Caenorhabditis elegans. Through genetic suppressor screens, we identify a gain-of-function mutation within rbm-39, an RNP-6-interacting splicing factor, which increases nuclear speckle formation, alleviates splicing defects and curtails longevity caused by rnp-6 mutation. By leveraging the splicing changes induced by RNP-6/RBM-39 activities, we uncover intron retention in egl-8/phospholipase C β4 (PLCB4) as a key splicing target prolonging life. Genetic and biochemical evidence show that neuronal RNP-6/EGL-8 downregulates mammalian target of rapamycin complex 1 (mTORC1) signaling to control organismal lifespan. In mammalian cells, PUF60 downregulation also potently and specifically inhibits mTORC1 signaling. Altogether, our results reveal that splicing fidelity modulates lifespan through mTOR signaling.Changes in splicing fidelity are associated with loss of homeostasis and aging, yet only a handful of splicing factors have been shown to be causally required to promote longevity, and the underlying mechanisms and downstream targets in these paradigms remain elusive. Surprisingly, we found a hypomorphic mutation within ribonucleoprotein RNP-6/poly(U)-binding factor 60 kDa (PUF60), a spliceosome component promoting weak 3′-splice site recognition, which causes aberrant splicing, elevates stress responses and enhances longevity in  Caenorhabditis elegans.RNA splicing has a role in aging and longevity, but the mechanisms involved are incompletely understood. Here the authors show that mRNA splicing components, RNP-6 and RBM-39, act in concert to regulate intron retention, inhibit mTORC1 signaling and prolong life in  Accordingly, mutations in core spliceosomal components that disrupt the splicing process can cause various pathologies, including muscular dystrophies, cancer and neurodegenerative and cardiovascular diseases3. Splicing has also been recently implicated in playing a role in the aging process located downstream of pro-longevity interventions such as diet restriction and mTOR inhibition5. However, the relationship between splicing and aging is still unclear and the detailed mechanisms of how the splicing machinery/splicing factors regulate longevity remain largely elusive.Pre-messenger RNA splicing is a major step of gene regulation that contributes to proteomic diversity in eukaryotes. In this process, multiple splicing factors and RNAs come together in an organized stepwise fashion to form the spliceosome, acting to remove introns and create alternative mRNAs. Ensuring splicing fidelity and isoform diversity is essential to proper growth, development and homeostasis7. PUF60 is required for cell viability, proliferation and migration in vitro. Its deficiency in humans causes developmental defects10 and overexpression is associated with tumorigenesis12, but a role in metabolism and aging is completely unknown. In a previous genetic screen for C. elegans longevity regulators using cold tolerance as proxy, we had identified a new mutation in the worm ortholog of PUF60, rnp-6, carrying a Gly281Asp substitution (referred to as rnp-6(G281D) hereafter) in the second RNA recognition motif (RRM), which alters resistance to multiple abiotic stresses and extends lifespan13.PUF60 encodes an essential splicing factor that binds polyuridine (U) tracts and promotes association of the U2 small nuclear RNP complex (U2 snRNP) with primary transcriptsrnp-6-induced physiology, we combined forward genetic screening, transcriptomic profiling and genetic epistasis analysis, and discovered that a RNP-6/RNA-binding protein 39 (RBM-39) spliceosome complex regulates longevity by modulating intron retention of the splicing target egl-8/PLCB4. Furthermore, we found that the RNP-6/EGL-8 axis acts mainly within the nervous system and downregulates mTORC1 signaling to confer longevity. In human cells, PUF60 knockdown leads to downregulation of mTORC1 signaling and re-localization of mTOR away from lysosomes, indicating its integral, evolutionarily conserved role in this pathway. Together, our results reveal a new mechanism by which the spliceosome complex regulates lifespan in a multicellular organism, partially through intron retention, and pinpoints potential targets to promote healthy aging.In the present study, to decipher the molecular and cellular mechanisms underlying rnp-6(G281D) in more detail and found that this mutation behaved as a recessive, hypomorphic allele, because: (1) rnp-6(G281D)/+ heterozygotes were as cold sensitive as wild-type (wt or WT) controls  enhanced cold tolerance in the wild-type background and caused developmental arrest in rnp-6(G281D) mutants  but not a rnp-6(G281D) transgene fully rescued the rnp-6(G281D) cold tolerance phenotype  transgene also fully rescued the longevity phenotype  using clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9. Consistent with its role as an essential splicing factor, rnp-6(wt) was ubiquitously expressed in all examined tissues and mainly localized in the nucleus  showed a similar expression pattern, but was present at significantly lower levels  caused changes in mRNA processing and transcription similar to, but not as extensive as, rnp-6i, including alternative splicing, intron retention and circular RNA formation  represents a reduction-of-function mutation. Notably, ~80% of the differentially expressed genes (DEGs)  and 3,730 of 4,707 genes in rnp-6i) were upregulated (Supplementary Table rnp-6(G281D) and rnp-6i  longevity. To bypass developmental defects, we performed rnp-6i during adulthood. Whereas rnp-6i from day 1 adults (coinciding with the onset of reproduction) decreased the lifespan  onward significantly extended it , suggesting that lifespan extension is independent of progeny production  exhibited a tight temperature-sensitive (ts) growth phenotype, which could be fully rescued by rnp-6(wt) overexpression  phenotypes. We screened ~20,000 genomes and isolated 13 mutants  and rbm-39(dh1183)  intragenic mutation led to a glutamate-to-lysine substitution (rnp-6(E161K)), which corresponds to Glu188 in human PUF60  domain, but additionally harbors an amino-terminal arginine–serine-rich (RS) domain  mutation caused a serine-to-leucine substitution (Ser294Leu) in the second RRM  Fig. . The rnpain Fig.  implicatRRM Fig. . This reRRM Fig. . It is iRbm-39(S294L) represents a semidominant allele because rbm-39(S294L)/+ heterozygotes partially suppressed the rnp-6(G281D) ts phenotype  ts phenotypes. Unlike rbm-39(S294L), rbm-39 RNA interference (RNAi) knockdown (rbm-39i) generally exacerbated rnp-6(G281D) phenotypes and caused a further decrease in body size, yet had little effect on wild-type controls 20 further delayed developmental rate, decreased body size at 20 °C and caused complete embryonic lethality at 25 °C  suppressor probably defines a specific change- or gain-of-function allele.rbm-39(S294L) largely reversed the ts defect  phenotypes visible at the permissive temperature (20 °C). Although showing little effect on its own, rbm-39(S294L) significantly restored body size  lifespan extension  ameliorates rnp-6(G281D) function. Rbm-39(S294L) did not affect rnp-6(G281D) cold tolerance -mediated suppression, we first examined whether rbm-39(S294L) restored reduced rnp-6(G281D) protein levels. Western blotting experiments showed, however, that rbm-39(S294L) had no impact on either rbm-39 or rnp-6 protein levels  altered the subcellular localization of RNP-6 or RBM-39. Endogenously mKate2-tagged RBM-39(WT) and RBM-39(Ser294Leu) were ubiquitously expressed and found mainly within the nucleus of various cell types, similar to RNP-6  might alleviate rnp-6(G281D) defects by enhancing splicing activity. To test this hypothesis, we performed RNA-seq analysis with wt, rnp-6(G281D), rbm-39(S294L) and rnp-6;rbm-39 double mutants. In accord with our idea, we observed that rbm-39(S294L) altered the transcriptional profile  mutant  significantly suppressed intron retention (115 out of 954 events) , and globally trended toward alleviating many such events. GO enrichment analysis showed that the rnp-6-dependent DEGs suppressed by rbm-39(S294L) were significantly enriched in the stress response category  ameliorates rnp-6(G281D) splicing changes.To address the potential mechanisms behind P-6 Fig. . It is iP-6 Fig. . These p rnp-6G28D proteints) Fig. , alternarnp-6(E161K) intragenic mutation was also a potent suppressor of rnp-6(G281D), which fully restored all measured phenotypes to wild-type levels  and restored by rbm-39(S294L). We narrowed down the list of candidates to 44 events by cross-referencing with the rnp-6(E161K) revertant and manual curation in the genome browser (Supplementary Table rnp-6(G281D). We performed RNAi knockdown to assess the impact on wild-type lifespan, reasoning that both RNAi and intron retention should result in partial loss of function. Of those genes tested, we found one candidate, egl-8, with a knockdown that yielded significant lifespan extension in wild-type, but did not further extend rnp-6(G281D) longevity  null allele  cold tolerance  altered the retention of several introns within egl-8  and rnp-6(E161K) suppressed intron 8 retention  compared with that in wild-type controls, and rbm-39(S294L) efficiently restored EGL-8 expression levels back to wild-type  and egl-8i  longevity  caused more pronounced changes in neuronal egl-8 intron retention and rbm-39(S294L) completely restored the defects  promotes longevity via intron retention of egl-8 within the nervous system. In addition, intestinal expression of rnp-6 also partially rescued the rnp-6(G281D) lifespan  in disrupting splicing, they nevertheless caused a modest but significant increase in intron 8 retention   and mTORC1 inhibition . We found that raga-1 but not daf-2 longevity was nonadditive with rnp-6  might work in the same pathway as mTORC1. In accord with this view, the raga-1 gain-of-function transgene, which shortens the wild-type worm lifespan33, completely abolished rnp-6(G281D) longevity  longevity  longevity was also nonadditive with dietary restriction . To obtain further evidence, we tested whether molecular outputs of mTORC1 signaling were also altered in the rnp-6(G281D) mutant. Downregulation of mTORC1 signaling results in enhanced nuclear accumulation of HLH-30 (helix–loop–helix protein 30)/TFEB (transcription factor EB)38 and increased levels of phosphorylated AAK-2/AMPK (AMP-activated protein kinase)39. Consistently, we observed a significant increase in both HLH-30 nuclear localization  mutants. Altogether, our results indicate that rnp-6(G281D) inhibits mTORC1 signaling activity through raga-1.To identify potential signaling pathways in which p-6 Fig. , suggestity Fig. , suggestion Fig.  and AAK-ion Fig.  in rnp-6rnp-6, we wondered whether it also interacts with the mTORC1 signaling pathway in regulating longevity. Similar to rnp-6(G281D), egl-8(n488) loss-of-function mutation significantly inhibited mTORC1 activity as indicated by increased HLH-30 nuclear localization . As rnp-6 regulates mTORC1 upstream of raga-1  RNA in HEK293FT cells, which show several characteristics that are normally observed in immature neuronsa-1 Fig. , we surma-1 Fig. , withoutE3) Fig. . As aminE3) Fig. . These f43, the underlying molecular mechanisms remain largely unknown. Our studies provide direct evidence that a genetic mutation within a core spliceosome component promotes longevity partially through intron retention in C. elegans, highlighting such events in the fidelity of information processing and stress response.Messenger RNA splicing is a fundamental cellular process which has recently emerged as important to organismal aging. Although specific splicing factors and splicing events have been shown to be associated with the aging processRnp-6 encodes an essential splicing factor and the null mutation causes lethality in C. elegans. However, rnp-6(G281D) shows no overt defects under standard growth conditions at 20 °C. Our RNAi, rescue and RNA-seq experiments indicate that rnp-6(G281D) is a unique change or selective loss-of-function allele, which is similar to, yet distinct from, canonical functional depletion by knockdown. Notably, rnp-6(G281D) mutants are long-lived. In comparison, rnp-6i during development and from D1 of adulthood onward impair essential functions and shorten lifespan, whereas rnp-6i from D4 of adulthood promotes longevity. In contrast to rnp-6 RNAi, mTOR RNAi extends life from D1 onward44. These findings imply that the activities of splicing factors are fine-tuned, and suggest an antagonistic pleiotropic trade-off that is beneficial early on but detrimental later in life, similar to other essential genes of autophagy15 and de-ubiquitination45 in C. elegans. Whether rnp-6(G281D) and D4 on rnp-6i work by the same precise mechanism is currently unknown.1. From our study, we identified a lesion in splicing factor RBM-39(Ser294Leu), which forms nuclear speckles, alleviates RNP-6(Gly281Asp) defects and reverses longevity phenotypes, giving us mechanistic insight into critical targets of splicing. It is interesting that in mammalian cells RBM39, PUF60 and the large subunit of the U2AF complex, U2AF65, share a similar domain architecture and work in close proximity to regulate 3′-splice site assembly. Notably, all three proteins interact with the U2 snRNP subunit, SF3b155, through their UHM domains, and U2AF65 and RBM39 have been shown to do so cooperatively46. Conceivably, RBM-39(Ser294Leu) ameliorates RNP-6(Gly281Asp) by altering interactions with other splicing factors within nuclear speckles. Whether RBM39 and PUF60 bind to SF3b155 in a cooperative or competitive manner remains to be seen, but their close proximity suggests possible modes of functional interaction and highlights the importance of this specific complex in stress signaling and lifespan control.Splicing factors form extraordinarily large and highly dynamic macromolecular assemblies to catalyze splicing. Similar to transcription factors, there are both positive and inhibitory regulators of this process23 and has recently been shown as a common feature in aging-related splicing changes25. Intron retention is also associated with longevity interventions, such as diet restriction, in both C. elegans and mice4. Several lines of functional evidence provide direct causal evidence that egl-8 intron retention in neurons contributes to organismal longevity. First, rnp-6(G281D) mutation causes egl-8 intron retention, reduced neural EGL-8 protein levels and longevity, in a manner restored by rbm-39(S294L). Furthermore, rnp-6(G281D) induces a level of egl-8 downregulation comparable to that of egl-8 RNAi, which similarly extends life. Second, cis-acting mutations that weaken the egl-8 intron 8 3′-splice site are sufficient to promote both intron retention and longevity, despite a smaller magnitude effect to rnp-6(G281D) itself. Third, neuronal expression of full-length egl-8 cDNA is sufficient to suppress rnp-6(G281D) longevity, although gut-specific egl-8 expression reportedly has little impact on lifespan30. By contrast, expression of rnp-6 in gut can rescue lifespan  longevity as well. In sum, we conclude that rnp-6(G281D) longevity arises at least in part from egl-8 intron retention, although other activities probably contribute.Intron retention is a major mechanism of gene expression regulationrnp-6(G281D) . In contrast to RNA-seq, the RT–PCR experiments showed greater levels of intron retention , rbm-39(syb1074), rnp-6(dh1127);rbm-39(syb1074), rbm-39(gk454899), rnp-6(dh1127);rbm-39(gk454899), egl-8(syb3661), rnp-6(dh1127);egl-8(syb3661), egl-8(syb4850), raga-1(ok701),rnp-6(dh1127);raga-1(ok701), egIs12, (rnp-6(dh1127), egIs12), daf-2(e1370), rnp-6(dh1127);daf-2(e1370), daf-16(mu86), rnp-6(dh1127);daf-16(mu86), , (rnp-6(dh1127), dhEx1132), , (rnp-6(dh1127), dhEx1139),  cDNA::unc-54 3′-UTR, myo-3::mCherry], (rnp-6(dh1127)), dhEx1147, , myo-3p::mCherry]), (rnp-6(dh1127), dhEx1159), , (rnp-6(dh1127) and dhEx1208). Detailed information for strains used in the present study can be found in Supplementary Table 59. For all experiments, synchronization of the animals was done through the egg laying.The following strains were used in the present study: N2 (wild-type), 2. Human female embryonic kidney HEK293FT  cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium , containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were purchased from Invitrogen before the start of the project. Their identity was validated by the Multiplex human Cell Line Authentication test (Multiplexion GmbH), which uses an SNP typing approach and was performed as described at www.multiplexion.de. All cell lines were regularly tested for Mycoplasma contamination using a PCR-based approach and were confirmed to be Mycoplasma free. Amino acid (AA) starvation/re-addition experiments were performed as described previously60. In brief, customized starvation media were formulated according to the Gibco recipe for high-glucose DMEM, specifically omitting the amino acids. The media were filtered through a 0.22-μm filter device and tested for proper pH and osmolality before use. For the respective AA-replete treatment medium, commercially available high-glucose DMEM was used . All treatment media were supplemented with 10% dialyzed FBS. For this purpose, FBS was dialyzed against 1× PBS through 3,500 molecular mass cutoff dialysis tubing. For AA starvation (–AA), culture media were replaced with starvation media for 1 h. For AA add-back experiments (+AA), cells were first starved as described above and then starvation media were replaced with +AA treatment media for 10 or 30 min.All cell lines were grown at 37 °C, 5% COrnp-6 rescue plasmid, rnp-6 promoter  was amplified from the N2 genome and inserted into a pDC4 vector-generated rnp-6p::FLAG::HA::GFP::::unc-54 3′-UTR construct. Then, rnp-6b cDNA was amplified from N2 cDNA and cloned into this plasmid to generate a rnp-6p::FLAG::HA::GFP::rnp-6b cDNA::unc-54 3′-UTR rescue plasmid. Site-directed mutagenesis ) was performed to incorporate Gly281Asp point mutation to generate rnp-6p::FLAG::HA::GFP::rnp-6b(G281D) cDNA::unc-54 3′-UTR plasmid. To generate neuronal rescue plasmid, rnp-6 promoter was replaced by neuronal-specific promoter rgef-1 . The unc-17p::gfp::egl-8 plasmid is a kind gift from S. Nurrish . The microinjection experiments were performed according to a standard protocol61: 10 ng μl−1 of plasmid of interest together with a 5-ng μl−1 of co-injection marker plasmid (myo-3p::mCherry) were injected into the gonads of young adult-stage worms. Positive offspring were singled to maintain stable lines. PCR primers related to these plasmids are available in Supplementary Table For rnp-6(G281D) had been performed previously13. The heat-sensitivity suppressor screen was done on an rnp-6(G281D) mutant background. Briefly, ~1,000 L4 larvae worms (P0) were exposed to 0.15% ethyl methane sulfonate  in M9 buffer for 4 h at room temperature. After overnight recovery, young adult P0 animals were transferred to new plates for egg laying at 20 °C. After 3 d of growing, adult F1 worms were bleached and eggs were seeded on nematode growth medium (NGM) plates and incubated at 25 °C. After 3 d, F2 worms that reached adulthood were singled and maintained at 20 °C. False-positive hits were excluded by re-testing heat sensitivity in F4/F5 generations. The rnp-6(G281D) animals were used as a negative control in all heat-sensitivity assays. Hawaiian SNP mapping and whole-genome sequence were used to map the causative mutation62. The rnp-6(G281D) mutation was first introduced to Hawaiian CB4856 by 6× outcrossing. Then, the EMS mutants were crossed with Hawaiian males that carry the rnp-6(G281D) mutation. Eggs of the F1 generation worms were grown at 25 °C and adult F2 were singled after 3 d. The heat-resistant strains were then pooled together, and genomic DNA was purified using Gentra Puregene Kit (QIAGEN). The pooled DNA was sequenced on an Illumina HiSeq platform (paired-end 150 nt). An MiModD pipeline (https://celegans.biologie.uni-freiburg.de/?page_id=917) was used to identify the mutations. The WS220/ce10 C. elegans assembly was used as reference genome for annotation. The causative mutations were confirmed by either CRISPR–Cas9 or multiple outcross. Dh1183 and dh1187 were identified as rbm-39(S294L) and rnp-6(E161K), respectively. The causative genes for the other 11 mutants remain unclear.The cold resistance-longevity screen that identified For cold tolerance assays, worms were synchronized and grown on standard NGM plates. When the worms reached the young adult stage, they were transferred to a 2 °C incubator for 24 h. The worms were recovered at room temperature for 4 h and the number of alive and dead worms were scored. The cold survival ratio was measured as the ratio of the number of live worms to the number of total worms. At least 60 worms from each genotype were used in the assay for each biological replicate. Three independent repeats were performed. For the heat-sensitivity assay, synchronized eggs from different genotypes were grown on standard NGM plates at 20 °C or 25 °C. After 3 d, images of the worms were captured. Body length or body area was measured by ImageJ software. At least 15 worms from each genotype were used for each biological replicate. Three independent repeats were performed except in Extended Data Fig. https://wormbase.org). A T-Coffee algorithm63 was used to align RNP-6, RBM-39 and their homologs from different species. Phylogenetic analysis of RBM-39 and its homologs was done with Phylogeny.fr64. Protein sequences of Homo sapiens RBM39 (UniProt: Q14498), C. elegans RBM-39 (UniProt: Q9N368) and Drosophila melanogaster Caper (UniProt: Q9VM49) were used in Fig. H. sapiens PUF60 (UniProt: Q9UHX1), C. elegans RNP-6 (UniProt: Q9N3S4) and D. melanogaster Hfp (UniProt: Q8T6B9) were used in Supplementary Fig. C. brenneri RBM-39 (UniProt: G0NLU2), C. elegans RBM-39 (UniProt: Q9N368), Strongyloides ratti SRAE (UniProt: A0A090LFF6), C. briggsae RBM-39 (UniProt: A8XIX5), C. japonica RBM-39 (UniProt: A0A2H2IF69), C. remanei RBM-39 (UniProt: E3MXT8), Brugia malayi RBM-39 (UniProt: A0A4E9ESP8), Trichus muris RBM39 (UniProt: A0A5S6R6A6), Xenopus tropicalis RBM39 (UniProt: Q566M5), Rattus norvegicus RBM39 (UniProt: Q5BJP4), Pan troglodytes RBM39 (UniProt: A0A2I3RX33), Schizosaccharomyces pombe Rsd1 (UniProt: O13845), Mus musculus RBM39 (UniProt: Q8VH51), H. sapiens RBM39 (UniProt: Q14498), Gallus gallus RBM-39 (UniProt: E1BRU3), D. melanogaster Caper (UniProt: Q9VM49), Bos taurus RBM39 (UniProt: A0A3Q1LWZ4), Anophelesgambiae RBM39 (UniProt: Q7PN29), Danio rerio RBM39 (UniProt: Q58ER0), Canis lupus RBM39 (UniProt: E2R4L0) and Pristionchus pacificus RBM39 (UniProt: H3FJ10) were used in Extended Data Fig. 16.Homologs of RNP-6 and RBM-39 were identified from Wormbase . Single guide (sg)RNAs were synthesized with EnGen sgRNA Synthesis Kit  by following the manufacturer’s protocol. CRISPR–Cas9 insertion was generated by following a co-CRISPR strategy65. Dpy-10 was used as a marker to enrich potential hits. RNP complexes containing sgRNA, Cas9 and repair templates were annealed at 37 °C for 15 min before injection. The primers used in the present study are listed in Supplementary Table rnp-6(syb645), rnp-6(syb626)), mKate2-tagged RBM-39 strains (rbm-39(syb1527), rbm-39(syb1545)), mNeonGreen-tagged EGL-8 strain (egl-8(syb3661)), rbm-39(S294L) mutant strain (rbm-39(syb1074)) and egl-8 intron 8 3′-splicing site-edited strain (egl-8(syb4850)) were generated by Sunybiotech (https://www.sunybiotech.com). All the strains were validated by Sanger sequencing.To generate hemagglutinin (HA)-tagged egl-8(n488), a final concentration of 50 µM FUDR was added to NGM plates. Survival was monitored every other day. Worms that did not respond to gentle touch by a worm pick were scored as dead and were removed from the plates. Animals that crawled off the plate or had ruptured vulva phenotypes were censored. All lifespan experiments were blinded and performed at least 3× independently unless otherwise noted. Graphpad Prism (9.0.0) was used to plot survival curves. Survival curves were compared and P values were calculated using the log(rank) (Mantel–Cox) analysis method. Complete lifespan data are available in Supplementary Table All lifespans were performed at 20 °C unless otherwise noted. Worms were allowed to grow to the young adult stage on standard NGM plates with OP50. For each genotype, ~150 young adults were transferred to NGM plates with OP50 supplemented with 10 µM FUDR. For lifespan experiments with Staphylococcus aureus (strain MW2) was grown in Tryptic Soy Broth medium at 37 °C with gentle shaking overnight. Then, 100 μl of the bacterial culture was seeded and spread all over the surface of the trypticase soy agar plate with 10 μg ml−1 of nalidixic acid. The plates were allowed to grow overnight at 37 °C. On the next day, the plates were left at room temperature for at least 6 h before the infection experiments. Around 25 synchronized young adult worms were transferred to the plates. Three technical replicate plates were set up for each condition. Worms were treated with 100 μM FUDR from L4 stage to prevent internal hatching during experiments. The plates were then incubated at 25 °C to initiate the infection experiment. Scoring was performed every day. Worms were scored as dead if the animals did not respond to gentle touch by a worm pick. Worms that crawled off the plates or had ruptured vulva phenotypes were censored from the analysis. All C. elegans killing assays were performed 3× independently unless otherwise stated. At least 60 worms per genotype were included at the start of the assay for each replicate. Genotypes were blinded for all C. elegans infection survival experiments to eliminate any investigator-induced bias. Results of each biological replicate of infection survival experiments can be found in Supplementary Table 13. E. coli HT115 and E. coli OP50(xu363) bacterial strains were used in the present study. The HT115 bacteria were from the Vidal or Ahringer library. The OP50(xu363) competent bacteria were transformed with double-stranded RNA expression plasmids, which were extracted from the respective HT115 bacterial strains. The RNAi bacteria were grown in lysogeny broth medium supplemented with 100 µg ml−1 of ampicillin at 37 °C overnight with gentle shaking. The culture was spread on RNAi plates, which are NGM plates containing 100 µg ml−1 of ampicillin and 0.4 mM isopropyl β-d-1-thiogalactopyranoside. RNAi-expressing bacteria were allowed to grow on the plates at room temperature for 2 d. RNAi was initiated by letting the animals feed on the desired RNAi bacteria. RNAi experiments related to Fig. OP50(xu363) bacteria. For RNAi lifespan experiments related to Fig. RNAi experiments were performed as previously describedC. elegans was lysed with QIAzol Lysis Reagent. RNA was extracted using chloroform extraction. The samples were then purified using RNeasy Mini Kit (QIAGEN). Purity and concentration of the RNA samples were assessed using a NanoDrop 2000c (peqLab). The cDNA synthesis was performed using an iScript cDNA synthesis kit (BioRad). Standard manufacturers’ protocols were followed for all mentioned commercial kits.66). DEGs between different samples were identified using Stringtie (v.1.3.0)67, followed by Cufflinks (v.2.2)68. The enrichment visualization was performed with WormCat 2.0 (ref. 69). P values were calculated from Fisher’s exact tests and adjusted with Bonferroni’s multiple hypothesis test. P < 0.05 was defined as significant. An SAJR pipeline70 was used for splicing analysis. Significant splicing changes were defined as those with P < 0.05 after adjusting for multiple testing using the Benjamini–Hochberg correction. For intron retention analysis, Bedtools coverage (v.2.29.0) was used to count intron and total gene expression. IBB 71 was used to calculate differential intron expression. DCC/CircTest (v.0.1.0) pipeline72 was performed to quantify circular RNA expression. Weblogo73 was used to generate intron 3′-splice site consensus sequence logos , libraries were pooled for quantification using the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems). The libraries were then sequenced with the Illumina HiSeq4000 sequencing system using paired-end 2× 100-bp sequencing protocol. For data analysis, the Wormbase genome (WBcel235_89) was used for alignment of the reads. This was performed with Hisat v.2.0.4 ) or visualized with ChemiDoc Imager  after staining with Roti-GelStain (Carl Roth). Sequences of the primers used in the RT–PCR assays are available in Supplementary Table Phusion Polymerase (Thermo Fisher Scientific) was used to amplify the C. elegans samples, animals were first washed with M9 buffer. Worm pellets were resuspended in radioimmunoprecipitation assay (RIPA) buffer supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) and snap-frozen in liquid nitrogen. Thawed samples were lysed using the Bioruptor Sonication System (Diagenode). Protein samples were then heated to 95 °C for 10 min in Laemmli buffer with 0.8% 2-mercaptoethanol to denature proteins. Samples were loaded on 4–15% Mini PROTEAN TGXTM Precast Protein Gels (BioRad) and electrophoresis was performed at a constant voltage of 200 V for around 40 min. After separation, the proteins were transferred to poly(vinylidene fluoride) membranes using Trans-Blot TurboTM Transfer System (BioRad). Then, 5% bovine serum albumin (BSA) or 5% milk in Tris-buffered saline and Tween-20 (TBST) was used for blocking of the membranes. After antibody incubations  1:2,000; anti-red fluorescent protein 1:2,000; anti-beta actin, 1:5,000; anti-FLAG, 1:2,000; anti-mouse horseradish peroxidase (HRP), 1:5,000; anti-rabbit HRP, 1:5,000; and anti-rat HRP, 1:5,000) and washing with TBST buffer, imaging of the membranes was performed with ChemiDoc Imager . Western Lightning Plus Enhanced Chemiluminescence Substrate (PerkinElmer) was used as the chemiluminescence reagent. For western blotting analyses using HEK293FT samples, cells were washed once in-well with serum-free DMEM  to remove FBS, and lysed in 250 µl of lysis buffer  for 10 min on ice. Samples were clarified by centrifugation  and supernatants were transferred to new tubes. The protein concentration was measured using a Protein Assay Dye Reagent . The protein samples were subjected to electrophoretic separation on sodium dodecylsulfate–polyacrylamide gel electrophoresis and analyzed by standard western blotting techniques. In brief, proteins were transferred to nitrocellulose membranes , stained with 0.2% Ponceau solution  to confirm equal loading. Membranes were blocked with 5% powdered milk in PBS-T  for 1 h at room temperature, washed 3× for 10 min with PBS-T and incubated with primary antibodies  rotating overnight at 4 °C. The next day, membranes were washed 3× for 10 min with PBS-T and incubated with an HRP-conjugated anti-rabbit secondary antibody  for 1 h at room temperature. Signals were detected by enhanced chemiluminescence (ECL), using the ECL Western Blotting Substrate  or SuperSignal West Femto Substrate  for weaker signals. Western blot images were captured on film . A list of antibodies is provided in Supplementary Table For 74. A solubilization buffer containing 0.5% NP40, 150 mM NaCl and 50 mM Tris, pH 7.4 supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) was used for immunoprecipitation. Flag immunoprecipitation was performed using Dynabeads Protein G (Thermo Fisher Scientific) and FLAG M2 mouse monoclonal antibody (Sigma-Aldrich), following the manufacturers’ protocols. Proteins were eluted from the beads by boiling with Laemmli buffer.Worms expressing HA::RNP-6, RBM-39::mKate2 or both were harvested and proteins were extracted using the following standard protocol75 was used for quantifying fluorescence intensity. For mNeonGreen::EGL-8 images, the head neuron region was selected for quantification. For HLH-30::mNeonGreen images, the nuclei of hypodermal cells were selected for quantification. For GFP::RNP-6 images, the whole worm was selected for quantification. To reduce bias, individual worms were randomly picked under a dissection microscope and imaged. At least 20 worms per genotype were picked for imaging and all the experiments were done 3× independently. Data from a representative experiment are shown in the figures for all the imaging panels.Analysis of worm reporters GFP::RNP-6, RBM-39::mKate2, mNeonGreen::EGL-8 and HLH-30::mNeonGreen was performed on a Zeiss Axioplan2 microscope  with a Zeiss AxioCam 506 CCD camera. Analysis of worm size was performed on a Leica stereo microscope  with Leica DFC3000G CCD. Fiji software (v.2.0.0/1.52p)Rotylenchulusreniformis luciferase gene (Horizon Discoveries) was used as control. In brief, HEK293FT cells were seeded in 12-well plates at 20% confluence and the following day transfected with 20 nM of the siRNA pool using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were harvested or fixed 72 h post-transfection and knockdown efficiency was verified by western blotting.Transient knockdowns were performed using a pool of four small interfering GENOME siRNAs (Horizon Discoveries) against PUF60, whereas an RLuc duplex siRNA that targets the 60. In brief, cells were seeded on fibronectin-coated coverslips and treated as indicated in each experiment. After treatment, cells were fixed for 10 min at room temperature with 4% paraformaldehyde in PBS. Samples were washed/permeabilized with PBT solution (1× PBS-T), and blocked with BBT solution . Staining was performed with the indicated primary antibodies in BBT (1:200 dilution) for 2 h at room temperature for mTOR and LAMP2 staining or overnight at 4 °C for TFE3 staining. Next, samples were washed 4× with BBT (15 min each), followed by incubation with appropriate highly cross-adsorbed, secondary fluorescent antibodies (1:200 in BBT) for 1 h at room temperature , using automatic Costes’ thresholding77. The MCC yields the fraction of the mTOR signal that overlaps with the LAMP2 signal. Subcellular localization of TFE3 was analyzed by scoring cells based on the signal distribution of TFE3, as shown in the example images in Fig. Ιmmunofluorescence/confocal microscopy experiments and quantification of colocalization were performed as previously describedn’ denotes the number of independent experiments performed, whereas ‘N’ indicates the total number of animals analyzed in each condition. All statistical analyses were performed in GraphPad Prism (v.9.0.0 (86)). Asterisks denote corresponding statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data distribution was assumed to be normal but this was not formally tested. No statistical method was used to predetermine sample size but our sample sizes are similar to those reported in our previous publications79. No data were excluded from the analyses. At least three independent experiments for each assay were performed to verify the reproducibility of the findings . For the worm experiments, sample preparations and data collection were randomized. For lifespan experiments, all the genotypes and RNAi treatments were blinded. For cold tolerance, developmental rate, body area, infection, western blotting and imaging experiments, the genotypes were not blinded before assay because mutant worms have obvious phenotypes that revealed the sample identity . However, worms were randomly picked and assigned to the different treatment conditions and the different conditions were assessed in random order. For RNA-seq experiments, the genotypes were not blinded before collecting samples. Once the RNA samples were ready, they were processed by staff at the Cologne Centre for Genomics in a blinded manner. For mammalian cell studies, no blinding was included in the data collection or analysis, because the method of quantification over multiple replicates and individual cells (for microscopy experiments) ensures unbiased processing.In all figure legends, ‘Further information on research design is available in the Supplementary InformationLegends of Supplementary Tables and Video.Reporting SummarySupplementary Table 1Supplementary table.Supplementary Video 1Supplementary video.


Anti-CRISPR proteins are very efficient for inhibiting CRISPR/Cas9-based genome editing tools in both herbaceous and woody plant species. Dear Editor,Saccharomyces cerevisiae), including AcrIIA4 (inhibits SpCas9), AcrVA1 (inhibits Cas12a), and AcrIIA5  (Nicotiana benthamiana) based on transient expression through leaf infiltration and viral delivery  and N. benthamiana, and the woody plant hybrid poplar “717” , using both leaf-infiltration and protoplast-based transient expression. The activity of AcrIIA4 on ABE7 was further investigated in Arabidopsis via Agrobacterium tumefaciens-mediated stable genetic transformation.Clustered regularly interspaced short palindromic repeats/CRISPR associated protein (CRISPR/Cas) systems have revolutionized genome engineering in plants  via specthologs) . Acr prothologs) . For exathologs) . Also, cthologs) . Howeverdelivery . AcrIIA5N. benthamiana, A. thaliana, and hybrid poplar “717” using a 35S promoter and codon optimized AcrIIA4 or AcrIIA5  and AGAMOUS (AG) (Supplemental Table S1;N. benthamiana. These results are consistent with a previous report in mammalian cells (To further evaluate the inhibition activity of Acrs to SpCas9/sgRNA directed target mutagenesis in plants, we tested the ability of AcrIIA4 and AcrIIA5 to prevent SpCas9-induced mutagenesis in an cells , suggestAgrobacterium-mediated co-transformation of the ABE7 and AcrIIA4-BS2 constructs (N. benthamiana (To test the Acrs activity following stable genetic transformation, the transcriptional units of AcrIIA4-BS2 and ABE7 were integrated into the Arabidopsis genome together via 2 independent constructs . The expnstructs . Among tnstructs . All transtructs . These rthamiana , and thuIn summary, we demonstrate that Acr proteins are effective in inhibiting CRISPR/Cas9-based genome editing tools in both herbaceous and woody plant species. As such, previously described biosensors for CRISPR-based tools  proved ukiad076_Supplementary_DataClick here for additional data file.


Cytokines act as potent, extracellular signals of thehuman immunesystem and can elicit striking treatment responses in patients withautoimmune disease, tissue damage, and cancer. Yet, despite theirtherapeutic potential, recombinant cytokine-mediated immune responsesremain difficult to control as their administration is often systemic,whereas their intended sites of action are localized. To address thechallenge of spatially and temporally constraining cytokine signals,we recently devised a strategy whereby recombinant cytokines are reversiblyinactivated via chemical modification with photo-labile polymers thatrespond to visible LED light. Extending this approach to enable bothin vivo and multicolor immune activation, here we describe a strategywhereby cytokines appended with heptamethine cyanine-polyethyleneglycol are selectively re-activated ex vivo using tissue-penetratingnear-infrared (NIR) light. We show that NIR LED light illuminationof caged, pro-inflammatory cytokines restores cognate receptor signalingand potentiates the activity of T cell-engager cancer immunotherapiesex vivo. Using combinations of visible- and NIR-responsive cytokines,we further demonstrate multiwavelength optical control of T cell cytolysisex vivo, as well as the ability to perform Boolean logic using multicoloredlight and orthogonally photocaged cytokine pairs as inputs and T cellactivity as outputs. Together, this work demonstrates a novel approachto control extracellular immune cell signals using light, a strategythat in the future may improve our understanding of and ability totreat cancer and other diseases. In contrast to visible light, near-infrared (NIR) photonscan more efficiently penetrate human tissues including bone, skin,and muscle up to several centimeters,19 and diagnostic methods relying on NIR light sources have demonstratedclinical utility in optical coherence tomography and NIR spectroscopyapplications.To address the challenge of improving the cell- and tissue-specificityof recombinant cytokines, we recently developed a strategy wherebycytokine activity  can be blunted viaconjugation with photo-labile polyethylene glycol (PEG) and recoveredvia brief, in this case blue, LED light exposure.18 to reversibly photocage immunostimulatory cytokines byusing photo-labile PEG that de-shields in response to NIR rather thanvisible light. We show that LED light exposure of NIR-photocaged cytokinescan be used to reversibly modulate cognate cytokine receptor signalingand enhance the activity of bispecific cancer immunotherapies thatrely on T cell-dependent cytolysis. Using combinations of visibleand NIR light-responsive cytokines, we further demonstrate orthogonal,multicolor photocontrol of T cell cytolytic activity ex vivo and thenovel ability to perform Boolean logic using multicolor light anddifferentially photocaged cytokines. This work expands the workingtoolbox for protein photocaging and may be extended in the futureto a range of other biological systems that rely on extracellularsignaling or synthetic cytokine/receptor pairs.Leveraging the ability of NIR photons to efficientlypenetratehuman tissues, here we advance our prior approachUnless otherwise specified,reagents were used as received without further purification. Recombinanthuman IL-2 , recombinant human IL-15 ,recombinant mouse scIL-12 , and recombinanthuman IL-12 . Sulfo-Cyanine7 NHS ester(Lumiprobe), DBCO-NHS ester , NHS-PEG-TCOester , poly(ethylene glycol)methylether DBCO , and poly(ethyleneglycol)methyl ether azide . Polyacrylamide gels . IFNγ ELISA . CD19 antibody(BD Biosciences Clone SJ25C1) and PD-L1 antibody (eBioscience CloneMIH1).HEK-Blue IL-12 cells (Invivogen)were cultured according to the manufacturer’s recommendationsin Dulbecco’s modified Eagle’s medium (Corning) supplementedwith 10% heat-inactivated fetal bovine serum (FBS) (VWR) and 100 IU/mLpen-strep (VWR). NALM-6 cells  were cultured in RPMI 1640 supplemented with 10% FBSand 100 U/mL penicillin and 100 μg/mL streptomycin. PrimaryCD8+ T cells were obtained from normal donor human buffy coats (LifeSouth)via Ficoll-Paque (Cytiva) gradient selection for peripheral bloodmononuclear cells followed by human CD8+ T cell negative magneticselection (Stem Cell Technologies). Primary CD8+ T cells were thawedin supplemented RPMI 1640 2–12 h prior to the start of experiments.E)-2-((E)-2-phenoxy)carbonyl)(ethyl)amino)ethyl)(ethyl)amino)-5-(2-azidoethoxy)-3-((E)-2--1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-7-sulfo-3-(4-sulfobutyl)-1H-benzo[e]indol-3-ium, cCy, was preparedat-scale by eMolecules  from 1,4-dioxaspiro[4.5]decan-8-oneand purified to >96% via preparatory high-performance liquid chromatography.cCy was structurally characterized by 1H NMR, 13C NMR, and UPLC–MS using an Acquity BEH C-18 column (1.7 μm):buffer A, 5 mM ammonium acetate in water; buffer B, acetonitrile;gradient was 10–90% A over 6 min at a 0.3 mL/min flow rate.See Supplementary Information for additionaldetails.2-((2). Data were fitted using a one-phasedecay model in Graphpad Prism.cCy was reconstitutedto 50 mM in dry dimethyl sulfoxide (Thermo Fisher Scientific) andfurther diluted 1000× in 1× phosphate-buffered saline (PBS)(Corning) for characterization studies. Absorption and emission spectrawere measured on a Spectramax iD3 plate reader (Molecular Devices)at 10 nm increments. Photolysis kinetics were monitored using a NanoDropOne spectrophotometer following irradiation with a 785 nm diode laser. The irradiated slide was thenwashed 5× with TBST and mounted with Prolong Diamond antifademedia (Life Technologies) for imaging. Patterned slides were imagedon an Odyssey CLX fluorescence imaging scanner. Image features werequantified and normalized using ImageJ.1 mm silicone spacerswith 9 mm diameter circular openings (Electron Microscopy Sciences)were adhered to amine-coated glass slides (Nanocs). The slides werefunctionalized with NHS-TCO at a 10:1 NHS/amine ratio overnight atRT. Wells were then washed 6× with deionized water, 3× with0.2% sodium dodecyl sulfate (SDS), and again 3× with deionizedwater, followed by conjugation with cCy at a 3:1 cCy/amine ratio overnightat RT in tris buffered saline with 0.1% Tween (TBST). Wells were thenwashed 5× with TBST (0.1% Tween 20) and covered in 50% glycerol.A chrome-coated quartz photomask was then placed on the silicone isolator,and a collimated 730 nm LED (ThorLabs) was used to irradiate the slidefor 30 min . Excess reactants wereremoved via 7 kDa size exclusion chromatography  in some experiments. IL-15-cCy-PEG was preparedusing methods identical to IL-12-cCy-PEG except for the use of a 10–20-foldmolar excess of NHS-TCO, a 40–80-fold molar excess of cCy,and a 160–320-fold molar excess of DBCO-mPEG20kDa (see the Supplementary Information).Cytokines were modified withcCy-PEG using carbodiimide and TCO–tetrazine chemistry, respectively,using methods adapted from Perdue et al.18 Briefly, rIL-2 was diluted in 150 mM sodium phosphate buffer (pH8.5) containing 0.5 mM SDS and sequentially reacted with an 80-foldmolar excess of cNB, followed by N3-mPEG20kDa at a 400-fold molar excess in PBS .rIL-2 was modified with cNB-PEGusing carbodiimide and azide–alkyne chemistry, respectively,using methods described in Perdue et al.Protein electrophoreticmobility was characterized via polyacrylamide gel electrophoresis(4–16% weight) under reducing conditions. Protein bands werestained with Coomassie G250 (Bio-Rad) and visualized using a LicorCLx imager. Coomassie was imaged in the 700 channel of the imager,while cCy was imaged in the 800 channel.2, 30 min). cCy-PEG conjugates were diluted to 0.125–0.15μg/mL in HEPES-buffered saline  containing0.1% bovine serum albumin and 0.02% Tween 20 prior to irradiation.Irradiation of IL-2-cNB-PEG was similarly performed except for theuse of a 365 nm LED  and pre-dilutionto 0.15 μg/mL in PBS (pH 7.4). Illumination was performed inall cases at 4 °C.Irradiation of IL-12-cCy-PEGwas performed in transparent film-covered U-bottom 96-well platesfollowing illumination with a 730 nm LED ,and placed at 37 °C for 0.25–3.0 h before reading absorbanceat 620 nm (Spectramax iD3 plate reader). Dose–response curveswere calculated using Graphpad Prism with a four-parameter fit andnormalized to background and signal from unmodified proteins.HEK-293 SEAP reportercells (Invivogen) were plated at 5 × 104 NALM-6 cells were platedwith 0.5–1 ng/mL of blinatumomab (Invivogen) and cytokine,if applicable, in a U-bottom 96-well plate for 72 h . Cytokine concentrations were in the ranges of 5–1000IU/mL (IL-2) and 7–400 ng/mL (IL-12). After 72 h, cells werestained with the Near-IR Live/Dead stain (Thermo Fisher Scientific)for viability and immediately assessed by flow cytometry. Specificlysis was calculated as % Specific Lysis = 100 × [(Treated CTV+ LD+) – (Untreated CTV + LD+)]/(100 – Untreated CTV+ LD+), where LD is the viability stain, CTV is CellTrace Violet,and the untreated condition received no blinatumomab. The supernatantfrom these cultures was immediately frozen at −80 ° C.For IFNγ ELISA (Biolegend), co-culture supernatants were diluted10–100-fold and assayed according to the manufacturer’sinstructions. During logic gate experiments, a positive output wasbased upon inter-donor variability and defined as cytolysis within50 % CI of values obtained from combined treatment with rIL-12, rIL-2,and blinatumomab. To assess changes in the tumor cell phenotype, cellswere stained for PD-L1 and CD19 for 1 h at a 1:100 antibody dilution,washed, and immediately assessed by flow cytometry.CD19+ NALM-6 cells (B cellacute lymphoblastic leukemia) were stained with CellTrace Violet (ThermoFisher Scientific), and primary human CD8+ T cells were stained witheither CellTrace Yellow (Thermo Fisher Scientific) or CFSE (TonboBiosciences) for ≥1 h prior to co-culture. During co-culture,CD8+ T cells and 5 × 10p-values were calculated using either one-way analysis ofvariance with Tukey post hoc correction or one-way T test (Graphpad Prism), depending on the number of experimental conditions.Unless otherwise noted, all 20 We surmised (i) thatsubstitution of this leaving group with a phenolic tetrazine wouldenable the photo-induced loss of a variety of compounds appended via trans-cyclooctene (TCO) click chemistry and (ii) that theaddition of a distal azido ethoxy group to the backbone cyclohexenewould enable the linker to be protein-anchored via orthogonal azide–alkyneclick chemistry.23 As anticipated, the subsequent heterobifunctionallinker (hereafter cCy) exhibited strong NIR absorption and fluorescence. Notably,the spectral blue-shift that we observe here following photo-irradiationstrongly resembles that reported by Gorka et al., who found that heptamethinecyanine photolysis occurs initially via cleavage of the pi-conjugateddye backbone.20 We hypothesize that thephotolysis of cCy here is mechanistically similar. To characterizethe spatial resolution in which cCy can be photocleaved, we next immobilizedit to TCO-functionalized glass slides via click chemistry and exposedthese slides to collimated NIR LED light (730 nm) under aqueous conditionsthrough a custom lithographic mask , and, using reporter cells that secretealkaline phosphatase in a pSTAT4-dependent manner, we identified cCyconjugation densities that increase the apparent molecular weightof rIL-12 while preserving its signaling activity  consistent with prior findingsthat PEG can sterically shield receptor binding interactions betweenproteins and their cognate receptors in a PEG density- and molecularweight-dependent manner.18 To confirm thegeneralizability of this approach to photocaging, we demonstrateda comparable increase and recovery in rIL-15 electrophoretic mobilityfollowing conjugation with cCy-PEG and NIR light exposure . Together, these data demonstratethat cCy-PEG reversibly modulates the size and ex vivo activity ofrIL-12 on T cells in an NIR photon-dependent manner.Having demonstrated that cCy undergoes rapid andspatially welldefined photolysis, we next investigated whether the conjugation ofpro-inflammatory cytokines with cCy-PEG modulates their activity onT cells. Using carbodiimide and TCO–tetrazine chemistry, wemodified surface primary amines of recombinant IL-12 to yield proteinsconjugated with either cCy or cCy-PEG20kDa S4, and, 20kDa S4, and, 30 To determine whether IL-12-cCy-PEG can likewise improve blinatumomab-inducedcytolysis in an NIR light-dependent manner, we treated co-culturesof labeled primary human CD8+ T cells and CD19+ NALM-6 B-ALL cellswith blinatumomab and rIL-12 or IL-12-cCy-PEG, with and without NIRlight exposure, and measured CD19-specific cell lysis and T cell activationvia flow cytometry and IFNγ ELISA, respectively .ectively 3a–c. As 33 To determine whether cooperativitybetween IL-12 and IL-2 can further enhance blinatumomab activity exvivo, we co-cultured CFSE-labeled primary human CD8+ T cells and CellTraceViolet-labeled CD19+ NALM-6 B-ALL cells with blinatumomab in the presenceor absence of each recombinant cytokine or their combination . Interestingly, while rIL-12 and rIL-2combined only sub-additively to alter T cell proliferation, they synergizedto enhance IFNγ secretion as measured by dye dilution and ELISAof co-culture supernatants, respectively  IL-2 treatmentupregulates IL-12R on T cells, (ii) IL-12 treatment upregulates IL-2R(CD25) on CD8+ T cells, and (iii) the combination of both IL-12 andIL-2 synergizes to improve tumor control in syngenetc mouse modelsof renal cell carcinoma.bination 4a. We obbination 4b, despibination S6. Intero-nitrobenzyl-linked PEG20kDa .18 Usingthis conjugate and the NIR-responsive IL-12 conjugate described here(IL-12-cCy-PEG), we constructed a Boolean circuit that combines ANDgates in which we digitize the presence or absence of NIR or visibleLED light, as well as the presence or absence of discrete concentrationsof IL-12-cCy-PEG or IL-2-cNB-PEG (S7). Usingthese components as inputs and blinatumomab-induced cytolysis levelas a digitized output, we observed full concordance with the expectedAND–AND gate truth table whereby above-threshold B cell lysiswas observed only in the presence of both caged cytokines, as wellas both visible and NIR LED light  tophotocontrol T cell cytokine signaling via tissue penetrant NIR lightand (ii) to orthogonally modulate cooperative cytokine signaling viacombined visible and NIR LED light. Using a photo-labile, heterobifunctionallinker based upon heptamethine cyanine fluorophores (cCy), we demonstratedrapid NIR photolysis kinetics with a photooxidation half-life of ∼25min, notably fast as compared with proteolytic activation methodsthat can exceed several hours in activation half-life. Using thisnovel photo-labile linker, we further observed a minimum lateral resolutionfor NIR photoactivation of ∼51 μm and anticipate furtherenhancements in resolution using focusing optics and/or multiphotonlaser excitation  as is commonin high-resolution microscopy applications.30 we demonstrated that rIL-12 couldpotentiate the therapeutic effects of the T cell engager blinatumomab(Blincyto) ex vivo, and here we further demonstrate the NIR photocontrolof this enhanced activity via NIR illumination of cCy-PEG-conjugatedrIL-12. This finding is significant as conventional rIL-12 therapiesare well known to be associated with severe hematologic and hepatictoxicities that may potentially compound with those observed fromblinatumomab when delivered in combination. Using the approach describedhere, we observed a >98% reduction in signaling EC50 thatwe anticipate would mitigate such adverse effects in vivo via sustainedor localized  delivery via NIR activation.In prior work,36 At the same time, there are now(four) clinically approved T cell bispecific immunotherapies thatinclude both hematologic cancers and solid tumors.38 Thus, these findings may support the future clinical developmentof combined, or chimeric, T cell bispecific and cytokine cancer immunotherapiesin the future.The observation that rIL-2, like rIL-12, potentiates the therapeuticactivity of blinatumomab is also significant as this is the firstreport to our knowledge that supports this effect from IL-2, as wellas the first to demonstrate cooperativity between these two cytokinesin enhancing the cytolytic activity of T cell bispecifics. Like IL-12,there are numerous IL-2-based clinical-stage drug candidates includingprotein fusions, PEG–cytokine conjugates, and mRNA.Finally, to exploit cooperativity between IL-12and IL-2 in enhancingthe ex vivo activity of blinatumomab, we constructed a Boolean circuitin which only 1 of 16 possible initial states—comprising cagedIL-12, caged IL-2, blue light, or NIR light—would correspondto above-threshold cytolytic activity. In the future, this approachmay be used to orchestrate or tune complex cytokine signaling networksusing cocktails of caged cytokines and time- and/or wavelength-modulatedlight sources.39 and light-dependentprotein dimerization40 necessitate theuse of live cells whose presence may be limiting in some systemsor applications.The finding that soluble proteins such as cytokinesmay be manipulatedusing exogenous light is also significant considering that the overwhelmingmajority of optical tools available to synthetic biologists such asphoton-gated ion channels42 In prior work, the addition ofjust a single PEG molecule to GM-CSF or interferon alfa-2b was foundto be sufficient to confer prolonged circulation and tissue-drug exposure,which in turn improves the therapeutic index.43 Here, we likewise anticipate that prolonged circulation from IL-12-cCy-PEGrelative to rIL-12 may enable it to more effectively elicit antitumorimmune responses both alone and in combination with T cell bispecifics,immune checkpoint inhibitors, chimeric antigen receptor T cells, andautologous gamma delta T cell44 or NK cell46 therapies in the future where it may potentiate improved treatmentoutcomes.As discussed above, one salient barrier tothe clinical applicationof recombinant cytokine immunotherapies in cancer is their relativelysmall size  and therefore rapid excretionthat necessitates frequent and/or high-dose and complex treatmentmanagement.49 we anticipate further extensionsof this approach in the future that will expand wavelength multiplexingcapabilities and further exploit the utility of high spatial and temporalcontrol over immune cell activation, migration, and differentiation.In summary, here we demonstrate abiologicalcontrol of cytokinesignaling networks using a multicolor light and prodrug strategy wherebyrecombinant cytokines are chemically modified with photo-labile polymersthat de-repress their activity in response to low-power LED light.Given recent advancements in the development of highly efficient andwavelength-variable photocages,


SARS-CoV-2 mRNA vaccines are administered as effective prophylactic measures for reducing virus transmission rates and disease severity. To enhance the durability of post-vaccination immunity and combat SARS-CoV-2 variants, boosters have been administered to two-dose vaccinees. However, long-term humoral responses following booster vaccination are not well characterized. A 16-member cohort of healthy SARS-CoV-2 naïve participants were enrolled in this study during a three-dose BNT162b2 vaccine series. Serum samples were collected from vaccinees over 420 days and screened for antigen (Ag)-specific antibody titers, IgG subclass distribution, and neutralizing antibody (nAb) responses. Vaccine boosting restored peak Ag-specific titers with sustained α-RBD IgG and IgA antibody responses when measured at six months post-boost. RBD- and spike-specific IgG4 antibody levels were markedly elevated in three-dose but not two-dose immune sera. Although strong neutralization responses were detected in two- and three-dose vaccine sera, these rapidly decayed to pre-immune levels by four and six months, respectively. While boosters enhanced serum IgG Ab reactivity and nAb responses against variant strains, all variants tested showed resistance to two- and three-dose immune sera. Our data reflect the poor durability of vaccine-induced nAb responses which are a strong predictor of protection from symptomatic SARS-CoV-2 infection. The induction of IgG4-switched humoral responses may permit extended viral persistence via the downregulation of Fc-mediated effector functions. The rapid development of novel vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has likely prevented millions of COVID-19-related deaths . A primaDespite the success of these mRNA vaccines, humoral responses decline by 6–8 months after the two-dose regimen and are less protective against emerging variants ,10,11. MA 16-member cohort of healthy SARS-CoV-2 naïve participants  were enrolled during a three-dose monovalent BNT162b2 vaccine series. Serum samples were collected with approval from the Institutional Review Board at Louisiana State University Health Sciences Center, New Orleans, LA (IRB no. 641). All subjects in the cohort stated that they were unaware of previous COVID-19 infection, remained asymptomatic during the course of the study, and were monitored for antibody responses to SARS-CoV-2 nucleocapsid (NC) protein. Recombinant Wuhan-Hu-1 RBD and spike (S) ectodomain proteins were expressed from NR-52309 and NR-52421 plasmids, respectively, obtained from BEI Resources . For variant RBD constructs, primers were designed to amplify RBD-coding regions from variant full-length spike plasmids (see pseudotyping methods below); PCR products were subcloned into the parent pcDNA3.4 expression vector. RBD- and S-encoding plasmids were transiently transfected into ExpiCHO-S cells  following the manufacturer’s protocol. His-tagged RBD and S were purified from clarified culture supernatants via immobilized metal affinity chromatography using HisPur cobalt resin (Thermo Fisher). RBD- and S-loaded resins were washed and eluted using a His-Tag buffer set . Elution fractions were concentrated and the buffer exchanged into 1× phosphate-buffered saline  using Amicon 30 kDa MWCO centrifugal filters  and stored at −80 °C.Recombinant RBD and S ectodomain proteins were diluted to 2 μg/mL (100 ng/well) in PBS and passively immobilized on 96-well high binding assay plates (Fisher Scientific) overnight at 4 °C. Wells were then washed to remove unbound Ag and blocked with 0.1 mL/well blocking buffer (2% milk/1 × PBS with 0.05% Tween-20). Sera were serially diluted 1:3 (starting at 1:60) in blocking buffer and incubated in Ag-coated plates for one hour at room temperature with shaking at 450 rpm. Assay plates were washed 5× and binding events were detected using HRP-labeled goat α-human IgG(H+L) and goat α-human IgA . For IgG subclass-specific assays, binding events were detected using HRP-labeled mouse α-human IgG1 Fc, IgG2 Fc, IgG3 hinge, and IgG4 Fc secondary antibodies . Plates were developed using KPL SureBlue TMB peroxidase substrate and stop solution  and the optical density at 450 nm was recorded. Sera were tested for the presence of antibodies against SARS-CoV-2 nucleocapsid (NC) protein as described elsewhere . 2 incubator. At 50–80% confluency, cell monolayers were transfected with 0.53 μg of pcDNA3.4-vectored spike-encoding plasmid, 9.2 μg of pHR’CMV-Luc , 9.2 μg of pCMV ΔR8.2 (VRC5602), and 0.16 μg of TMPRSS2-encoding plasmid (VRC9260). These four plasmids were delivered using jetPRIME transfection reagent  according to the manufacturer’s protocol. At 96 h post-transfection, culture supernatants were harvested, clarified, and concentrated using Lenti-X Concentrator solution (Takara Bio) according to the manufacturer’s protocol. Concentrated pseudovirus lots were stored at −80 °C.Lenti-X 293T cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 1× MEM non-essential amino acids, and 1% penicillin/streptomycin in T75 tissue culture flasks  in a humidified 37 °C 5% COPlasmids encoding spike from the ancestral Wuhan-Hu-1 strain (Genbank no. NC_045512) and the B.1.351 (GISAID accession number EPI_ISL_1046568), B.1.617.2 (EPI_ISL_3431049), B.1.621 (EPI_ISL_4079202), and B.1.1.529  SARS-CoV-2 variants were created via gene synthesis  and expressed from the pcDNA3.4 mammalian expression vector. These spike genes were modified in two ways: (1) the native viral leader sequence was replaced with a human CD5 leader sequence and (2) the native cytoplasmic domain was replaced with a truncated HIV-1-related sequence . 5 cells/well in 96-well black wall clear-bottom assay plates (Corning). Sera were serially diluted 1:3 in culture medium (starting at a 1:30 dilution) in sterile 96-well round-bottom tissue culture plates (Corning). SARS-CoV-2 spike pseudovirus was added 1:1 (v/v) to diluted sera and incubated at 37 °C for one hour. Permissive cell seeding volumes were aspirated from the assay plates and the pseudovirus and serum mixtures were transferred to appropriate assay wells. At 24 h post-transduction, all assay wells were replenished with fresh culture medium (0.2 mL/well). Assay plates were developed at 72 h post-transduction using the ONE-Glo Ex Luciferase Assay Kit  according to the manufacturer’s protocol. Luciferase reporter signaling events were recorded using the Cytation1 multi-mode reader .Permissive HEK-293T-ACE2 cells  were seeded at 3.0 × 10GraphPad Prism (version 9.5.1) was used for statistical analyses as specified in figure legends. We used two-way ANOVA with Tukey’s multiple comparisons of the mean area under the curve to compare RBD-specific and neutralizing antibody responses and Pearson correlation coefficients for linear correlations. We followed our cohort longitudinally from the baseline through multiple post-vaccination time points and analyzed serum samples for spike RBD-specific antibody responses and nAbs. Demographic information for the cohort is shown in Vaccinees donated sera prior to vaccination (pre-vax), three weeks following the initial dose (post-vax #1), three weeks and four months after the second dose , and three weeks and six months following the third dose  A. To measure antigen (Ag)-specific humoral responses, sera were screened against recombinant SARS-CoV-2 spike receptor-binding domain (RBD) protein (Wuhan-Hu-1) by ELISA. Significant increases in α-RBD IgG levels were detected in vaccinee sera after the first and second vaccine doses B. Serum The three-dose vaccine series induced significant changes in RBD-reactive serum IgG subclass distribution. Following the second dose, IgG1 and IgG3 subclasses were predominant while IgG2 and IgG4 levels were negligible A. IgG1 a50) values increased significantly after two vaccine doses although with some heterogeneity (1:450 to 1:6500) but had decayed to pre-immune levels at four months  pseudotyped virus inhibition assays to detect nAb responses. Mean serum 50% neutralization titer  were rescreened in binding assays using recombinant RBD proteins from B.1.351 (Beta), B.1.617.2 (Delta), B.1.621 (Mu), or B.1.1.529 (Omicron) variants. Compared with Wuhan-Hu-1 RBD binding, vaccinee serum IgG reactivity declined significantly against each of the variant RBD proteins A,B. NotaVaccinee sera were also screened in inhibition assays using variant spike pseudotyped viruses to evaluate the impact of SARS-CoV-2 evolution on BNT162b-induced nAb responses. Sera with peak nAb responses against Wuhan-Hu-1 (post-vax #2A and #3A) showed significantly reduced neutralization activity against all variant pseudoviruses A,B. VaccThis report describes longitudinal analyses of serum antibody responses in a three-dose BNT162b2 mRNA vaccinee cohort. High and sustained levels of serum RBD-specific IgG and IgA were detected following two and three vaccine doses. While Ag-specific antibody levels peaked at three weeks following the second dose, α-RBD IgG and IgA titers declined by four months. The detection of vaccine-induced Ag-specific IgA is noteworthy as this isotype has been reported to dominate the early serum antibody response to SARS-CoV-2 infection . VaccineCγ3-Cγ1-Cγ2-Cγ4-3′) on human chromosome 14 supports a hypothesis of progressive CSR-mediated IgG4 expression in response to a third vaccine dose [In addition to establishing persistent α-RBD IgG titers, serum IgG subclass profiles evolved through the three-dose regimen. The detection of inflammatory IgG1 and IgG3 responses in two-dose sera was expected given previous reports of elevated Ag-specific IgG1 and IgG3 titers during early humoral responses to SARS-CoV-2 infection and vaccination ,26,27. Tine dose ,30,31. Dine dose ,20. UnexSerum nAb responses, while heterogeneous, peaked at three weeks after the second and third vaccine doses and correlated strongly with RBD-specific IgG titers. Recent serum antibody mapping analyses involving members of this cohort revealed donor-to-donor variability in the breadth of epitope targeting through two and three vaccine doses . These v50 values for variant S pseudoviruses were significantly lower than against vaccine strain-matched Wuhan-Hu-1 S pseudovirus, with the observed decline in serum NT50 values for B.1.617.2 S pseudovirus likely due to post-boost narrowing of nAb responses towards variant-specific mutations in the spike glycoprotein [The dynamics of immune escape by RNA viruses limits the efficacy of vaccine-induced protective immune responses ,33. In poprotein . While testing for mucosal humoral immunity or T cell responses to BNT162b2 vaccination was beyond the scope of the present study, it is important to note that both may play an important role in host defense against respiratory pathogens. Secretory IgA may prevent SARS-CoV-2 adhesion to target epithelial cells via neutralization of the coronavirus spike glycoprotein  althoughIn summary, longitudinal analysis of the dynamics of BNT162b2-induced humoral immunity through a three-dose mRNA vaccine series over a period of 420 days suggests poor durability of BNT162b2-elicited nAb responses. A potential limitation of our study is the possibility of asymptomatic infection with SARS-CoV-2 that could impact the antibody responses that were detected in our cohort. We did, however, monitor antibody responses against the SARS-CoV-2 viral nucleocapsid protein in the cohort and saw no clear spikes in reactivity during the course of the study. It should also be noted that low-level responses against the nucleocapsid may be a result of SARS-CoV-2 infection but also, potentially, of previous coronavirus infections due to the cross-reactive nature of this viral protein . Our stuIn a longitudinal study of the dynamics of BNT162b2-induced humoral immunity through a three-dose mRNA vaccine regimen, we have demonstrated that while vaccine boosting restored peak α-RBD IgG and IgA antibody titers and generated strong antiviral neutralization responses, these nevertheless decayed to pre-immune levels within six months. Boosters enhanced serum reactivity and nAb responses against Beta, Delta, Mu, and Omicron SARS-CoV-2 strains; however, all variants that were tested showed resistance to two and three vaccine-dose immune sera. Our data indicate poor durability of vaccine-induced nAb responses while the induction of markedly elevated levels of antibodies of the IgG4 subclass could potentially facilitate extended viral persistence via downregulation of Fc-mediated effector functions.


Small vessel disease (SVD) causes most spontaneous intracerebral haemorrhage (ICH) and is associated with widespread microstructural brain tissue disruption, which can be quantified via diffusion tensor imaging (DTI) metrics: mean diffusivity (MD) and fractional anisotropy (FA). Little is known about the impact of whole-brain microstructural alterations after SVD-related ICH. We aimed to investigate: (1) association between whole-brain DTI metrics and functional outcome after ICH; and (2) predictive ability of these metrics compared to the pre-existing ICH score.Sixty-eight patients (38.2% lobar) were retrospectively included. We assessed whole-brain DTI metrics (obtained within 5 days after ICH) in cortical and deep grey matter and white matter. We used univariable logistic regression to assess the associations between DTI and clinical-radiological variables and poor outcome . We determined the optimal predictive variables (via LASSO estimation) in: model 1 (DTI variables only), model 2 (DTI plus non-DTI variables), model 3 (DTI plus ICH score). Optimism-adjusted C-statistics were calculated for each model and compared (likelihood ratio test) against the ICH score.p = 0.010) and white matter MD  were associated  with poor outcome. Discrimination values for model 1 (0.67 [95% CI 0.52–0.83]), model 2 (0.71 [95% CI 0.57–0.85) and model 3 (0.66 [95% CI 0.52–0.82]) were all significantly higher than the ICH score (0.62 [95% CI 0.49–0.75]).Deep grey matter MD (OR 1.04 [95% CI 1.01–1.07], Our exploratory study suggests that whole-brain microstructural disruption measured by DTI is associated with poor 6-month functional outcome after SVD-related ICH. Whole-brain DTI metrics performed better at predicting recovery than the existing ICH score.The online version contains supplementary material available at 10.1007/s00415-023-11592-7. The ICH score was designed to predict short-term (30-day) mortality and has limited predictive performance for longer-term functional outcome, with C-statistics ranging from 0.74 to 0.82 for 6-month functional outcome ), have no prior history of stroke and had small ICH volumes (median volume 7.1 ml [IQR 3.2–13.7]). We found that although DTI metrics were associated with excellent (6-month mRS 0–1) outcome prediction, they did not statistically improve prediction over the ICH score alone. Despite no major differences in terms of pre-ICH mRS between included and excluded patients Below is the link to the electronic supplementary material.


After this article  was publeat-4(-) panel in daf-28(tm2308) panel in The control RNAi/tfg-1 (RNAi)/Phsp-16.2::gfp panel in Phsp-16.2::gfp panel in The right side of the The wild type panel in Fig 2A is duplicated as the wild type panel in eat-4(-) panel in daf-28(tm2308) panel in tfg-1 (RNAi)/Phsp-16.2::gfp panel in Phsp-16.2::gfp panel in In response to queries about the experiments in Figs 4D, S1, and S4C in [In response to queries about the experiments in Figs The corresponding author provided the below updated versions of Figs daf-28(sa191) mutants was increased by approximately 4 fold compared to wild-type animals (daf-28(tm2308) null strain, which is deficient in daf-28 and does not produce the toxic insulin peptide which induces ER stress (tfg-1 RNAi treatment of the two daf-28 mutant strains increased the number of germline corpses in daf-28(tm2308) null strain (P<0.05), but did not further increase germline apoptosis in the daf-28(sa191) strain Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(ZIP)Click here for additional data file.S4 FileSince images include multiple worms, images were independently measured several times to achieve measurements for distinct worms in one shared image. Only animals whose body was fully imaged (at least from front to back intestine) were measured. Exposure levels were maintained constant per worm strain.(ZIP)Click here for additional data file.S5 File(ZIP)Click here for additional data file.S6 File(ZIP)Click here for additional data file.S1 Figtfg-1 RNAi. Bar graph presents relative fluorescence +/-SEM of the indicated genotypes . Phsp-4::gfp is induced in response to ER stress, Pgst-4::gfp and Psod-3::gfp are induced in response to oxidative stress, Phsp-6::gfp is induced in response to mitochondrial stress and Phsp-16.2::gfp is induced in response to heat shock. Asterisk marks One sample-test value of P<0.05 compared to control RNAi. No increase in the levels of any of the cellular stress reporters was seen upon treatment with tfg-1 RNAi except for the Phsp-4::gfp ER stress response reporter.Representative fluorescence micrographs (100-fold magnification) of adult transgenic animals expressing a GFP reporter fused to promoters, whose activity is induced in response to a variety of cellular stresses. Animals were treated with control RNAi or with (TIF)Click here for additional data file.S2 Figxbp-1 or by blocking protein export from the ER by inactivation of tfg-1 . No germ cell corpses were detected in either ced-3(n1286), xbp-1(tm2457); ced-3(n1286) or tfg-1(RNAi); ced-3(n1286) backgrounds. Bar graph shows average number +/-SEM of apoptotic corpses per gonad arm (n = 60 per genotype). Asterisk marks One-way ANOVA followed by Tukey’s multiple comparisons test values of P<0.05 compared to ced-3-deficient animals.Representative fluorescence micrographs (200-fold magnification) of SYTO12-stained germ cell corpses in day-2 adults. Arrows point at SYTO12-labeled germ cell corpses. ER stress was induced by inactivation of the UPR gene (TIF)Click here for additional data file.S4 Fig(A) Representative fluorescence micrographs (400-fold magnification) of GFP-expressing ASI neurons driven by the daf-7 promoter. The overall pattern of the ASI neurons was similar in control RNAi and the tfg-1 RNAi treated animals. (B-C) Bar graph and representative fluorescence micrographs (200-fold magnification) of germline corpses in unc-13(e51), unc-64(e246) unc-31(e928) and eat-4 (ky5) day-2 mutants are presented. The average number of apoptotic corpses per gonad arm was scored by SYTO12 staining . Asterisk marks One-way ANOVA followed by Šidák multiple comparisons test values of P<0.05. Error bars represent SEM +/-. Note that although these strains have a severely defective nervous system, they display normal basal levels of germline apoptosis, which increase in response to ER stress. These results uncouple general neuronal dysfunction and the responsiveness to ER stress-induced germline apoptosis.(TIF)Click here for additional data file.


Youth with special healthcare needs or on immunosuppression are at increased risk for severe COVID-19, yet evaluation of the immunogenicity of SARS-CoV-2 vaccines in this population has been limited. A quarter of individuals with inflammatory bowel disease (IBD) are diagnosed in childhood and require chronic immunosuppressive therapy. Describing the quality and duration of the humoral and cellular responses to novel mRNA vaccines in this population can improve implementation and protection for this vulnerable population.This ongoing single-center, prospective, longitudinal cohort study enrolled children aged 6–21 years at-risk for severe COVID-19 beginning in January 2023. On enrollment and at pre-specified times after vaccination peripheral blood samples, epidemiologic and clinical data were obtained. Total SARS-CoV-2 spike (S) and receptor binding domain (RBD)-specific IgG antibodies, as well as endemic coronavirus (eCoV) antibodies, were measured using a multiplex microsphere immunoassay. SARS-CoV-2 specific T cell responses were measured with intracellular cytokine staining (ICS) after peptide stimulation.11 SARS-CoV-2-immunized children with IBD treated with anti-tumor necrosis factor (TNFα) therapy were enrolled (Table 1). The seroconversion rate was 100% . Anti-S IgG levels increased with more exposures (r =.81 p =.003) and decreased over time with a negative correlation between anti-S IgG and days since last exposure (r = -.691 p =.02), as well as a significant difference in anti-S IgG when grouped by time since last exposure (p =.02) . ECoV antibody levels did not significantly correlate with anti-S IgG. 63% and 81% of participants developed SARS-CoV-2 specific CD4+ and CD8+ T cell responses, respectively, though response magnitude was low . A majority of participants demonstrated measurable antibody (100%) and T cell (90%) responses > 12 months after their last vaccination.Table 1Demographics of Enrolled Research SubjectsFigure 1Cohort Humoral and Cellular Responses. Antibody levels (left) of SARS-CoV-2 wild type plus Delta and Omicron variants in BAU/mL. ECoV antibody levels in U/mL. T cell response (right) evaluated by frequency of CD4+ and CD8+ IFN-γ+ T cells following stimulation with wild type SARS-CoV-2 S, N, M peptides.Figure 2Humoral Immune Durability and Response. Significant correlation between SARS-CoV-2 S IgG and days since last exposure (left) and SARS-CoV-2 S IgG and number of exposures (right). Exposure = vaccination or infection. Shaded region 95% CI, n=11.Our small, ongoing cohort study demonstrates both SARS-CoV-2 antibody and T cell responses can develop following vaccination in children with IBD on anti-TNFα therapy. While the humoral response decreased over time, a durable cellular response persisted at 12 months in most participants.All Authors: No reported disclosures


Dear Editor,2 Among SARS-CoV-2 proteins, the N protein can be detectable in damaged tubules in COVID-19 patients with AKI.3 However, the role and mechanisms of N protein-induced AKI in diabetes remain unclear. By using ultrasound-microbubble-mediated kidney-specific gene transfer technique, we found that SARS-CoV-2 N protein overexpression could dose-and time-dependently induce AKI in non-diabetic mice (db/m), showing tubular necrosis with renal dysfunction including a marked increase in blood urea nitrogen (BUN) and creatinine, which was further increased in diabetic db/db mice at age of 8 weeks and became much more severe in those with older age at 16 and 32 weeks with the development of DKD  in critically ill COVID-19 patients, especially in the elderly and diabetic patients with diabetic kidney disease (DKD).DKD Fig.  and S2. DKD Fig. , althougDKD Fig. . These f5 here we also uncover that the N protein can cause AKI in diabetes by inducing tubular cell death via the Smad3-receptor interacting protein kinase 3 (Ripk3)/Mixedlineage kinase domain-like protein (MLKL) necroptosis pathway. Indeed, Smad3 is markedly activated in the diabetic kidney in response to TGF-β1, advanced glycation end products (AGEs), and angiotensin II (Ang II).6 Thus, once SARS-CoV-2 N protein is overexpressed, it could bind and promote further Smad3 signaling and Smad3-dependent RIPK3/MLKL necroptosis pathway, resulting in progressive AKI in diabetic mice, particularly in those with DKD  with a Smad3 inhibitor SIS3 or control DMSO at dosages of 5, 10, or 15 mg/kg body weight intraperitoneally (ip) from the day before the SARS-CoV-2 N gene transfer. Compared to DMSO control, SIS3 treatment dose-dependently attenuated SARS-CoV-2 N protein-caused AKI by markedly inhibiting tubular necrosis, reducing serum creatinine and BUN, and suppressing tubular Kim1 expression in both db/m and db/db mice, with a better therapeutic dose at 10 mg/kg body weight Fig. . HoweverAs expected, SIS3 treatment also resulted in a marked inhibition of SARS-CoV-2 N-triggered Smad3-Ripk3/MLKL signaling in db/m and db/db mice Fig. , demonst5In summary, we identified that SARS-CoV-2 N is pathogenic and can cause severe AKI in diabetic mice via the Smad3-Ripk3/MLKL necroptosis pathway, specifically in those with older age and DKD. Targeting this pathway with a Smad3 inhibitor SIS3 can attenuate SARS-CoV-2 N-induced AKI in db/db mice, suggesting SIS3 as a novel therapeutic agent for COVID-19 AKI in diabetic patients (supplementary Fig. Supplementary materials and Figures


To describe humoral immune responses to symptomatic SARS-CoV-2 infection, we assessed immunoglobulin G binding antibody levels using a commercial multiplex bead assay against SARS-CoV-2 ancestral spike protein receptor binding domain (RBD) and nucleocapsid protein (N). We measured binding antibody units per mL (BAU/mL) during acute illness within 5 days of illness onset and during convalescence in 105 ambulatory patients with laboratory-confirmed SARS-CoV-2 infection with Omicron variant viruses. Comparing acute- to convalescent phase antibody concentrations, geometric mean anti-N antibody concentrations increased 47-fold from 5.5 to 259 BAU/mL. Anti-RBD antibody concentrations increased 2.5-fold from 1258 to 3189 BAU/mL. Humoral immune responses to infection with SARS-CoV-2 include production of immunoglobulin G (IgG) antibodies that bind to spike (S) glycoprotein, including the receptor binding domain (RBD) within S, as well as the nucleocapsid (N) protein. Virus neutralization titers and antibodies that bind to RBD and other S protein epitopes have been associated with protection against symptomatic infection with ancestral and pre-Omicron SARS-CoV-2 variants –4. BecauWe previously showed that anti-N antibody seropositivity modified COVID-19 mRNA vaccine effectiveness against symptomatic SARS-CoV-2 infection with SARS-CoV-2 Delta and Omicron variants [Between December 2021 and June 2022, respiratory swabs and acute-phase dried blood spots on filter paper were obtained <5 days after symptom onset from ambulatory patients with respiratory illness enrolled in the US Influenza Vaccine Effectiveness Network, as previously described , 10. Pat™ SARS-CoV-2 Human IgG Antibody Test, Tetracore, Rockville, MD) on a Luminex MAGPIX instrument with LX200 flow analyzer . Eluted specimens were diluted 1:300 and individual specimen median fluorescence intensity (MFI) ratios were calculated compared to antigen-specific human IgG calibrator serum. MFI units were standardized to binding antibody units per mL (BAU/mL) against World Health Organization (WHO) international standards [Methods for estimating SARS-CoV-2 binding antibody concentration from blood spots have been previously published , 12. Dritandards , 13.Antibody concentrations in BAU/mL were log-transformed (log 10) for estimation of geometric mean concentrations (GMC) and 95% confidence intervals (CI) and back-transformed for plots by BAU/mL concentrations. Geometric mean fold rise in bAb concentration was estimated as the geometric mean ratio of bAb concentrations measured at enrollment and follow-up. Fold rise in anti-N and anti-RBD antibody concentrations represented response to acute SARS-CoV-2 infection. Associations between antibody fold rise and patient characteristics, vaccination status, baseline N antibody serostatus, and prior positive COVID-19 test were assessed by t-test or ANOVA. Statistical analysis was performed using R version 4.0.3 .A total of 105 SARS-CoV-2 positive participants had acute-phase blood specimens collected a median of 2 days (range: 0–5) and convalescent-phase specimens collected a median of 36 days (range: 23–61) after symptom onset. Of 48 SARS-CoV-2 positive specimens with genomic sequencing, 43 (90%) belonged to Omicron BA.1 (n= 5) or BA.2 (n=38) lineages, with five sequences belonging to BA.2.12.1 (n=3), BA.4 (n=1) and BA.5 (n=1) lineages. Among 105 case patients with paired specimens, 36% were aged <40 years, 17% were >65 years, 57% were female and 23% had an underlying health condition (Table 1).Among 7 (7%) patients with ≥1 electronic health record documented prior COVID-19 positive laboratory PCR test, median time since most recent positive COVID-19 test was 351 days (range: 64–758). Among 101 patients who had received at least two doses of COVID-19 mRNA vaccine, median time since receipt of most recent mRNA vaccine dose was 175 days (range: 33–382); 11 had received a booster dose <90 days prior to their enrollment in the study (measured between the date of booster and date of illness onset).Anti-N and anti-RBD geometric mean fold increases were not associated with days from symptom onset to specimen collection (data not shown). For both antigens, geometric mean fold rise from acute- to convalescent-phase bAb concentrations were similar by patient age, sex, or presence of underlying medical conditions.p-value=0.007). Among four unvaccinated case patients, anti-N bAb concentrations increased 116-fold (95% CI: 7–1998). Patients with prior documented positive COVID-19 tests trended towards having higher convalescent-phase anti-N bAb concentrations than patients without prior positive COVID-19 tests  and 3189 (CI: 2639–3853) in acute- and convalescent phase specimens, respectively; mean fold RBD bAb rise was 2.5 (CI: 1.9–3.3; Among symptomatic patients infected with SARS-CoV-2 Omicron variant viruses, most of whom had been previously vaccinated, we observed marked differences in antibody levels against nucleocapsid protein and ancestral spike RBD antigen measured during acute illness and convalescence. The 47-fold increase in IgG antibodies against SARS-CoV-2 nucleocapsid protein indicated a strong humoral response to infection with Omicron variant viruses, mainly BA.1 and BA.2. Baseline seronegative patients had a higher mean fold rise in anti-nucleocapsid antibodies than seropositive patients. Increased convalescent anti-N bAb levels among baseline seropositive patients suggests boosting of immune responses acquired from past infection; this boosting was also observed among patients with ≥1 documented prior COVID-19 positive test. In contrast, we observed modest 2.5-fold increase in antibodies against ancestral spike RBD antigens. Among vaccinated cases, lower baseline anti-RBD antibody concentrations among patients who had received 2 versus ≥3 mRNA vaccine doses were associated with greater antibody response but similar convalescent antibody concentrations. While anti-RBD bAb levels correlate with protection from infection with pre-Omicron variants , 14.Dried blood spots for SARS-CoV-2 serology were collected as part of a COVID-19 vaccine effectiveness study using a test-negative design. Because antibody levels measured during acute illness reflect an estimation of antibody levels close to the time of infection rather than response to current infection, comparison between antibody levels among case patients and SARS-CoV-2 uninfected patients may provide a measure of correlates of risk . HoweverThe study had several limitations. The serologic assay used in our study contained ancestral SARS-CoV-2 antigens and serum was quantified using the WHO international serum standards from early in the COVID-19 response. Against pre-Omicron variants, virus neutralization titers and IgG antibody concentrations were associated with protection; however, antibody binding and neutralization activity was lower against Omicron variants , 18. In Serologic assays that quantify anti-SARS-CoV-2 specific antibody levels during and following acute infection may provide information on incidence and recency of infection. As new SARS-CoV-2 variants emerge, frequent updates to serologic antigens will be needed to quantify binding IgG antibody levels that correlate with immune protection . Observa


Dear Editor,1 Those features make CLDN a potential therapeutic target. However, monoclonal antibody targeting CLDN induce limited antitumor immune responses in clinical trials and fusion of strong immunomodulators might be needed to enhance its efficacy. High dose IL-2 activates tumor infiltrating lymphocytes (TILs), but the severe toxicity and poor tumor targeting limits its use.2CLDN18.2 (CLDN), a member of tight junction protein family, is strictly limited to express on differentiated epithelial cells of the gastric mucosa and abnormal overexpression has been found in many cancers, especially in digestive system malignancies.+ T cells and expression level of IL-2 in the tumor microenvironment (TME) were associated with better survival in several human cancers (Supplementary Fig. + T cells. Indeed, when IL-2 signaling was blocked by anti-IL2Rβ (Supplementary Fig. + T cells were dramatically decreased (Supplementary, Fig. We first discovered that the abundance of CD8We evaluated the characteristic of CLDN-ProIL2 in vitro and found that CLDN-ProIL2 can specifically bind to MC38-CLDN tumor cells with similar affinity to CLDN-Fc (Supplementary Fig. + T cells in the peripheral blood were dramatically increased (Supplementary Fig. To explore whether CLDN targeting is necessary for Pro-IL2 in vivo to act efficiently, we compared the antitumor efficacy of different constructs. Pro-IL2 has no anti-tumor activity, whereas CLDN-ProIL2 and CLDN-IL2 exhibited a superior antitumor efficacy in MC38-CLDN tumor model Fig. . HoweverWe next measured the tumor-targeting capacity of CLDN-ProIL2 using a bilateral tumor model and confirmed that CLDN-ProIL2 can substantially accumulate in CLDN positive tumor (Supplementary Fig. + T cells in the tumor tissues were increased after CLDN-ProIL2 treatment (Fig. Rag1−/− mice but had remained anti-tumor effect in WT mice after NK cells depletion (Supplementary Fig. + T cells depletion (Fig. + T cells were sufficient and essential for tumor control.We discovered that the total number of CD8ent Fig. . Then, wion Fig. . To studion Fig. , suggest3 CLDN-ProIL2 reduced the frequency of Treg cells in the tumor but unexpectedly increased it in the spleen (Fig. + T cells were decreased (Supplementary Fig. + cells in PD1+TIM3+CD8+ T cells were also decreased after CLDN-ProIL2 treatment (Supplementary Fig. Treg cells limit immune responses.een Fig. . Such in4 We observed tumor-specific CD8+ T cells (Supplementary Fig. +TIM3−CD8+ T cells were increased whereas that of PD1+TIM3+ CD8+ T cells were decreased (Supplementary Fig. + T cells (Supplementary Fig. Tumor-specific T cells are required for the memory T cells formation and establishing systemic protective immunity against relapse.+ T cells (Supplementary Fig. +TIM3− CD8+ T cells (Supplementary Fig. + T cells (Supplementary Fig. +TIM3-CD8+ T cells, the absolute number of PD1+TIM3-CD8+ T cells were still increased even after FTY720 plus CLDN-ProIL2 and CLDN-ProIL2 treatment (Supplementary Fig. +TIM3-CD8+ TILs.Moreover, the frequency of proliferating CD8We next explored if CLDN-ProIL2 can generate systemic antitumor effects after local treatment in a bilateral tumor model (Supplementary Fig. 5 To explore if CLDN-ProIL2 can also serve as an effective neoadjuvant therapy to control metastasis. 4T1-CLDN tumor-bearing mice were preoperatively treated with CLDN-ProIL2 followed by surgical resection (Supplementary Fig. Emerging evidence showed that neoadjuvant immunotherapy has emerged as a promising treatment against metastasis lesion.supplemental data