TEER) at 12hpi . Since the transepithelial transport of ions is mediated by pores formed by integral membrane proteins termed claudins [, we observed a change in claudin-1 distribution in RV-C15-infected cells at 12hpi by IF . To determine if this translocation was specific to claudin-1, we characterized Zona occludens 1 (ZO-1), a cytoplasmic tight junction-associated protein. Similar to claudin-1, with the exception of one donor at 48hpi, ZO-1 protein levels for both subunits (ZO-1 +α and ZO-1 -α) did not vary in RV-C15-infected HAE compared to the negative control , while ZO-1 disposition was more widespread in dsRNA(+) cells . To quantify the effect of RV-C15 infection on ZO-1 disposition over time, we utilized the Junction Analyzer Program (JAnaP) [Fig 7F). The increase of discontinuous ZO-1 quantified in this assay  indicates the ZO-1 translocation in HAE  parallels the progression of RV-C15-infection. Additionally, two profiles of discontinuous ZO-1 were evaluated, and ZO-1 was found in a more perpendicular than punctual disposition in RV-C15- infected HAE .Our results to this point highlight RV-C15-induced cytopathic effects at the single-cell level in HAE  –46, whicS6 Fig). The transport of microspheres indicated a slight increase in MCC in HAE inoculated with PBS and a similar, albeit significant, elevation in microsphere transport rate in UV-inactivated RV-C15-inoculated cultures at 12hpi , likely attributable to the addition of fluid on the apical surface after the T = 0 time point. Despite this, RV-C15 infection resulted in a significant decrease in MCC at both 12 and 24hpi  indicating viral replication impairs this innate host defense mechanism.Given the ciliated cell tropism RV-C15 and the important role of these cells in promoting mucociliary clearance (MCC), we also assessed the global impact of RV-C15 infection on mucus transport. HAE cultures were equilibrated at 34°C and the transport rate of red-fluorescent microspheres in the extracellular mucus gel was calculated immediately prior to inoculation, and up to 48hpi. IF confirmed viral replication at 12hpi in HAE inoculated with viable, but not UV-inactivated, RV-C15  and peak viral release at 24hpi  in agreement with a previous report characterizing RV-C11 and RV-C15 infection in bronchial cells [RV is becoming increasingly recognized as a cause of both upper and lower respiratory tract infection –8. Furthlication ,25,27. Hal cells .Fig 1C–1E). Interestingly, we did not observe any evidence of cytosolic CDHR3 in non-infected cells, differing from previous immunofluorescence data [Figs S1F) without altering levels of acetylated alpha-tubulin or FoxJ1 . Unlike ICAM-1 (the receptor for major-group RVs) and LDLR (the receptor for minor-group RVs), the dynamics of CHDR3 during infection and associated pathway(s) of RV-C entry remain unknown. After endocytosis of RV-A and RV-B particles, ICAM-1 and LDLR are either degraded in the lysosome with empty capsids, or LDLR is trafficked from the endosome to be recycled [In well-differentiated HAE, we detected CDHR3 on the apical surface of ciliated cells . Prior reports identified the induction of replicative complexes derived from the ER by the poliovirus 2BC and 3A proteins [Figs  suggesting RV-C impacts Golgi membranes similar to other picornaviruses. However, despite the strong correlation between dsRNA and fluorescence levels of both giantin and PI4P , colocalization analysis indicated neither giantin- nor PI4P-positive vesicles are the main site for RV-C15 genome replication . Similar observations were made in RV-A16-infected HAE  although dsRNA fluorescence levels only strongly correlated with giantin and not PI4P , and neither the significant increase in levels of giantin nor insignificant increase in PI4P correlated strongly with viral dsRNA . It is important to highlight that our study evaluated rhinovirus replication in HAE at a later time point than other reports (3-8hpi) [Notably, our TEM analysis also identified smaller, double-membraned vesicles, similar to those identified during replication of other RVs and related picornaviruses –31 that proteins ,49 and tproteins . Still, proteins ,31,34. Fproteins , and fraproteins . In this(3-8hpi) –52; thusFig 4A and 4B) and a high ratio of dsRNA/calnexin colocalization in cells infected with RV-C15, but not RV-A2 and RV-A16 , identifying the ER as the main site for RV-C15 replication. The increase in calnexin levels observed in this study could be the result of ER stress in infected cells, as reported for RV-A1B and RV-A16 [Fig 4E–4G).Most notably, our results demonstrated elevated levels of calnexin in RV-infected HAE , as expected [Fig 5E). The results obtained in RV-C15-infected HAE were similar to RV-A2, indicating the induction and manipulation of the autophagy pathway (5C and 5E). Use of the autophagy pathway to release progeny would explain the absence of cytopathic effects visible under light microscopy in HAE cultures infected with RV-C15. To our knowledge, this is the first time the autophagy pathway has been analyzed during RV-C infection.In the final stages of the viral life cycle, progeny picornavirus virions are released through cell lysis or by usurping the autophagy pathway –39. Expl6D and 6F) which correlated with levels of dsRNA  and VP1 protein . This correlation, however, was not related to a close interaction between STING and dsRNA, as the median colocalization ratio at the spatial level was lower than 5% . Additionally, viral genome replication was impacted in the absence of STING, resulting in significantly lower intracellular viral RNA levels at 12hpi for RV-C15 and RV-A16, but not RV-B14 . These data, which assayed endogenous STING expression in RV-infected HAE, corroborate published data in undifferentiated cells indicating STING expression is important for RV-A and RV-C genome replication [Fig 6H) is surprising and indicates that either genome replication of RV-C15 occurs at different sites on ER membranes (STING-negative) or most of STING had already been translocated to ERGIC. RV-C15 replication also induced autophagy in HAE, though more experiments are needed to relate this phenomenon to STING activation.The identification of ER as a site for genome replication and induction of autophagy during RV-C15 infection of HAE led us to investigate STING, which has been associated with the induction of ER stress and autophagy ,56. In tlication . STING ilication . The pholication , while tFig 7A), in agreement with viral cellular tropism for ciliated epithelial cells that face the luminal surface of the airway  [Fig 7B) which may be due, at least in part, to claudin-1 translocation . Additional claudins  that are also expressed in the airway epithelium and known to impact transcellular transport were not assayed here [Fig 7C and 7E). Despite being a tight junction protein, ZO-1 interactions are more important to cell polarization and paracellular permeability [Fig 7F–7I), possibly impacting cellular organization. Nonetheless, the loss of TEER and changes in claudin-1 and ZO-1 observed in this study for RV-C15 were less dramatic than previous reports for RV-A and RV-B [Given the observed impacts of RV-C15 replication in infected cells, we further probed the effects of viral infection on the epithelium functionality, including barrier permeability and mucociliary clearance. The level of cytotoxicity of RV-C15 infection in HAE, measured by LDH release, increased overtime in the apical compartment only  ,25. The   . A previ  . Similaryed here ,60. Stileability . Althougand RV-B ,39,58. Tand RV-B .Fig 7J). We speculate that the temporary increase in MCC observed in cultures inoculated with UV-inactivated virus is due to the addition of fluid on the apical surface. However, as this increase was not significant for the negative control, it may also indicate that the host response to incoming viral particles stimulates MCC. The coordinated beating of ciliated cells drives the basal MCC rate of ~5.5 mm/min, which can be altered by mucus composition, temperature, and humidity [Fig 1D and 1F) which likely indicates impaired cilium structure and function in these cells. Whether the progressive loss of cilia function and eventual death of infected cells contributes to our observed change in MCC during RV-C15 infection is not clear.Beyond epithelial barrier integrity, we also identified an impact of RV-C15 on mucociliary clearance functionality. MCC decreased significantly after 12hpi in HAE infected with RV-C15 but not UV-inactivated virus, similar to a previous study that reported an impact on MCC 5 days post-infection , and indhumidity –66. Indehumidity , which hhumidity . In thisIn conclusion, our present study expands our current understanding of RV replication in a physiologically-relevant setting, substantiating previous observations related to virus-induced membrane reorganization, and demonstrating that RV-C15 displays many typical features of picornavirus infection. In addition, we identify RV-C15 replication in association with ER membranes unlike previously characterized RVs. While we speculate that these data identify a unique feature of RV-C replication, our study is limited by the use of only one RV-C genotype; thus, further analysis to confirm these observations using additional RV-C isolates is important. Notwithstanding, our findings underscore the fact that virus-host interactions critical for rhinovirus replication are not always conserved, possibly contributing to their different clinical profiles, and supporting further investigation into this group of pathogens.4 cells/well) on rat-tail collagen type 1-coated permeable transwell membrane supports . HAE cultures were grown in Pneumacult-Ex basal medium , or Pneumacult-ALI medium  with provision of an air-liquid interface for approximately 6 weeks to form differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium. CDHR rs6967330 genotyping was achieved by sequencing using the following primers: 5’-GAAAGAAGGCCCGCCAGAACCC-3’ (forward) and 5’-TGGGGCTGGAATCAAGGTCGGT-3’ : donor 1 –heterozygous G/A  and HEK-293T  cells were cultivated in Dulbecco’s Modified Eagle Medium  supplemented with fetal bovine serum  and 1% penicillin/streptomycin . All cell cultures were maintained at 37°C with 5% CORV-C15, RV-A2, RV-B14, and RV-A16 were rescued from infectious clones kindly donated by Drs. James Gern and Yuri A. Bochkov, (pRV-C15), Dr. Wai-Ming Lee (pRV-A2) and Dr. Ann Palmenberg (pRV-B14 and pRV-A16) from the University of Wisconsin–Madison. Rescue of infectious RV was done according to published protocols ,70 in H1Single guide RNAs (sgRNA) targeting STING or no known target (NTC) and flanked by restriction sites for cloning into the pLentiCRISPRv2 backbone  with eGF2 for 6hr. The inoculum was removed and replaced with fresh Pneumacult-Ex Plus media (StemCell Technologies). At 60–80% confluence, eGFP-positive cells were enriched by fluorescence-activated cell sorting on a BD FACSAria-II Cell Sorter . These sorted BCi-NS1.1 cells were then expanded, seeded at 3.3x104 cells/well on 6.5mm rat-tail collagen type I-coated transwell membranes, and cultured at air-liquid interface as previously described for HAE.For target cell transduction, lentivirus-containing supernatants were applied to BCi-NS1.1 cells , mainta2. After 1 hour incubation at room temperature (RT), the inoculum was exchanged for a semi-solid overlay composed of 0.6% of bacteriological agar  in DMEM-F12 , supplemented with 1% FBS (Genesee), 1% penicillin/streptomycin (Gibco), 1% L-glutamine , and 30mM MgCl2 (Sigma-Aldrich). The plate was incubated at 34°C for 72 hours and the cells were fixed with 4% formaldehyde . The fixing solution was removed together with the overlay after a 24-hour incubation at RT and plaques visualized by staining the monolayer with 0.1% Crystal Violet  diluted in a 20% ethanol (200 proof) solution.RV-A2, RV-B14, and RV-A16 stocks were titrated by plaque assay method using H1 HeLa cells plated in 24-well dishes and a published protocol with modifications . Brieflyin vitro-transcribed pRV-C15, pRV-A16, and pRV-B14.RV-C15 was titrated by qPCR, which was also used to quantify RV-A16 and RV-B14 in experiments done in CRISPR/Cas9-modified HAE. To quantify viable viral particles, 10μl of RV-C15 stock or 100μL of experimental sample was first treated with 1u RNase A  and 1u DNase I  for 1hr at 37°C. Viral RNA was then extracted using a QIAamp Viral RNA Mini Kit ; total RNA was extracted from cells using a RNeasy Mini Kit . Next, up to 1μg of RNA was used in a reverse transcription reaction , with random hexamer primers, as per the manufacturer’s protocol. qPCR was done using 3μl of cDNA, 10pM HRVTF63 forward (5’–ACMGTGTYCTAGCCTGCGTGGC–3’) and HRVTR reverse (5’–GAAACACGGACACCCAAAGT GT–3’) primers, 10pM HRVTF probe (5’–FAM/TCCTCCGGCCCCTGAAT/BHQ1–3’), and LuminoCTT aqman Master Mix  according to the manufacturer’s protocol. For absolute quantification, a standard curve was delineated using cDNA generated from ten-fold serial dilutions of 10 copies of RNA), RV-A16 (5x105 PFU), RV-B14 (5x105 PFU), or RV-A2 (5x105 PFU) and incubated at 34°C with 5% CO2 until sample collection. In the replication curve and cytotoxicity assays, the inoculum was removed, and the apical surface rinsed with PBS, at 4hpi.One week before the experiment in HAE, cells were washed 3 x 30 minutes with 100μl of phosphate buffered saline  at 37°C to remove excessive mucus. In CRISPR/Cas9-modified BCi-NS1.1-derived HAE an additional wash was performed immediately before inoculation. HAE cultures were inoculated on the apical surface with 10μl of PBS (negative control), sucrose-purified RV-C15  was added to the apical chamber and harvested after a 30-minute incubation at 34°C; basolateral samples (500μl ALI media) were collected directly from underneath the culture. Cells were harvested from the transwell membranes using 350μl RLT buffer  for downstream RNA extraction, or 75μl of Radio Immuno-Precipitation Assay (RIPA) buffer  plus protease inhibitors  for subsequent Western blot (WB) assays. The aliquots for RNA extraction and WB were stored at -80°C and -20°C, respectively. Alternatively, cultures were fixed with 4% (w/v) freshly-prepared paraformaldehyde , Hatfield, PA) for immunofluorescence (IF) assays or 2% (w/v) glutaraldehyde in 0.1M cacodylate buffer for downstream transmission electron microscopy analysis.4Cl diluted in PBS) before being washed twice for 3 minutes each with PBS. Permeabilization of cell membranes was done using 0.2% Triton-X diluted in PBS; after a 15-minute incubation at RT, the cells were washed 3 x 3 minutes with PBS. A blocking solution  or normal goat serum  diluted in PBS with 0.2% Tween-20) was added and only removed from the apical side after a 10-minute incubation at RT. The apical surface of the culture was washed 3 x 3 minutes with PBS. Primary antibody diluted in PBS with 0.2% Tween-20 and 1% Bovine Serum Albumin  was added to the apical chamber and incubated overnight at 4°C, protected from light. The apical surface was washed 3 x 5 minutes with PBS and incubated with the secondary antibody diluted in PBS with 1% BSA. After a 2-hour incubation at RT, protected from light, the secondary antibody solution was removed and the apical surface of HAE was washed 3 x 5 minutes with PBS. If necessary, additional stains were done by repeating this protocol; reducing the secondary antibody incubation to 20 minutes when the next primary antibody applied was generated in the same species as another primary antibody used in the same experiment. Fluorescent-conjugated primary antibodies were applied during the final round of staining, where applicable. Nuclei were visualized in the final step by adding 1ng/ml of Hoechst 33342  diluted in PBS with 0.2% Tween-20 to the apical surface. After a 5-minute incubation at RT, the cultures were washed 3 x 5 minutes with PBS, after which the transwell membrane was separated from the plastic holder and mounted on a glass slide using VectaShield Antifade mounting medium  and 1.5mm-thick cover glass.HAE cultures were fixed with 100μl  and 500μl  4% (w/v) freshly prepared paraformaldehyde . After a 15-minute incubation at RT, cultures were washed once for 3 minutes with PBS and then incubated for 10 minutes at RT with a quenching solution  equipped with EC-Plan-NEOFLUOR 20x/0.5NA Ph2 and 40x/0.75NA Ph2 air objectives. Higher resolution images and z-series optical sections  were acquired with a LSM710 Zeiss confocal microscope , zoom factor of two, optimal acquisition mode, AIM-Zen 2009 software) equipped with a 63x/1.4NA Oil DIC Plan Apo objective at the Imaging Core, University of Maryland, College Park. All images were analyzed using Fiji–ImageJ v.2.1.0/1.53c software . Z-serieTables 2).The IF protocol described above was used in all assays with the following exception: for PI4P detection, permeabilization was done using a 20mM Digitonin solution (diluted in PBS). Antibodies used in this study are listed below  method . Total CTo evaluate the ratio of colocalization between two markers, the background was subtracted from ROI single-cell images following application of a threshold model and watershed filter to better identify the centroids. The final images were first used in colocalization analysis by pixel intensity-based methods , followed by spatial analysis based on the distance between centers of mass . All col4) in 0.1M cacodylate buffer plus 1% potassium ferricyanide (K3Fe(CN)6). Two percent uranyl acetate (diluted in distilled H2O) was used as post-fixative. The membrane was then incubated in propylene oxide before being embedded in Spurr’s Resin. The samples were sectioned to 60–90nm with a diamond knife (DiATOME) and ultra microtome (Reichart-Jung) and two slices were placed per copper grid (EMS). The images were obtained using a Hitachi S-4700 Field Emission Scanning Electron Microscope with transmitted electron detector in the Laboratory for Biological Ultrastructure, University of Maryland, College Park.The TEM protocol used in this study was based on published methods  with modTable 3). Following a series of washes in TBS-T, membranes were incubated for 1hr at RT with the appropriate peroxidase-conjugated secondary antibody. All antibodies were diluted in 5% non-fat milk TBS-T solution. Blots were visualized using SuperSignal West Femto Maximum Sensitivity Substrate  or SuperSignal West Dura Extended Duration Substrate . Western blot imaging was performed on an iBright 1500 (Thermo Fisher) and densitometry carried out with ImageJ software.Total protein in cell lysates (stored at -20°C in RIPA buffer) were quantified by BCA Protein Assay . A total of 20μg per sample  was then separated on a NovexWedgeWell 4–20% Tris-Glycine gel  and transferred to a PVDF membrane. Membranes were blocked with 5% non-fat milk solution ) or 3% BSA (diluted in TBS-T) for the detection of the tight junction proteins and incubated overnight at 4°C with the primary antibody  following the manufacturer’s protocol.TEER was quantified in HAE using the Millicell Electrical Resistance System (ERS)-2 (Sigma) after adding 100μl of Pneumacult-ALI medium to the apical surface and incubating cultures for 30 minutes at 34°C. Statistical analysis of resulting data was done by ordinary one-way ANOVA and Tukey’s multiple comparison test methods using Prism GraphPad v.9 software (GraphPad).https://github.com/StrokaLab/JAnaP along with the JAnaP program in its entirety. Junction characterization was performed by calculating the length of each individual junction piece that coincides with the perimeter as well as the relative aspect ratio (RAR) with respect to the cell perimeter. A junction was classified as continuous if its length was greater than 15 pixels, otherwise it was deemed discontinuous and further separated into perpendicular or punctate if the RAR was greater or less than 1.2, respectively. Statistical analysis was done by one-way ANOVA and Dunnett’s multiple comparison test using Prism GraphPad v.9 software (GraphPad).Junction coverage and characterization were quantified using the Junction Analyzer Program (JAnaP) as previously described –46. In sMucociliary transport was measured based on the transport of 2μm red-fluorescent polystyrene microspheres (Sigma-Aldrich). Five microliters of microsphere suspension  was added on top of the native mucus; after a 24-hour incubation at 34°C, videos of three regions were recorded using a Zeiss Axio Observer 3 Inverted fluorescence microscope  equipped with a 10x/0.25NA Ph1 air objective. Images were collected at a frame rate of 0.5 Hz for 10 seconds on the plane of the mucus gel. Images were acquired centrally within cultures and away from the edges, where mucus tends to accumulate. The microsphere tracking data analysis was based on an image processing algorithm that was custom written in MATLAB (The MathWorks). Briefly, the analysis software computes the XY-plane trajectories of each fluorescent microsphere in each frame. Using the trajectory data, displacement of microspheres was computed, and transport rate was calculated by dividing the displacement of microsphere by total time elapsed.S1 FigA: Immunofluorescence to detect dsRNA (red) in RV-C15 (1010 RNA)- infected HAE . B-C: Spearman correlation analysis  between fluorescence levels of CDHR3 and FoxJ1 (B) or acetylated α-tubulin (C) in non-infected HAE at 12hpi. Each dot represents the fluorescence levels of CDHR3 and FoxJ1 or acetylated α-tubulin quantified in the same Z-slice. D: Orthogonal XY view from non-infected and RV-C15-infected  HAE  stained by immunofluorescence for FoxJ1 (gray) and acetylated α-tubulin (green) at 12hpi . E and F: Fold change graph represents FoxJ1 (E), acetylated α-tubulin (E), and CDHR3 (F) protein levels normalized to the endogenous control (actin) and compared to non-infected cultures. Data shown are from two independent donors, represented by circles and triangles. Blot above is from the donor represented by circles (E) and triangles (F); VP1 protein of RV-C15 was detected in cultures from the donor "triangle" used for both assays . G-H: Quantification of fluorescence levels (CTCF) for acetylated α-tubulin (G and FoxJ1 (H) in non-infected and RV-C15-infected (dsRNA+) HAE at 12hpi. Dots represent the total CTCF per cell and the line represents the mean. Statistical analysis was done using Mann-Whitney U test .(TIF)Click here for additional data file.S2 FigA-D: Orthogonal views  from the RV-A16 (A-B) and RV-A2 (C-D) -infected HAE immunostained for dsRNA  at 12hpi (z-stacks at 1μm of thickness). Z-stacks at 1μm of thickness shows two profiles for dsRNA (red) detection by immunofluorescence at perinuclear  or close to the plasma membrane in a ring-like disposition  at 12hpi . E-F: Transmission electron microscopy of HAE infected with RV-A16  or RV-A2  at 12hpi. Visualization of ciliated cells (CC) with large, double-membrane vesicles  located above (E–left panel) or below the nucleus (E–middle panel); and the fusion of small vesicles to a larger vesicle . N = nucleus; Gol = golgi; ER = endoplasmic reticulum; Aut = autophagosome.(TIF)Click here for additional data file.S3 Fig(A). B: XZ orthogonal views  showing the differences in giantin (gray) and PI4P (green) distribution in RV-C15-infected cells with dsRNA (red) with either a perinuclear (cell I) or ring-like profile (cells II and III) . C-D: Spearman correlation analysis  between giantin and PI4Pfluorescentlevels (CTCF) in RV-A16 (C) or RV-A2 (D) -infected HAE at 12hpi. Each dot represents the fluorescence levels of giantin and PI4P quantified in the same Z-slice.Maximum Z-projection for dsRNA (red), PI4P (green), and Giantin (gray) detection by immunofluorescence in non-infected and RV-infected HAE  (TIF)Click here for additional data file.S4 FigA: Fold change graph represents calnexin protein levels normalized to the endogenous control (actin) and compared to non-infected cultures. Data shown are from two independent donors, represented by circles and triangles. Blot above is from the donor represented by triangles, which includes the detection of VP1 of RV-C15. B: Orthogonal XZ views show the perinuclear (cell-I) and ring-like (cell-II and cell-III) pattern of dsRNA (red) and calnexin (green) in RV-C15 infected HAE .(TIF)Click here for additional data file.S5 FigA-B: Spearman correlation analysis  between dsRNA and Lamp-1 or LC3b fluorescence levels (CTCF) in RV-A16 (A) or RV-A2 (B) -infected HAE at 12hpi. Each dot represents the fluorescence levels of dsRNA and Lamp-1 or LC3b quantified in the same Z-slice.(TIF)Click here for additional data file.S6 FigA: Immunofluorescence detection of dsRNA  in non-infected HAE or HAE inoculated with UV-RV-C15 or RV-C15 at 12hpi . B: Mean transport rate of mucus in non-infected HAE compared to HAE inoculated with UV-RV-C15 or RV-C15 at 24hpi. Box represents the 10–90 percentile values of MCC quantified in the same culture/condition at different time-points. Statistical analysis was done using Kruskal-Wallis followed by Dunn’ multi-comparison test .(TIF)Click here for additional data file.S1 video(AVI)Click here for additional data file.S2 video(AVI)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.S14 Table(DOCX)Click here for additional data file.S15 Table(DOCX)Click here for additional data file.S16 Table(DOCX)Click here for additional data file.S17 Table(DOCX)Click here for additional data file.S18 Table(DOCX)Click here for additional data file.S19 Table(DOCX)Click here for additional data file.


Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK’s catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting. Hyperactivation of components of the MAPK pathway contribute to the etiology and progression of cancer. At the plasma membrane, the RAS-GTPase family members HRAS, NRAS, and KRAS function as molecular switches for the activation of the downstream kinase cascade ,2,3,4. IIn recent years, the pharmaceutical targeting of the deregulated activities of the interconnected MAP-kinases BRAF, MEK, and ERK have been intensified. In a subset of tumors harboring kinase cancer driver mutations, the selective and direct blocking of mutant BRAF-dependent downstream signaling to MEK-ERK is a remarkable feature of BRAFi ,15. TragS218D/S222D double-mutant kinase in particular. Thus, we propose that activated MEK1 KinCon biosensors have the potential to be used for efficacy validations of MEKi in different cell systems at expression levels below the endogenous kinase. It is of interest that several MEKi such as trametinib, cobimentinib, refametinib, and selumetinib are not ATP-competitive and act allosterically . They biBenzyl-coelenterazine ; Trametinib , Cobimetinib , Refametinib , Selumetinib , PLX8394 .® M2-tag .HEK293T (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-11268) and A2058 (ATCC CRL-11147) melanoma cells were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfections of HEK293T cells were performed with Transfectin reagent . For transient overexpression of KinCon reporter constructs in the melanoma cell line A2058 , 4 million cells were seeded on 10 cm dishes and cultivated overnight. Transfection was performed with jetPRIME transfection reagent  according to the manufacturer’s instructions. Twenty-four hours post transfection, the cells were passaged and split on 96-well plates at a maximum density of 40,000 cells per well. At 48 h post transfection, drug exposure experiments and bioluminescence measurement were performed as described below. Primary antibodies used were the rabbit anti-GAPDH , rabbit anti-P-ERK1/2 , mouse anti-ERK1/2 , rabbit anti-MEK1/2 and anti-P-MEK1/2 , mouse anti-FLAGMEK1 gene (MEK1: NM_002755.3), we fused it N-terminally with -F[1] and C-terminally with -F[2] of the Rluc -PCA (pcDNA3.1 backbone vector) as previously described -MEK1-F[2] [The dual-specificity kinases MEK1/2 are the downstream targets of physiological and pathological RAF activities A 8]. Th. Th8]. TEK1-F[2] ,38 whichS218A/S222A, and MEK1 KinConS218E/S222E showed no major reporter dynamics upon MEKi exposures for 1 h. The BRAFi PLX8394 acting as negative control had no effect on the tested MEK1 conformations  represents the active kinase state, we transiently overexpressed the flag-tagged wild-type version and the S218D/S222D mutated MEK1 flag-fusion proteins in HEK293T cells. Following 48 h of expression, we detected that flag-tagged MEK1marginal A. Dose-dmarginal A. Next, marginal B. Next, tectable C. S218A/S222A reporter, the wild-type and the activated MEK1S218D/S222D KinCon reporters responded to drug exposure by promoting the more closed kinase conformation (MEK kinases are activated as a result of RAF-mediated phosphorylation of, e.g., MEK1 at the positions S218 and S222 of the kinase activation loop . Thus, wormation E, right.The explicit inhibition of MEK activities causes qualitative alterations of MAPK signal propagation. The integration of the phospho-mimetic mutations S218D/S222D triggered the opening of the kinase conformation as we have tracked it in intact cells using KinCon reporter. Thus, we set out to validate our cellular MEK1 reporter measurements with simulations of structure dynamics in the presence and absence of MEKi and the respected MEK1 mutations of S218/S222. S218D/S222D, MEK1S218E/S222E, MEK1S218A/S222A and dephosphorylated WT (S218D/S222D dynamics as measured by root mean square fluctuation (RMSF, S218E/S222E variant. In particular, we investigated individual segments of MEK1 and identified the distal A-helix (S218D/S222D exhibits significantly broader distributions of the angles describing their orientation relative to the kinase core. The variances of MEK1S218D/S222D A-helix angles range from 2.1 to 2.6 degrees, whereas variances for MEK1S218E/S222E are approximately half, at 1.2–1.6 degrees. Similarly, the variances of MEK1S218D/S222D alpha-C helix angles range from 3.4 to 4.5 degrees, whereas variances for MEK1S218E/S222E are considerably lower at 2.4–3.2 degrees, excepting RDEA119-bound complexes, which are highly variable across all systems. Such an increase in mobility is indicative of an enhanced propensity for structural rearrangement as indicated by the KinCon reporter system, and hence the findings are consistent with experimental evidence of structural change.In order to identify the molecular origin of the observed differences, we conducted extensive molecular dynamics simulations of the WT, MEK1lated WT . The stron RMSF, B exceed  A-helix A and the A-helix B as majo-S218D/S222D KinCon reporters occupy an active and opened kinase conformation, which is in accordance with the shown structural MD simulation data. Treatment with allosteric MEKi exclusively affected the activated, phosphorylated and thus opened conformation states of the MEK1 wild-type reporter and the respective phospho-mimetic MEK1-S218D/S222D mutant.In summary, we successfully extended the KinCon biosensor concept to the dual-specificity kinase MEK1. The data obtained by the biosensor experiments suggest that BRAF-phosphorylated MEK1 along with the MEK1Signaling units of the MAPK pathway are among the most frequently mutated oncogenic proteins. Therapeutic strategies to combat different types of cancer involve the inhibition of the participating kinases using defined RAFi and MEKi or their combinations ,8,24,42.S218D/S222D, MEK1S218E/S222E and MEK1S218A/S222A revealed the molecular origin of the observed differential behavior. MEK1S218D/S222D exhibited increased dynamics in both the A-helix and alpha-C helix compared particularly to the superficially analogous MEK1S218E/S222E variant, as evidenced by a broadening in angle distributions under apo and ligand-bound conditions. Such broadening is consistent with behavior observed for the simulated di-phosphorylated system. Increased dynamics in the simulation showed that conformational stability for the MEK1S218D/S222D variant is reduced, which resulted in an enhanced propensity for reporter activation. It would furthermore appear that ligand binding particularly affects MEK1S218D/S222D in the region of the alpha-C helix with a more pronounced conformational shift observed for MEKi as compared to apo or any complex of MEK1S218E/S222E, which corresponded accurately with the reporter activation. Whereas our simulations show a consistent difference in behavior for the two phospho-mimetic mutations, we could not consistently observe a difference between WT and MEK1S218D/S222D. The cell-based demonstration of MEK1 kinase dynamics was affirmed in structural simulations, underlining that the S218D/S222D variant of MEK1 follows similar dynamics as the phosphorylated counterpart. Simulations of MEK1 WT, MEK1S218D/S222D KinCon reporter. Moreover, we managed to track BRAF-V600E-mediated MEK1 KinCon activity states at expression levels of the KinCon well below the endogenous kinase. Thus, this approach seems to be suitable for kinase-targeted and cellular drug profiling efforts. This observation should be helpful for determining efficacies of drugs which interfere with the MAPK pathway using the MEK1 biosensor as read out in different cell settings. The complexity of kinase cascade regulations and the high incidence of cancer mutations require new means for recording cellular kinase rearrangements upon lead molecule binding to allosteric or ATP-competitive binding pockets. Such an approach might be suitable to track context-dependent kinase dynamics directly in the cell line of choice. The intact cell setting not only takes the whole spectrum of possible protein interactors into account, but also allows the imitation of the respective tumor microenvironment. Further, KinCon allows the implementation of patient mutations to assist in analyses of specific drug efficacies. The current focus of drug discovery and repurposing highly favors poly-pharmacological approaches. Here, KinCon offers high adaptability, practicability, and qualifies for high-content screening concepts for unveiling novel drug combinations.MEK1/2 relay physiological but also oncogenic input signals to ERK1/2. This function of MEK at a central crossroad of signal propagation has fostered drug discovery efforts to identify efficient MEKi. This led to the approval of a collection of non-ATP competitive MEKi. Amongst others, trametinib and cobimetinib found the way into the clinic for cancer therapy ,24. In cAspects of the present study are subject of pending patent applications.


Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na Hydrogen sulfide has recently been gaining significance as a novel gas mediator . It is eSpinocerebellar ataxia (SCA) is a group of autosomal dominant neurodegenerative diseases characterized by progressive cerebellar ataxia and cerebellar atrophy ,4. AlthoWe recently reported that D-cysteine ameliorates impaired dendritic growth in primary cultured PCs expressing several SCA-causing proteins . Additio2S), on CMA and mA using a CMA/mA activity marker that we have previously established  sig sig2S onincrease C,D. In Aeased it A,B. An ieased it  20] sig sig2S ond it  that is activated by hydrogen sulfide. We previously demonstrated that CMA and mA activities are separately evaluated in cells with siRNA-mediated knockdown of an mA-related protein, TSG101, or a CMA-related protein, LAMP2A, respectively . Na2S siwn cells . This re2S in AD293 cells (We then explored the mechanism of how hydrogen sulfide activates CMA. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) upregulates LAMP2A, leading to CMA activation . Hydroge93 cells C,D.2S tended to enhance it H quinone dehydrogenase 1 (NQO1), a downstream protein of the Nrf2 pathway  Figure , suggestIntracellular protein degradation systems are crucial for maintaining intracellular protein homeostasis, especially in the neurons . The autIn the present study, we first revealed that hydrogen sulfide activates CMA. Compared with MA, CMA and mA have been studied less . Hsc70 i2S rapidly increased the amount of Nrf2 in AD293 cells. However, this effect did not last 24 h after the treatment (2S, it can rapidly modify the cysteine residues of Keap1, leading to the dissociation of Nrf2 from Keap1 and subsequent Nrf2 elevation and activation [2S-treated AD293 cells and D-cysteine-treated cerebellar lysates (2S (The treatment with Nareatment . Since rtivation . Activattivation . LAMP2A tivation . In the  lysates . Taken tates (2S , Nrf2 acIn the present study, we revealed that in vivo long-term treatment with D-cysteine activates Nrf2 in the mouse cerebellum . This fiSeveral D-amino acids are endogenously generated in mammals and have roles different from those of L-amino acids . Among DIn addition to the cerebellum, DAO is also expressed in the liver and kidney ,12. TherOur study suggests that D-cysteine activates CMA in cerebellar PCs via the generation of hydrogen sulfide and the activation of Nrf2. Since several findings indicate that CMA impairment is related to SCA pathogenesis ,15,17, t


Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers. Prostate cancer (PC) is a frequent cause of cancer among men worldwide, second only to lung tumors, and is the fifth-leading cause of cancer-related death in men ,2; it isGM-CSF, approved against metastatic cutaneous melanoma [Oncolytic viruses (OVs) or onco-immunotherapeutic viruses (OIVs) are promising candidate anticancer therapeutics in the preclinical and clinical stages of development ,8. OVs emelanoma ,11,12. Omelanoma ,  is a tumor-associated antigen (TAA) highly expressed in prostate adenocarcinoma cells regardless of androgen status , in the We recently engineered an oHSV retargeted to PSMA, which was capable of infecting and in vitro killing PSMA-positive cells. The objective of this study was to evaluate the in vitro susceptibility of human PC cells to the PSMA-retargeted oHSV, called R-405, to provide the first proof-of-principle evidence that R-405 exerts antitumor efficacy and long-term protection in vivo against PSMA-positive tumors in syngeneic murine models, and to determine whether the treatment elicited a specific adaptive immune response and in situ vaccination against distant challenge tumors.PC3 (human prostatic adenocarcinoma), LNCaP (human prostate adenocarcinoma), 22Rv1 (human prostate carcinoma), SK-OV-3 (ovary adenocarcinoma), LLC1 (Lewis Lung carcinoma), and Renca cells  were purchased from ATCC  and cultured as indicated by ATCC. J cells, their derivatives expressing Nectin1, HVEM and PSMA ,37 and PACCTTCCGGTCCTGGACCAGCTGACCCCTCCGGGGGTCCGGCGCGTGCCTGTTGACAATTAATCATCGGCA and gD39GalKRev ATCGGGAGGCTGGGGGGCTGGAACGGGTCTGGTAGGCCCGCCTGGATGTGTCAGCACTGTCCTGCTCCTT and using pGalK as template. Recombinants were checked by PCR with primer galK_129_ForThe R-LM5 virus was described . The HSVACCTTCCGGTCCTGGACCAGCTGACCCCTCCGGGGGTCCGGCGCGTGGAGGTGCAGCTGCAGCAGTCAGGACC and gD39_J591Rev ATCGGGAGGCTGGGGGGCTGGAACGGGTCTGGTAGGCCCGCCTGGATGTGGGATCCACCGGAACCAGAGCCACCGC using BAC R-593 as template [GAGCCATGGAAAGAGCCTTGAGTGG and J591_503_527_Rev AGTTTAGGAGATTGTCCTGGTTTCT. After reconstitution of R-405 virus in SK-OV-3-PSMA cells, single plaque purification was performed [ACAATCTCTGTTTGCCAACGCATTTGG and galK_417_Rev CATTGCCGCTGATCACCATGTCCACGC. The J591 cassette with homology arms to gD was amplified with primers gD37_J59For erformed ,43. ViruSK-OV-3, LLC1 and Renca cells were made transgenic for the expression of a truncated form of human PSMA, in which the 4 N-terminal amino acids (WNLL) of the human-PSMA gene were deleted. The pIRES-PSMA plasmid  was amplJ cell derivatives, PSMA-positive  and PSMA-negative cancer cells  were infected with R-405 virus at an input multiplicity of infection of 10 PFU/cell as titrated in SK-OV-3-PSMA, for 90 min at 37 °C. Pictures were taken 24 h after infection by Nikon Eclipse TS100 fluorescence microscope. For the neutralization assay, replicate monolayers of the PSMA-positive  and PSMA-negative cancer cells (SK-OV-3) seeded in 96-well plates were preincubated with increasing amounts of MAb D2B or with mouse IgG for 60 min at 37 °C. R-405 (3 PFU/cell) was added to the MAb-containing medium for additional 90 min. Alternatively, R-405 and R-LM5 virions (3 PFU/cell) were pre-incubated with increasing amounts of HSV-1-neutralizing MAb 52S for 1 h at 37 °C, and then allowed to adsorb to cells for 90 min . Viral inoculum was removed, and cells were overlaid with medium containing MAbs for 18 h. The extent of infection was measured by FACS as the percentage of EGFP-positive infected cells. Western blot assay was performed as described .3 cells/well and infected with R-405 or mock-infected. The input multiplicity of infection was 0.05 PFU/cell as titrated in the corresponding cell line. AlamarBlue  was added to the culture media (10 μL/well) at the indicated times after infection and incubated for 4 h at 37 °C. Plates were read at 560 nm and 600 nm with GloMax Discover System . For each time point, cell viability was expressed as the percentage of AlamarBlue signal reduction in infected versus uninfected cells, after subtraction of the background value .The number of encapsidated/enveloped infectious and non-infectious viral particles in R-405 stocks was determined as described  and expr6 cells/mouse. Where indicated, mice were subcutaneously engrafted with LLC1-WT or Renca-WT cells in the right flank, in 100 μL of serum-free medium, 1 × 106 cells/mouse . Tumor volumes were scored 3 times weekly by measuring the largest and the smallest diameter by means of a caliper. Tumor volume was calculated using the formula: largest diameter × 2 × 0.5. Mice were sacrificed within one or two days after their tumors reached a volume of about 1500 mm3, ulceration occurred, or animals exhibited distress or pain. For virus treatment, when the tumor volumes averaged 70–100 mm3 , mice received 2–4 intratumoral injections of R-405  or vehicle (50 µL PBS), at 2–7 days intervals. Mice which survived the primary tumor were engrafted with challenge tumors: C57BL/6 mice were challenged with LLC1-PSMA (left flank) and LLC1-WT (right flank), BALB/c mice were challenged with Renca-PSMA (left flank) and Renca-WT (right flank). The challenge tumors were not treated. Where indicated, mice received 4 intra peritoneal (i.p.) injections of anti-mouse CTLA4 antibody  or anti-mousePD-1 antibody , or vehicle (50 µL PBS) at 3–4 days intervals. C57BL/6 and BALB/c mice were obtained from Charles River Laboratories  and bred in the facility of the Department of Veterinary Medical Sciences, University of Bologna . Both female and male mice were randomly used throughout experiments, according to a request from the Ethical Committee. Retrospective evaluation of results based on sex-based stratification of mice showed no sex-related difference. LLC1-PSMA or Renca-PSMA cells were implanted subcutaneously in the left flank of 7–12-week old C57BL/6 and BALB/c mice, respectively, in 100 μL of serum-free medium, 1 × 104Cl, 10 mM NaHCO3, 1 mM EDTA), samples were pelleted and resuspended in flow cytometry buffer. For each sample, 2 × 106 cells were blocked with α-CD16/32 Ab , and then reacted with the antibodies CD45-FITC , CD45-PE-Cy7 , CD4-FITC , CD8a-PE , CD335-PE , CD11b-FITC , MHC-I(H-2Kb)-APC , FoxP3-PE , IFNγ-APC , and CD69-PercP . To detect Human PSMA, samples were reacted with MAb D2B (10 µg/mL), washed twice, then reacted with Alexa Fluor™ 488 anti-mouse IgG H+L . Staining reactions were performed on ice for 30 min. Data were acquired on BD C6 Accuri. Only samples which provided at least 100,000 events were included in the analysis.Single cell suspensions were prepared from freshly isolated LLC1-PSMA tumors and spleens at sacrifice. Tumors were minced in small pieces and digested with collagenase (1 mg/mL) for 30 min at 37 °C. The resulting cell suspensions were passed through 70 μm cell strainer and rinsed with flow cytometry buffer (PBS + 2% FBS). Spleens were processed directly through the cell strainer, and the red blood cells in splenic specimens were lysed by means of ACK buffer (150 mM NHIl12a), mm01168134_m1 (Ifnγ), mm00444662_m1 (Cxcl11), mm00443258_m1 (Tnfα), mm00475162_m1 (FoxP3), mm00450960_m1 (Tbx21/T-bet), Mm03048248_m1 (Pd-l1), gC of HSV-1 . The levels of expression were determined using the ΔΔCt method, normalized relative to the Rpl13a housekeeping gene.A few mgs of the LLC1-PSMA tumor homogenates were employed for RNA purification with the Nucleospin RNA kit (Macherey-Nagel), including the on-column DNaseI treatment. Two μg of total RNA was employed for the cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit  in a 20 μL reaction. qRT-PCR reactions were performed in a StepOnePlus System (Applied Biosystems) using 0.5 μL of cDNA for each assay, and the TaqMan probes employed for the assay are: mm00434169_m1 (6 cell/well) were incubated either with 1 × 105 LLC1-WT or LLC1-PSMA cells in 0.5 mL medium, and cocultured for 72 h. Media were collected and the amount of secreted IFNγ was quantified by ELISA .Freshly explanted spleens were smashed through a 70 μm cell strainer in PBS to isolate splenocytes. Red blood cells in spleens were lysed with ACK buffer, while the splenocytes were resuspended in medium , counted and seeded in 24 well plates. Suspensions of LLC1-WT and LLC1-PSMA cells were treated with mitomycin 15 μg/mL  for 2 h at 37 °C, then washed 3 times with fresh medium. Splenocytes . Data were acquired on BD C6 Accuri.To detect the serum antibodies to cancer cells, LLC1-WT and LLC1-PSMA cells were trypsinized and resuspended in flow cytometry buffer. For each sample, 0.25 × 10We engineered a novel PSMA-retargeted oHSV, called R-405, the backbone of which was similar to that of R-337, the prototype of third generation retargeted oHSVs . R-405 gTo verify whether R-405 could cause a bona fide infection of PSMA-positive human prostate tumor cells, PSMA-positive LNCaP cells derived from human androgen-dependent prostate adenocarcinoma lymph node metastasis , and PSMTo express human PSMA in additional human and murine tumor cells, a form of PSMA deleted of the endocytosis motif located in the N-terminal cytoplasmic tail (PSMAΔ) was engineered to maximize and stabilize its cell surface expression. The PSMAΔ was expressed in SK-OV-3 cells, a human tumor cell line routinely utilized in our laboratory for the high titer production of HSV stocks, and in the murine tumor LLC1 and Renca cells, syngeneic with C57BL/6 and BALB/c mice, respectively, thus generating LLC1-PSMAΔ and Renca-PSMAΔ . The extent of PSMAΔ expression is shown in HIGH and PSMALOW human PC cells, respectively [The extent of R-405 replication in the above cells was quantified in a virus yield experiment at 24, 48, and 72 h after infection. Infection was carried out at 0.1 plaque-forming unit (PFU)/cell, according to the R-405 titer in each cell line A. R-405 ectively .HIGH and PSMALOW human PC cells, respectively.To provide the first proof-of-principle evidence that R-405 exerts antitumor efficacy against PSMA-positive tumors in syngeneic murine models, we made use of the LLC1-PSMA and Renca-PSMA tumor cells described above, syngeneic with C57BL/6 and BALB/c mice, respectively. Two systems were utilized since it has been well established that tumors vary greatly in their mechanisms of immune response and evasion, also called immune editing. On one extreme are tumors which are very poor in eliciting the immune response; they are designated as immunologically cold, or, in more severe examples, immunologically desert. At the other extreme are tumors which are competent in eliciting an immune response  which they dampen by a variety of immune suppressive mechanisms ,52,53. I7 PFUs for each dose. This dose was chosen based on our previous study regarding the efficacy of the HER2-retargeted oHSV called R-337 against HER2-positive LLC1 tumors [In the first series of in vivo experiments, LLC1-PSMA tumor cells were implanted subcutaneously (s.c.) in syngeneic C57BL/6 mice. The experimental design is shown in 1 tumors . Ex vivoSubsequently, all the R-405-treated mice were simultaneously implanted with two challenge tumors in the right and left flanks, made up of LLC1-PSMA or wt LLC1 (herein LLC1-wt) cells, respectively. gC glycoprotein and of the R-405-encoded Il12a  were increased as was the T-bet transcription factor, which favored Th1 polarization, and the T-regulatory cell FoxP3 factor  resulted in a CR in 100% of the mice. The lowest dosage 2 × 105 PFUs protected only 1 out of six mice. The 3 × 106 and 1 × 106 PFU dosages induced a CR in 80% and 33% of the mice, respectively.We subsequently asked whether R-405 was sensitized to anti-PD-1 therapy. This experiment required a dose of R-405, which protected approximately 50% of the mice, and not 100% of the mice, as was the case in the experiment in 105 PFUs A. The do6 PFU dosage for each injection; it induced a CR in 50% of the mice  in 25% of the mice. The reduction in tumor volume at d 37 and the Kaplan-Meier survival curve showed highly significant differences between the untreated and R-405-treated mice .As an immunologically warm model, Renca-PSMA cells implanted s.c. and treated i.t. with R-405 were utilized A. Upon ited mice E,F. The ted mice B, in theThe mice which survived the primary tumor were simultaneously implanted with two s.c. challenge tumors made up of Renca-PSMA and Renca-wt cells. The extent of distant protection is reported in In the final experiment K, the efWe report on a first proof-of-principle study of the properties and in vivo efficacy of a fully virulent oHSV retargeted to PMSA as an example of precision medicine and in situ vaccination approach against PC. The key features were as follows. (i) The PSMA-retargeted IL-12-armed oHSV, called R-405, infected exclusively PSMA-positive cells and spared PSMA-negative cancerous or non-cancerous cells. It infected the cells and caused cytopathic death of the PSMA-positive human prostate cancer LNCaP and 22Rv1 cells, spared the PSMA-negative human prostate cancer PC3 cells, but infected the PC3-PIP cells transgenically expressing PSMA. The PSMA served as the port of entry of the R-405 into such cells. Thus, the human PSMA-positive PC cells were specific targets for R-405. With respect to murine tumor cells, the efficacy of the R-405 infection, cell-to-cell spread, and virus-induced cell death were higher in LLC1-PSMA than in Renca-PSMA cells; these features likely resulted from the intrinsic properties of the two murine tumor cells and, possibly, also from the different extent of PSMA expression. (ii) The in vivo key features of R-405 were its high efficacy against primary tumors , and the anti-tumor in situ vaccination effect which resulted in protection against distant challenge tumors. Notable, the immune protection was exerted not only towards the PSMA TAA but, in part, also towards the LLC1 tumor antigens, confirming that the retargeted oHSVs primed for antigen-agnostic antitumor immune response. (iii) The bases of the immune protection were the anti-tumor Th1 polarization of the TME , the development of systemic tumor-specific T-lymphocytes (splenocytes), infiltration of the tumor masses by CD8+ and activated NK cells, and the inhibition of the mechanisms of tumor tolerance, exemplified by the depletion of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and the restoration of MHC-I expression in tumor cells. (iv) R-405 sensitized tumors to a checkpoint blockade-based therapy. The virus treatment inflamed the TME through infection and direct oncolysis, activation of innate and adaptive immunity, and expression of pro-inflammatory cytokines (including the virus-encoded IL-12 and the subsequent IFN cascade). Nevertheless, these modifications also triggered some tumor defenses against immune system, including the increase of PD-L1 expression and the tumor infiltration by Tregs. Both effects were caused by higher IFN levels ,61 and o(i)Previous attempts to generate OVs specific for PC cells have included conditionally replicating transcriptionally retargeted adenoviruses (AdVs) carrying key viral genes under prostate specific antigen (PSA) or PSMA enhancer elements ,63, an a(ii)High PSMA expression is frequent (75.21%) in metastatic castration-resistant prostate cancer patients and portends a poor prognosis ,32,33. A(iii)The importance of PSMA as a target for precision medicine is strengthened by investigations regarding PSMA-targeted chemical antigen receptor T cells (CAR-Ts) . Clinica(iv)Very few trials address anti-PC vaccination. Sipuleucel-T is a dendritic cell (DC)-based vaccine recommended as first-line therapy for the treatment of metastatic castration-resistant prostate cancer . It mustMetastatic castration-resistant prostate cancer remains incurable and fatal, despite the availability of multiple classes of therapy which delay disease progression and prolong life . Beyond Given the slow progression typical of human PCs, an approach capable of eliciting a long-term adaptive immune response appears to be appropriate. An off-the-shelf therapeutic treatment capable to induce in situ vaccination would respond to this need and would be of practical application and at the reach of a high number of patients. The properties of the PSMA-retargeted oHSVs described in the current proof-of-principle study addressed these challenges. The target patients should include early and advanced stage patients for the purpose of slowing down progression to more advanced stages and to metastatic disease as well as metastatic disease patients. The PSMA-oHSV might ultimately be combined with PSMA T-cell-based therapies to achieve long-term immunoprevention and immunotherapy. We concluded from this proof-of-principle study that PSMA-retargeted oHSVs are worth being additionally investigated as anticancer agents and as candidate therapeutic in situ vaccines against prostate cancer.Herpes Simplex Virus (HSV) with modified tropism, uses and process of preparation thereof-WO2009144755 Retargeted herpesvirus with a glycoprotein H fusion-WO201612849Herpesvirus with Modified Glycoprotein B-WO2017211941Herpesvirus with Modified Glycoprotein D-WO2017211944Herpesvirus with Modified Glycoprotein H for Propagation in A Cell-WO2017211945


Ig) and T cell receptor (TCR) genes, which enables the recognition of highly diverse antigens and the elicitation of antigen-specific immune responses. This process is mediated by recombination-activating gene (Rag) 1 and Rag2 (Rag1/2), whose expression is strictly controlled in a cell type-specific manner; the expression of Rag1/2 genes represents a hallmark of lymphoid lineage commitment. Although Rag genes are known to be evolutionally conserved among jawed vertebrates, how Rag genes are regulated by lineage-specific transcription factors (TFs) and how their regulatory system evolved among vertebrates have not been fully elucidated. Here, we reviewed the current body of knowledge concerning the cis-regulatory elements (CREs) of Rag genes and the evolution of the basic helix-loop-helix TF E protein regulating Rag gene CREs, as well as the evolution of the antagonist of this protein, the Id protein. This may help to understand how the adaptive immune system develops along with the evolution of responsible TFs and enhancers.Adaptive immunity relies on the V(D)J DNA recombination of immunoglobulin ( TCR) and immunoglobulin (Ig) genes from arrays of variable (V), diversity (D), and joining (J) gene segments. These assembled antigen receptors are able to recognize highly diverse antigens and elicit antigen-specific immune responses .,22.−CD8−Rag1/2 gene expression is stringently controlled in a cell type-specific manner. The first wave of RAG expression is required for the recombination of TCRβ and Igh genes in pro-T and pro-B cells, respectively. After the β-selection of pro-T cells and pre-BCR selection of pro-B cells, Rag expression is transiently downregulated during the developmental transition toward precursor stages (DP and pre-B cells). In precursor cells, Rag1/2 genes are upregulated for the VJ recombination of TCRα, Igκ, and λ genes. Following successful TCR and Ig gene recombination, Rag1/2 gene expression is completely suppressed in mature T and B cells [Rag gene expression and functions results in severe combined immunodeficiency, resulting from developmental arrest at pro-T and pro-B cell stages [Rag expression led to profound immunodeficiency in mouse [Rag1/2 genes are stringently regulated during T and B lymphopoiesis. There have been in vivo and in vitro studies that attempted to identify the CREs associated with Rag1/2 expression [Erag, which is 23 kb upstream of the Rag2 gene, caused impaired Rag1/2 expression in pro-B cells and a moderate developmental block at the pro-B stage but did not affect the Rag gene expression in T cell development [R1B and R2B , resulted in a developmental arrest at the pro-B stage, indicating the enhancer redundancy of Rag gene in B cells [Rag gene enhancer R-TEn, which is included in ASE, is critically important for Rag1/2 expression and TCR recombination during thymocyte development, and its deletion led to a developmental block at the DN3 cells and DP cells in fetal and adult thymus without affecting B cell development [Rag gene expression is tightly regulated in a cell type-specific manner, and T and B cells use distinct enhancer regions for Rag gene expression. B cells ,14,24,25l stages ,27. Furtin mouse . Thus, Relopment . Interes B cells . On the elopment ,21,29. TRag genes? Notably, mutating the E-box motifs in the R-TEn enhancer (R-TEn-E-box-mutant) in mice, in which the binding of E2A is blocked, resulted in an impaired Rag1/2 expression and blocked thymocyte development, as well as deletion of the entire R-TEn region. Of note, the R-TEn-E-box-mutant abolished chromatin accessibility throughout the entire Rag gene locus. These results indicated that E-protein binding to the T cell-specific Rag gene enhancer is required for T cell-specific spatial interactions to enhance Rag1/2 expression [Rag1 gene promoter region (R1pro) by generating E-box motif mutations alone resulted in the complete loss of Rag1 expression without affecting Rag2 expression in both developing T and B cells, leading to developmental arrest at the pro-T and pro-B cell stages [Rag1 promoter depend on the binding of E2A to these regions and that E2A is a core TF that specifies the adaptive lymphoid cell identity through the regulation of Rag gene expression.Previous reports and ChIP-seq showed that many T cell- or B cell-specific TFs bind to these enhancer regions  ,29,30,31pression ,21. NotaRag gene enhancers reflect the evolution of Rag gene regulation. Although Rag1/2 genes are well known to be conserved among jawed vertebrates, the conservation of Rag gene enhancers had not been investigated. Thus, we investigated the conservation of R-TEn, R1B, and R2B regions and E-box motifs in these regions [R-TEn and R2B are readily observed among mammals, most birds, and reptiles; however, sequence similarities of these enhancers are not noticeable in the corresponding genomic regions of amphibians and fishes  and Caenorhabditis elegans gene product HLH-2 are involved in other developmental pathways. Da is essential for both neurogenesis and sex determination in D. melanogaster embryonic development [C. elegans somatic gonad [D. melanogaster gene product extramacrochaete (emc) [E proteins are basic helix-loop-helix (bHLH) transcription factors involved in multiple developmental processes. E proteins include E12, E47, E2-2 (TCF4), HEB (TCF12), daughterless (Da), and HLH-2 . E proteelopment . HLH-2 iic gonad . Id protic gonad . When thic gonad . Id protte (emc) .E2A (Tcf3) is critically required for B cell lineage commitment [It is well established that a majority of adaptive lymphocyte development trajectories require regulation by E and Id proteins ,39,41. Emmitment ,43 and tmmitment ,45. In lmmitment ,46. Uponmmitment . Likewismmitment , and E2Ammitment . Interfemmitment ,50,51.Id2 and Id3 are involved in both T and B cell development to modulate E protein DNA binding activity. Id2 is particularly important for ILC, NK, and LTi cell development through the suppression of E protein activity [Id3 is first upregulated by pre-TCR signaling in DN3 cells and further upregulated upon positive selection of TCR signaling in DP cells [Id3 abundance and E2A-Id3-Tcf1 transcription axis control γδ T cell development and effector function [Id3 plays a key role in follicular helper T (TFH) and follicular cytotoxic T(TFC) cell development through the regulation of CXCR5 expression [Id2 and Id3 have distinct roles in the differentiation of CD8 cytotoxic T cells toward effector and memory T cells [Id2 and Id3 expression to suppress systemic TH2 inflammation and function as a gatekeeper for follicular regulatory T (TFR) cell [activity ,52. In Bactivity . Id3 is activity ,55. In TDP cells ,57. Simifunction ,59. Furtpression ,61,62. N T cells ,64. In aFR) cell . Taken tDa gene expression in Drosophila. Coupled transcriptional feedback loops maintain the widespread Emc expression that restrains Da activity to induce neurons [Drosophila.In this section, we address the question of how the E–Id axis was evolutionally developed. Emc is a negative feedback regulator that prevents runaway self-stimulation of  neurons , suggestPetromyzon marinus) (Three E protein homologs and two Id protein homologs were found in the lamprey (marinus) . A reconmarinus) . A reconmarinus) . LampreyIg, TCR, Rag1 and Rag2, and MHC class I/II genes have an integral role in AIS in jawed vertebrates. Homologs of these genes have been identified in all extant classes of jawed vertebrates [Ig, TCR, Rag1/2, or MHC genes [TCR and Ig, different types of antigen receptors, which are known as variable lymphocyte receptors (VLRs), composed of highly diverse leucine-rich repeat (LRR) modules, have been identified in lampreys, and, like Rag-mediated recombination of TCR and Ig in jawed vertebrates, VLRs are generated by cytidine deaminase (CDA) 1 and 2 [Rag1 and Rag2 genes that are essential for AIS in jawed vertebrates are proposed to have arisen from a transposable element and transmitted vertically through chordate and jawed vertebrate evolution [Rag gene during T and B cell development [Rag genes contributed to the genesis of the AIS in jawed vertebrates. The two rounds of WGD are considered important for the acquired immunity of jawed vertebrates [Jawed vertebrates (gnathostomes) possess AIS that can recognize and initiate a protective response against invading pathogens. AIS in jawed vertebrates is centered on T and B lymphocytes bearing TCR and B cell receptors , which are generated through the V(D)J recombination mediated by Rag1 and Rag2 . The TCRtebrates . On the tebrates ,73,74. Mtebrates . HoweverHC genes ,76,77. I 1 and 2 ,35,73,78 1 and 2 ,79. The  1 and 2 . Additiovolution . This scelopment  and Id2 elopment ,23. Thertebrates ,83. Prevtebrates . E-Id patebrates ,65,85,86tebrates .