Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the E P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain’s adaptation to rabbits (ATR) . However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein  lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs. Viruses have a predefined host spectrum . However6–rns, E1, and E2) and eight nonstructural proteins   is a devastating infectious disease of pigs caused by classical swine fever virus (CSFV), which often leads to huge economic losses to the pork industry . CSFV isnd NS5B) . The Ernith LamR . The E2 ith LamR , 15 and ith LamR . Meanwhi in pigs –22.In general, CSFV infects only domestic pigs and wild boar . Howeverrns carrying the Erns of C-strain in the background of the highly virulent CSFV Shimen strain is not adaptive to rabbits, indicating that the Erns of C-strain alone could not render the Shimen strain adaptive to rabbits  or crucial mutation sites (E2P108L-T109I) on E2DomainI of the Shimen strain do not adapt to rabbits, while the chimera harboring E2 domain II (E2DomainII) of the Shimen strain is adaptive to rabbits (DomainI (E2P108-T109) of C-strain are responsible for its ATR, whereas E2DomainII containing three different residues  of C-strain are not determinants of its ATR. Because of the above findings, we further investigated the effects of these critical amino acids of C-strain on the Shimen strain ATR and the virulence in pigs.Our previous study found that vSM-HCLVE rabbits . In cont rabbits . TherefoP108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) of C-strain confers adaptation to rabbits on the Shimen strain. Remarkably, our data revealed that E2P108-T109-Erns of C-strain promotes viral entry during infection in the target cells using a series of pseudotyped viruses. Finally, we demonstrated that E2P108-T109-Erns of C-strain, responsible for the CSFV ATR, does not affect viral virulence in pigs, whereas E2DomainII-Erns, which is not associated with the adaptation, attenuates the Shimen strain in pigs.In the present study, we demonstrated that E2P108-T109 or E2DomainI but not E2DomainII of C-strain is necessary for conferring adaptation to rabbits on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns. Since vSM-HCLVErnsE2, harboring the Erns and E2 glycoproteins of C-strain, showed better adaptation to rabbits than vSM-HCLVE1E2, containing the E1 and E2 glycoproteins of C-strain, according to the reproductive animal experiments in our previous study  were constructed based on the infectious clone pSM-HCLVErns containing Erns of C-strain in the backbone of the Shimen strain   . CollectP108-T109-Erns of C-strain adapts the Shimen strain to rabbits, five groups of 24 rabbits were inoculated with different chimeric viruses , and no mutation occurred, as confirmed by sequencing , anti-E2 antibodies were detected in the remaining two rabbits of each group  or the parental virus C-strain increased over time. In contrast, the viral genome copy numbers of non-rabbit-adaptive CSFVs (vSM-HCLVErnsE2DomainII and the Shimen strain) decreased over time  or C-strain (HCLVpps) were generated to investigate whether their differences occur in viral entry. We infected rabbits with SMpps or HCLVpps and isolated the spleen lymphocytes at 48 hpi. To amplify the fluorescence signals, an anti-EGFP (enhanced green fluorescent protein) antibody was used to measure EGFP expression. Meanwhile, mouse IgG was used to exclude nonspecific signals. We detected EGFP in the spleen lymphocytes from the rabbits inoculated with HCLVpps but not SMpps . Clinical signs and rectal temperatures were monitored. All the pigs showed typical clinical signs  with vSME2al signs  of CSF so 16 dpi . Howevern strain  and B. Ial virus , while vK6 cells .L108P-I109T-HCLVErns, vSM-HCLVErnsE2DomainI, or the Shimen strain displayed CSF-specific pathological changes  (rnsE2DomainII (The pigs inoculated with vSME2 spleen) . In contDomainII .rnsE2DomainII showed slight histopathological lesions, including depletion in lymph nodes and reactive hyperplasia in spleens. However, the pigs inoculated with vSM-HCLVErnsE2DomainI or vSME2L108P-I109T-HCLVErns showed histopathological lesions similar to those in pigs inoculated with the Shimen strain in tonsils (lymphopenia), kidneys , and bladder   .P108-T109-Erns of C-strain contributes to the ATR but does not alter the virulence of the Shimen strain in pigs, whereas E2DomainII-Erns of C-strain attenuates the Shimen strain, suggesting that there are different molecular determinants of CSFV ATR and virulence in pigs.Taken together, these observations indicate that E2rns or E1 of C-strain. Furthermore, P108 and T109 in E2DomainI are crucial amino acids for C-strain to be adaptive to rabbits  alters the tropism to different cells in contrast to the parental BVDV , 27. Recrns glycoprotein of pestiviruses attaches to the virion envelope by directly interacting with the E2 glycoprotein to form Erns-E2 heterodimers and is indispensable for virus attachment and infection of target cells  1640 medium  supplemented with 10% FBS, 1% antibiotics-antimycotics , 1% l-glutamine, and 0.20 ng/ml interleukin 2 (IL-2)  (AY805221) and Shimen strain (GenBank no. AF092448.2) were used for the construction of infectious cDNA clones.SK6 and PK-15 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 5% heat-inactivated fetal bovine serum (FBS)  in a 37°C incubator with 5% CO; Abcam) . Primaryrns, which harbors Erns of C-strain in the background of the Shimen strain . All these constructed infectious cDNA clones were identified by PCR, enzyme digestion, and sequencing.Based on the infectious cDNA clone pSM-HCLVEn strain , we tookrns glycoprotein by a CSFV antigen test kit . The positive samples were identified by RT-PCR, sequencing, and IFA.Three chimeric viruses were rescued as described previously with a slight modification . Six mic50) per milliliter  of 0.1. Two hours later, the supernatants were removed, and the cells were washed three times with PBS. Then, fresh DMEM supplemented with 2% FBS was added to each well, and the cells were cultured at 37°C and 5% COTotal RNA from tissues or cells was extracted using RNAiso Plus  according to the manufacturer’s protocol. cDNA synthesis was processed in a 20-μl volume with avian myeloblastosis virus (AMV) reverse transcriptase XL . The copy numbers of the CSFV genome were determined using a previously described RT-qPCR assay .pro protein (between the amino acids 13 and 14) based on C-strain was generated. Rabbit spleen lymphocytes infected with vHCLV-NanoLuc or C-strain or treated with DMEM only were lysed with 100 μl of passive lysis buffer  and incubated on a shaker for 1 h at 4°C. The supernatants were collected by centrifuging at 12,000 × g for 10 min at 4°C. NanoLuc activities were measured with EnVision multilabel plate readers (PerkinElmer).The reporter virus vHCLV-NanoLuc expressing the NanoLuc protein fused with the NThe spleens from the rabbits inoculated with C-strain were collected after rabbits were euthanized at 3 dpi, and spleen cells were obtained by smearing the organs on 200-mesh copper wire mesh with RPMI 1640 medium. The red blood cells were lysed using red blood cell lysis buffer , and the lymphocyte suspensions were washed with PBS. The following antibodies were used for flow cytometry, according to the manufacturers’ instructions: goat anti-rabbit IgM μ chain preadsorbed to secondary antibody (DyLight 488)  for isolating B cells, mouse anti-rabbit T lymphocytes  for isolating T cells, and Alexa Fluor 633 goat anti-mouse IgG (heavy plus light chain [H+L])  as the secondary antibody. Stained mononuclear cells from the spleens of the rabbits infected with C-strain were added to cytometry tubes and sorted with a high-speed cell sorter .2 incubator, the infected cells were washed three times with PBS and further incubated for 4, 24, 48, or 72 h  via the marginal ear vein with the viruses indicated in 6 cells in each sample were permeabilized with 0.15% Triton X-100. The cells were further incubated with primary antibody for 1 h and washed three times for 5 min with PBS. Then the cells were incubated with secondary antibody for 45 min and washed three times for 5 min with PBS. The fluorescence signal was analyzed with an Accuri C6 Plus flow cytometer (BD Biosciences).The rabbits were inoculated i.v. with different chimeric pseudotyped viruses. Spleen lymphocytes were isolated at 48 hpi. Mouse anti-coral green fluorescent protein (cGFP)-tagged monoclonal antibody (MAb)  was used to detect the expression of EGFP in lymphocytes, and irrelevant mouse IgG  was used as a negative control. As the secondary immunoreagent, fluorescein isothiocyanate-labeled goat anti-mouse IgG  was used. All antibodies mentioned were diluted at 1:200 in PBS. For flow cytometry analysis, 10n = 3; group 4, n = 2), and each group was housed in an individual room. Group 1 was inoculated i.m. with vSM-HCLVErnsE2DomainI, group 2 was inoculated i.m. with vSM-E2L108P-I109T-HCLVErns, group 3 was inoculated i.m. with vSM-HCLVErnsE2DomainII, and group 4 was inoculated i.m. with the Shimen strain. The inoculation dose of the virus for each group was 105 TCID50  of the Chinese Academy of Agricultural Sciences (CAAS) .P value of <0.05 was considered significant.SPSS 22.0 software was used to analyze all data. An unadjusted


J-coupling based experiments offer the possibility to distinguish them from backbone amide groups if the spin-echo lifetimes are long enough, which turned out to be the case for the glutamine side-chains, but not for the asparagine side-chains forming asparagine ladders. We explore the sensitivity of NMR observables to asparagine ladder formation. One of the two possible asparagine ladders in HET-s(218–289), the one comprising N226 and N262, is assigned by proton-detected 3D experiments at fast MAS and significant de-shielding of one of the NH2 proton resonances indicative of hydrogen-bond formation is observed. Small rotating-frame 15N relaxation-rate constants point to rigidified asparagine side-chains in this ladder. The proton resonances are homogeneously broadened which could indicate chemical exchange, but is presently not fully understood. The second asparagine ladder (N243 and N279) in contrast remains more flexible.Asparagine and glutamine side-chains can form hydrogen-bonded ladders which contribute significantly to the stability of amyloid fibrils. We show, using the example of HET-s(218–289) fibrils, that the primary amide side-chain proton resonances can be detected in cross-polarization based solid-state NMR spectra at fast magic-angle spinning (MAS).  We discuss NMR spectral and relaxation parameters determined for asparagine side-chains forming asparagine ladders, such as high-frequency shifted proton resonances and small rotating-frame 15N relaxation-rate constants.Solid-state nuclear magnetic resonance (NMR) spectroscopy is a suitable technique to identify asparagine/glutamine ladders in amyloid fibrils: the gen bond  whereas gen bond . Infraregen bond  and solugen bond   fibrils (abbreviated in the following with DUL), recorded at a MAS frequency of 110 kHz, are shown in 15N and 1H shifts although the regions overlap. Some of the observed N/Q side-chains  lead to two proton correlation peaks with a common 15N frequency indicating no or only a slow rotation around the Cγ -Nδ2 (N) or Cδ -Nε2 (Q) bond as shown in solution-state experiments .The side-chain amide protons of asparagine (N) and glutamine (Q) side-chains can be observed in proton-detected fast MAS experiments. CP hNH correlation spectra of deuterated and 100% back-exchanged, as well as uniformly eriments . The sidspectrum . In totaspectrum . The sidJ-coupling based INEPT-hNH experiments are also suitable to detect N/Q side-chains, similar to 15N,1H HSQC experiments applied in solution-state NMR. The well-known differences in the magnetization-transfer curves of NH and NH2 side-chain resonances in a refocused INEPT-based hNH experiment  are visible in INEPT experiments (see We next address the question whether periment  could ths see τN  to 2.7 mctra see . For HETents see . None oux– and DNA). τN = 1.2 ms (cyan) and τN = 2.7 ms , two asparagine ladders (one comprising N226 and N262 and the other N243 and N279) have been postulated from the structure obtained by solid-state NMR. N226 and N262 point to the inside of the fibril and are located next to the hydrophobic core, whereas N243 and N279 are outside of the core see . The NMR bonding  characte bonding . As illue vector , while ipt fixed . Both ca protons . We spec2 protons are thus shifted in different directions with respect to their average chemical-shift values over all deposited asparagine residues as extracted from the BMRB database (7.3 and 7.1 ppm)1.Experimentally, for N226 we find a large difference between the two proton resonances resonating at 8.4 and 6.5 ppm. For N262 the less-shielded value is 7.6 ppm compared to 6.8 ppm for the more-shielded one. The two NH2 group, but also probe the spatial proximity between such side-chains. A 9 ms 13CO-1H CP-step was employed to achieve a polarization transfer to both NH2 protons of the neighboring asparagine side-chain within the same ladder. Similar experiments have been described recently to identify backbone hydrogen bonding patterns in SH3 and GB1  and N226 (114.8 ppm) clearly show cross peaks to the proton shifts of the NH2 group of N226 and N262, respectively (see 3D hNCOH experiments  do not o and GB1 . And indhomo) which is determined by the geometry of the proton dipolar network  values. The pulse sequences used to measure Δhomo values are shown in The visibility of the proton resonances in INEPT-type experiments is related to the homogeneous proton linewidth . Δhomo for N279 is even larger (543 ± 140 Hz). All these values are significantly larger than the corresponding values of the backbone amide protons, which have been reported before, showing on average 22 ± 1 Hz at 100 kHz MAS for DUL HET-s(218–289) . As an additional reference we also measured the homogeneous proton linewidths for asparagine and glutamine side-chains in DnaB. In accordance with the experimental observation of glutamine side-chains in INEPT spectra, the Δhomo are indeed smaller and amount on average to 31 ± 5 Hz (For HET-s(218–289) fibrils, Δrved see  which ex218–289) . The glu1 ± 5 Hz .homo values observed for the asparagine side-chains are related to coherent broadening mechanisms, we have numerically simulated the 1H solid-state NMR spectrum using the geometry of the six NH2 proton spins of N262-N226-N262 taken from the PDB file  fibrils. Typical rigid backbone nitrogen atoms possess R1ρ values of around 2 s−1  pointing to a higher degree of flexibility of these side-chains. Based on the measured R1ρ values, the side-chain of Q240 is similarly rigid as N226 and N262, whereas Q259 is more flexible and shows weaker correlations in 13C-detected 3D spectra  experiments  on a millisecond timescale to explain the broader-than-expected lines. Such processes have been observed for asparagine side-chains in T4 lysozyme  or in coeriments , but wercess see . Howeverperiment . Whether2 are observed in the CP and INEPT-spectra , no ladder formation is possible from the structure . Both gl-spectra , 3D. For2 side-chains proton signals in fast MAS spectra of HET-s(218–289) in its amyloid form. The spectral and relaxation properties are described and the influence of the formation of ladders is investigated. 2 protons, (ii) a large chemical-shift difference between the two protons, (iii) correlations in 3D through-space hCONH experiments, and (iv) small 15N R1ρ relaxation-rate constants. These properties characterize the N226-N262 asparagine ladder. In addition (v) a large homogeneous proton linewidth and (vi) a large 1H R1ρ relaxation-rate constants were found. 15N rotating-frame relaxation rate constants indicate that the side-chains involved into the N226-N262 asparagine ladder are more rigid than for the N243-N279 ladder which appear more dynamic not forming a strong restraining hydrogen network. The large homogeneous proton linewidths for asparagine side-chains involved in the ladder, not fully understood yet, could be related to millisecond timescale chemical exchange characteristic for protons engaged in an asparagine ladder in HET-s(218–289) fibrils. The glutamine side-chain amide protons of HET-s(218–289) not participating in such interactions are less homogeneously broadened and have linewidths comparable to the backbone NH protons. Fast MAS NMR experiments thus probe protein side-chain hydrogen bonding, in particular in the context of asparagine/glutamine ladder formation. While more systems must be investigated for a full understanding, the results on HET-s(218–289) clearly show the potential of the approach.We detected asparagine and glutamine NHHpDnaB:ADP:AlF4–:DNA (2O in presence of deuterated 13C-glucose (2 g/L) and 15N-ammonium chloride (2 g/L) as sole sources of carbon-13 and nitrogen-15. In case of HET-s(218–289), fibrillization was performed in protonated solvent (50 mM citric acid at pH 5 in H2O) for 24 h which yields 100% back-exchanged fibrils  is described in reference 2O instead of D2O and protonated 13C-glucose (2 g/L) and 15N-ammonium chloride (2 g/L).The preparations of the DUL lF4–:DNA  and DUL lF4–:DNA  samples  fibrils . For Dnahttps://doi.org/10.3929/ethz-b-000430151.Solid-state NMR spectra were acquired at 20.0 T static magnetic-field strength using a Bruker 0.7 mm probe. The MAS frequency was set to 110 kHz. The 2D spectra were processed with the software TOPSPIN  with a shifted (3.0) squared cosine apodization function and automated baseline correction in the indirect and direct dimensions. The sample temperature was set to 293 K . All spe1H MAS spectra were performed with SIMPSON . The orientation-dependence of the 1H chemical-shift values was studied by moving the two molecules relative to each other along the N-H… O distance vector (The DFT calculations were performed with TURBOMOLE  version e vector  or movine vector . No addiAll datasets generated for this study are included in the article/RC prepared the samples. TW and AM performed the NMR experiments. TW, AM, ME, AB, and BM analyzed the data. TW and ME performed the simulations. TW, AB, ME, and BM designed and supervised the research. All authors contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SARS-CoV-2 causes the expansion of colony formation by erythroid progenitors and can be detected in these cells after 2 weeks of the initial infection. Our findings constitute the first report of SARS-CoV-2 infectivity in erythroid progenitor cells and can contribute to understanding both the clinical symptoms of severe COVID-19 patients and how the virus can spread through the circulation to produce local inflammation in tissues, including the bone marrow.We document here that intensive care COVID-19 patients suffer a profound decline in hemoglobin levels but show an increase of circulating nucleated red cells, suggesting that SARS-CoV-2 infection either directly or indirectly induces stress erythropoiesis. We show that ACE2 expression peaks during erythropoiesis and renders erythroid progenitors vulnerable to infection by SARS-CoV-2. Early erythroid progenitors, defined as CD34  •COVID19 severe patients show severe anemia and increase in nucleated red blood cells•Erythroid progenitors co-express ACE2 and TMPRSS2•Early and mid-late erythroid progenitors could be infected by SARS-CoV-2•SARS-CoV-2 can be detected in erythroid colonies 14 days after the initial infection H. Huerga Encabo and colleagues demonstrate that SARS-CoV-2 can infect erythroid progenitors without impairing their functionality, rather causing an expansion of colonies where the virus can be detected. These findings shed light into the patients with severe COVID-19 who suffer from an aberrant increase of nucleated red blood cells concomitant with reduced levels of hemoglobin. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected and spread globally among humans, causing a pandemic crisis. The resulting Coronavirus Disease 2019 (COVID-19) is a spectrum of responses to SARS-CoV-2 infection, from asymptomatic individuals to severe disease and mortality .The mechanism of infection by SARS-CoV-2 is fairly well characterized. For viral entry, the binding between the viral surface spike glycoprotein (S) and target cell surface angiotensin-converting enzyme-2 (ACE2) followed by cleavage of S by the transmembrane protease serine 2 (TMPRSS2) are required . ACE2 exFurther reports demonstrate ACE2 expression in cells from other tissues such as intestinal epithelial cells, hepatocytes, and neurons, making these cells vulnerable to be infected by SARS-CoV-2 . In linePatients treated for severe COVID-19 (as defined by requirement for intensive care admission) at King's College Hospital  show a rapid and profound decline in hemoglobin following admission A. Interein vitro human erythropoiesis . Intrigued by this observation, we used detailed datasets of opoiesis  to charaopoiesis C. Duringopoiesis C. Intereopoiesis D. S1 difopoiesis  as expecopoiesis .+CD117+CD71+CD235a−, ERP-S2 as CD34−CD117+CD71+CD235a− and ERP-S3 as CD34−CD117−CD71+CD235a+  and ERP-S3 (CD71+CD235a+), by SARS-CoV-2. Especially relevant is the high ability (or facility) of the virus to infect ERP-S2, the most vulnerable erythroid progenitor population. Based on previous works and our observations, ERP-S2 population includes CFU-E cells and early pro-erythroblast cells  do not express ACE2 and TMPRSS2 at the RNA or protein levels. Consequently, we show that HSPCs are not infected by SARS-CoV-2.In contrast to the observations in erythroid progenitors, we report that bone marrow HSPCs (CD34+CD235a−). Importantly, our results might also have an impact in our understanding of the transmission and incubation period of SARS-CoV-2, as different levels of infected progenitors in the bloodstream could greatly alter such parameters. Finally, identification of infected ERP-S2 populations in hospitalized patients could help identify those patients who will suffer from severe hematopathology, potentially allowing preemptive management strategies to improve outcomes.In this report, we also provide clinical data of a cohort of 30 patients with COVID-19 who were treated in intensive care units (ICU) at King's College London. We show that the decline in hemoglobin levels coincides with an aberrant increase of nucleated red blood cells in circulation. These nucleated red blood cells correspond to ERP-S3 cells . SimilarA sample of 30 patients who were being treated for COVID-19 in ICUs at King's College Hospital on May 1, 2020, and who had first tested positive for SARS-CoV-2 by PCR within 7 days of admission to hospital, was identified using information from the hospital's Business Intelligence Unit and electronic patient record (EPR) system. Values for peripheral blood hemoglobin concentration, white blood cell count, platelet count, and nucleated red blood cell percentage on the day of presentation to hospital and subsequent 28 days were collected from the EPR system. In cases where multiple tests were performed in the same day, the first value was collected.GSE118537).RNA sequencing dataset was downloaded from GEO database . The collection and use of these samples was approved by the East London Research Ethical Committee (REC:06/Q0604/110) and in accordance with the Declaration of Helsinki. Mononuclear cells were isolated by density centrifugation using Ficoll-Paque . Human bone marrow mononuclear cells were obtained from StemCell Technologies (Cat# 70001). Peripheral blood was isolated from consenting unscreened healthy adult volunteers following approved protocols by the ethics board of the Francis Crick Institute and the regulations of the Human Tissue act 2004. Peripheral blood mononuclear cells were isolated by centrifugation over a Histopaque-1119 gradient .Total RNA was extracted using the RNeasy Microkit , and the RNA was retrotranscribed using the SuperScript III First-Strand Synthesis system . For real-time qPCR, PowerUP SYBR Green , MicroAmp Optical 384-Well Reaction Plate , and the Applied Biosystems QuantStudio 7 were used according to the instructions provided by the manufacturers. For the detection of SARS-CoV-2 we used previously validated primers . See priAll experiments were analyzed at the Flow Cytometry core facility of The Francis Crick Institute using the LSR FORTESSA (BD Biosciences) equipped with a 488-nm laser, a 561-nm laser, a 633-nm laser, and a 405-nm laser. For sorting, cell suspensions were filtered through a 35-μm nylon mesh  and sorted in a BD FACS FUSION cell sorter equipped with 488-nm, 561-nm, 633-nm, and 405-nm lasers. See antibodies used in the Polyclonal goat anti-ACE2 antibody  at 15 μg/mL and polyclonal rabbit anti-TMPRSS2  at 15 μg/mL were used for the primary antibody incubation. Donkey anti-goat Alexa 647  and donkey anti-rabbit Alexa 488  were used at 10 μg/mL for the secondary antibody staining. Fluorescence imaging was performed at a Leica TCS SP5 inverted confocal microscope using the sequential scan in between frames mode with a ×63 objective. Background signal was accounted for by performing secondary only staining controls. Fiji/ImageJ version 2.0.0 software was used for image analysis. Fluorescence intensity quantifications were done by selecting individual cells using an intensity-based mask at the DAPI channel of at least five different representative regions of interest per group of cells. This mask was then applied for the quantification of fluorescence intensity in the 488 channel (TMPRSS2) and the 647 channel (ACE2).Vero E6 cells  were maintained in DMEM modified with high glucose, L-glutamine, phenol red and sodium pyruvate  supplemented with 10% fetal calf serum (FCS). SARS-CoV-2 strain BetaCoV/England/02/2020  was propagated at 37°C on VeroE6 cells in DMEM supplemented with 2% FCS. The titer was determined by plaque assay as follows: confluent monolayers of VeroE6 cells grown on six-well plates were incubated with 200 μL of a 10-fold serial dilution of virus stock in DMEM supplemented with 10% FCS for 1 h at room temperature. The cells were then overlaid with 0.5× DMEM supplemented with 1% FCS and 1.2% Avicel . After 4-d incubation at 37°C, cells were fixed with 4% formaldehyde in PBS followed by staining with 0.1% toluidine blue . The titer was calculated as plaque-forming units (PFU) per mL. Hematopoietic cells were infected with SARS-CoV-2 at a MOI of 5 PFU per cell. Cells were washed three times with PBS to remove unbound virus prior to lysis for RNA isolation.After 24 h of infection, cells were seeded for 2 weeks in methylcellulose-based medium with recombinant cytokines for human cells . Two independent experiments with two to three biological donors in each experiment were analyzed to assess the number of CFUs in each cell population.Statistical methods relevant to each figure are outlined in the figure legend. Sample size was not predetermined. Data are presented as means with SD to indicate the variation within each experiment. A two-way ANOVA test was used for the comparison between the different cell populations.Conceptualization: H.H.E., W.G., and D.B.; Methodology: H.H.E., W.G., R.U., and I.V.A.; Analysis and interpretation: H.H.E., W.G., M.G.A., H.W., R.U., I.V.A., and D.B.; Patient data analysis: A.G.K., G.M., and H.W.; Writing paper: H.H.E. and D.B.; Review & Editing paper: H.H.E., W.G., M.G.A., H.W., V.P., R.B., and D.B.; Funding Acquisition: D.B.


Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains. This sequence of events, called homing, is initiated by the interaction of selectins  with their ligands (–The multistep paradigm of cell migration outlines a sequence of events that leads cells ( ligands –3. Althox) GlcNAc1-R) cNAc1-R) , which icNAc1-R) –9. StudiNAc1-R) –, but lesThe selectins are structurally composed of five distinct domains: an N-terminal extracellular C-type lectin-like domain, followed by an endothelial growth factor (EGF)–like domain, a defined number of short consensus repeats (SCRs) with 60 amino acids per motif, a transmembrane domain, and a C-terminal cytoplasmic tail that is likely involved in signal transduction regulation –15. Alth–17–20–Studies using P-selectin suggest that it adopts a bent (lower affinity) conformation in the absence of its sulfated ligand but in its presence shifts toward an extended (higher affinity) conformation . MoreoveA) secreted by a novel silkworm expression system that included dimeric and monomeric forms harboring varying numbers of SCR domains as well as an enforced extended conformer. These E-selectin proteins allowed us to demonstrate the influence of the various structural domains and conformational extension on the overall ability of E-selectin to bind its ligands. By employing a comprehensive set of assays under static and physiological flow conditions, we showed that SCR domains, the conformational extension of the lectin and EGF domains, and protein dimerization contribute to the efficient binding of E-selectin to its glycosylated ligands. Moreover, these three structural elements collectively contribute to the association rate, whereas the dissociation rate was mainly controlled by the lectin and EGF domains. These findings correlate, for the first time, a very important physiological role for the association rate in the functional binding of the selectin to its ligands.In this study, we generated five recombinant E-selectin proteins see A secreteN-glycosylation events that take place in insect cells are mainly pauci-mannose whereas in mammalian cells, they are more complex . The differences in glycosylation patterns that result can affect the activity, stability, and solubility of the protein . This observation highlights the suitability of silkworm as an alternative system for expressing E-selectin.A Western blot analysis comparing the binding of two common E-selectin ligands, CD44/HCELL (hematopoietic cell E- and/or L-selectin ligand) and PSGL-1 (P-selectin glycoprotein ligand-1) –33, to d–A): i) E-S6-IgG, which is a full-length dimeric protein that consists of a lectin domain followed by an EGF-like domain; six SCR domains; and an Fc region of IgG that aids in the dimerization of the molecule; ii) E-S6, which is a monomeric version of E-S6-IgG lacking the Fc region; iii) E-S2, which is a truncated protein with only the first two SCR domains; and iv) E-S0, which lacks any SCR domains. These protein constructs were successfully purified and did not appear to aggregate (B). On the other hand, the dimeric E-selectin, E-S6-IgG, was primarily dimerized (as facilitated by the Fc region) with a small amount showing up as a monomer . These observations suggest an absence of aggregation among the E-selectin proteins purified from the silkworm system.To address the role of SCR domains and protein dimerization in the binding activity of E-selectin, various E-selectin constructs with varying numbers of SCR domains were generated A: i) E-Sggregate B. On the+) cells, which are able to bind E-selectin (A), then analyzed by flow cytometry. As illustrated in Fig. 2A, E-S6-IgG, E-S6, E-S2, and E-S0 were all capable of binding the KG1a cells to varying degrees. As shown in Fig. 2B, E-S6-IgG bound to the ligands on 96% ± 0.8 of the live KG1a population, whereas E-S6 and E-S2 bound to significantly fewer . Interestingly, we found that the construct containing only the lectin and EGF domains (E-S0) bound the lowest percentage of KG1a cells . E-S6-IgG displayed the strongest fluorescence signal, followed by E-S6, E-S2, and E-S0 . Additionally, samples stained in the presence of EDTA, which removes divalent cations and inhibits the binding of selectins to their ligands, were included as controls to confirm binding specificity . Furthermore, we used several increasing concentrations of each construct to test if the binding ability of the weaker constructs would improve upon increasing their concentrations in solution. The results confirmed that the binding trend of each construct remained the same and did not improve for the weaker constructs to the degree that is comparable with the stronger-binding constructs .We used flow cytometry to confirm the binding functionality of our recombinant E-selectin proteins on known E-selectin ligands under native conditions. In these experiments, KG1a cells were used to represent HSPC-like  and PSGL-1  compared with the other constructs. E-S6 exhibited visibly stronger staining to both ligands compared with E-S2 or E-S0 . Furthermore, identical blots were stained with E-selectin constructs in the presence of EDTA to chelate out the calcium that is essential for binding and binding was abrogated . Overall, these results suggest that the strongest ligand binding is to the dimeric form of E-selectin and that the SCR domains contribute to this interaction. Because the binding of the E-selectin proteins to PSGL-1 and CD44/HCELL was similar, we chose to focus on the latter for further characterization of the E-selectin proteins.Western blot analysis was conducted to determine the ability of the recombinant E-selectin protein constructs to bind common E-selectin ligands. To this end, PSGL-1  and CD44Fig. 2A). An overall decrease was observed in the binding of E-selectin to different glycans upon reducing or omitting the SCR domains (B). In particular, shorter E-selectins (E-S2 and E-S0) showed a gradual decrease in the binding to sLex -GlcNAc-β) and to two sLea structures -GlcNAc-β- and Neu5Gc-α-2,3-Gal-β-1,3--GlcNAc-β)) compared with E-S6-IgG and E-S6 (C). This indicates that the recognition level of each E-selectin construct toward sLex/a structures is similar to that toward intact ligands. A list of all the glycans within a microarray is shown in Fig. S5.Furthermore, glycan-binding microarray analysis was conducted for each E-selectin construct A. An ove domains B. In parand E-S6 C. This iA). Each channel was coated with equal amounts of protein A, followed by an antibody against the histidine tag (A), which was found to consistently immunoprecipitate each recombinant E-selectin . The rolling velocities (B) and the numbers of rolling cells (C) were determined for each E-selectin protein and found to be consistent with our observations above. An analysis of the rolling velocities revealed that E-S6-IgG, followed by E-S6, supported the slowest rolling velocities with 0.15 ± 0.03 μm/s and 0.55 ± 0.05 μm/s, respectively, whereas the shorter proteins supported much faster rolling velocities (1.28 ± 0.26 μm/s for E-S2 and 4 ± 0.16 μm/s for E-S0) (B). Longer constructs with six SCR domains supported the highest number of rolling KG1a cells with 161 ± 22 rolling cells on E-S6-IgG and 117 ± 15 on E-S6; significantly fewer cells rolled on the truncated constructs  (C). Moreover, similar results were obtained when Fab fragments of the anti-histidine antibody were used in place of the intact antibody . Interestingly, forced dimerization, induced by using the intact antibody, effectively reduced the rolling velocities for the shorter constructs compared with the Fab fragments, further supporting the influence that dimerization of E-selectin has in promoting binding to its ligands.We next characterized the differences in binding among the E-selectin protein constructs using a physiological flow-based binding assay. A modified form of the parallel-plate flow-based binding assay –42 was u–Fig. 1dine tag A, which locities B and theng cells C were deor E-S0) B. Longer; n = 3) C. MoreovB). Additionally, the fluorescence signal of E-S2-A28H (1689 ± 352.5) was only ∼2-fold lower than that of E-S6, whereas the signal of E-S2 was ∼3-fold lower than that of E-S6 , making the difference between the fluorescence signals of E-S2-A28H and E-S6 insignificant compared with the significant difference observed between E-S2 and E-S6 . Moreover, flow-based experiments evaluating the rolling velocities of KG1a cells on immobilized E-S2-A28H showed a significant reduction in the rolling velocity (0.33 ± 0.06 μm/s)  compared with E-S2 (rolling velocity = 1.28 ± 0.26 μm/s), and even approaches that of E-S6 (B). Together, these data illustrate the importance of the SCR domains, conformational extension, and dimerization in E-selectin binding behavior and in mediating slower rolling velocities.Previous studies of the interaction between selectins and their ligands improved our understanding of the mechanical behavior of selectin-ligand interactions , 43–55. 43–Fig. 2 of E-S6 B. TogethA). Then, the E-selectin proteins were injected in sequential concentrations at similar flow rates to determine the equilibrium dissociation binding constant (DK) and to estimate the association (kon) and dissociation rate constants (koff). The captured CD44 bound various forms of recombinant E-selectin constructs, with a DK of 170 ± 20 nm for the full-length dimeric E-S6-IgG and significantly higher DK of 2040 ± 490 nm, 5680 ± 1170 nm, and 17250 ± 2540 nm for E-S6, E-S2, and E-S0, respectively  (B–D and F–G). To better understand the reason for these differences among the DK values of each construct, we determined the kon and koff. At the dissociation phase, all of the E-selectin proteins tested showed similar koff values, demonstrating that neither dimerization nor the SCR domains are required for the stable binding of E-selectin with CD44/HCELL. Strikingly, in contrast to the dissociation rate, the association rate constants revealed different binding kinetics. Full-length E-S6-IgG exhibited a kon (950 ± 110 m−1s−1) that is ∼9-fold higher  than that of monomeric full-length E-S6 (110 ± 15 m−1s−1). Moreover, shorter constructs displayed significantly lower kon values; E-S2 exhibited kon values ∼4-fold lower than E-S6 (30 ± 5 m−1s−1), and E-S0 exhibited kon values ∼28-fold lower than E-S6 (4 ± 1 m−1s−1) . Furthermore, we confirmed that the binding of the shortest construct (i.e. E-S0) at high concentration does not seem to be a result of aggregation .We used our previously described real-time surface plasmon resonance (SPR) binding assay  to quantFig. 5DK for the binding of E-S2-A28H (E and G) to immobilized CD44/HCELL was 2500 ± 660 nm. This DK is ∼2-fold higher than E-S2 (DK = 5680 ± 1170 nm). On the other hand, the difference between the affinities of E-S2-A28H and E-S6 (DK = 2040 ± 490 nm) decreased by only ∼1-fold compared with the ∼3-fold difference between E-S2 and E-S6. The kon for E-S2-A28H was ∼3-fold higher (80 ± 6 m−1s−1) than the kon for E-S2 (30 ± 5 m−1s−1) and approached that of E-S6 (kon = 110 ± 15 m−1s−1)  (G). Interestingly, the koff was not influenced by the mutation in E-S2-A28H (E and G). These findings indicate that binding can be improved by extending the lectin and EGF domains, even under conditions where the binding via the SCR domain is weakened. They also suggest that the extended structure contributes to the on rate of E-selectin binding, similar to the role of dimerization and the SCR domains. Consistent with the physiologically relevant flow data above, the SPR results further support that the SCR domains are indeed intrinsically important to the improved binding affinity of E-selectin. They also reveal the critical roles of the SCR domains, protein extension, and oligomerization in influencing the on rate, but not the off rate, of E-selectin binding to its ligands.The -S2-A28H , E and G; n = 3) G. Intere-S2-A28H , E and GEmploying a novel silkworm expression system, we were able to purify five different E-selectin proteins in high yield and high purity. We showed that the binding activities of E-selectin to its ligands were improved by increasing the number of SCR domains, by forming an extended lectin and EGF domain structure, and by dimerizing/oligomerizing the protein. These observations were consistently verified by the analysis of these constructs in a number of static and flow-based assays. Our findings further unveiled that all three of these factors contribute to the on rate of E-selectin binding to its ligands, but do not significantly influence its off rate.The contribution of the SCR domains to ligand binding has been shown previously for P-selectin . In thatTo begin to understand how the SCR domains influence the binding affinity of an E-selectin, it is interesting to note that quite a low conservation of SCR domains exists (only 35%) among the selectins , which cx-expressing ligand, whereas the SCR domains contribute more to increase the association rate by interacting with other regions of the ligand. It is also possible that the SCR domains might influence the conformation of the selectin to better expose the lectin and EGF domains to facilitate binding to its ligand.Structurally, SCR domains are indeed important for sufficiently extending the lectin domain of E-selectin to access the ligands on cells traveling through the blood. A simple model proposes that the full-length E-selectin tends to be in closer proximity to cells in the blood flow than the truncated constructs, leading to a higher frequency of interactions with their ligands. Additionally, our observations suggest that SCR domains may play a more direct functional role in improving the binding affinity of E-selectin; when E-S6-IgG, E-S6, E-S2, and E-S0 were introduced in solution to their ligands, they each exhibited distinctive binding characteristics. It is likely that the lectin domain is more involved in the actual binding to its sLex and sLea units (x/a) varies depending on the dimerization and the number of SCR domains. These findings are more likely to support the role of SCR domains in presenting the lectin and EGF domains in the proper conformation and, therefore, supporting the binding functionality of E-selectin. However, further detailed structural studies are needed to confirm this hypothesis.Testing the binding activity of our constructs toward different glycan structures revealed that all the constructs recognize sLeea units , albeit x was introduced by soaking it into the bent conformer crystals, it bound to both selectins without any observed changes to the conformation. However, co-crystals formed following the binding of a sulfated fragment of PSGL-1 with P-selectin, or the binding of a glycomimetic of sLex with E-selectin, resulting in an extended conformation of both selectins , except for a single mutation that removed the free cysteine residue  to eliminate crosslinking. Then, the construct was synthesized and obtained at the institutional core facility, Bioscience Core Lab (KAUST BCL). The C-terminal His6 tag (8 (6), and an XhoI restriction site. Similarly, E-S0 was produced in a PCR reaction identical to the E-S2 PCR synthesis, with the replacement of the reverse primer CCATCATGCAAAGCTtccggcctgaacgac with CCCAACTGTGAGCAAtccggcctgaacgacatcttcgaggctc. Subsequently, the E-S2 and E-S0 constructs were subjected to a second PCR reaction to add Strep-tag, followed by a second polyhistidine, (His6), with the forward primer ATGAATGCCTCGCGCTTTCTCTCT and the overlapping reverse primers CCAAGCTCTTGAatgatgatggtgatgatgttcgt, catTCAAGAGCTTGGCGTCATCCGCAGTTCGGTGG, tcaGTGATGGTGATGGTGATGACCACCGAACTGCGga, and aggttaCTCGAGtcaGTGATGGTGATGGTGATGAC. The TEV and Avi tags were included for future work and are not utilized in the current study.A E-S6-IgG chimera common construct, containing a full-length extracellular region of mouse E-selectin and an Fc region of human IgG1 attached with HisHis6 tag  was replHis6 tag , 74 comp6 tag , whereas the pENTR11 (Invitrogen) vector was digested with XhoI and XmnI (GAAnn∧nnTTC) (New England Biolabs). The fragments were directionally cloned into pENTR11 by ligation using T4 DNA ligase (Invitrogen). E-S2-A28H was created by subjecting E-S2-pENTR11 recombinant vector to site-directed mutagenesis. A PCR reaction was performed using the forward primer TACACACATCTGGTGcacATTCAGAACAAGGAA and reverse primer TTCCTTGTTCTGAATgtgCACCAGATGTGTGTA. The GatewayTM LR recombination reaction was performed using LR clonase to produce recombinant pDEST8 expression vectors  containing the different forms of E-selectins under the transcriptional regulation of a PPH Polyhedrin promoter. Design of all constructs and primers was performed using ApE-A Plasmid Editor.Truncated E-selectin constructs were cloned into a pENTR11 (Invitrogen) vector using the GatewayRecombinant pDEST8 expression vectors, constructed as described above, were used to generate recombinant viral DNAs using a BmNPV/T3 bacmid system, as described previously . The virm 1-phenyl-2-thiourea. Following brief centrifugation, they were stored at −80 °C until use.The recombinant E-selectins/BmNPVs were infected into the hemocoel of 2-day-old fifth instar silkworm larvae  using a microliter syringe with a 30-gauge needle (Hamilton Co.). At 4 days post infection, the sera from the silkworm larvae were collected in a 15-ml tube containing 20 mm Tris-HCl, pH 7.4, 0.5 m NaCl, 1 mm PMSF) was purified by nickel affinity chromatography with a HisTrap Excel column  and eluted by increasing the concentrations of imidazole solution buffers (100–500 mm imidazole). Then, the eluted solution was concentrated  and diluted with the binding buffer B ) (Roche). A final 50 ml diluted solution was applied to the StrepTrap HP column , followed by elution with buffer B, which contained 2.5 mm desthiobiotin. Finally, the purified recombinant E-selectins were dialyzed against 1× PBS (pH 7.4), separated through 10% SDS-PAGE electrophoresis and quantified by Pierce BCA Protein Assay kit.Purification of the recombinant E-selectins was modified from our previously published protocol . Briefly+ HSPCs (ATCC). The cells were cultured in RPMI 1640 media (Gibco) supplemented with 10% fetal bovine serum (FBS) (Cellgro) and 100 units/ml of HyClone penicillin/streptomycin (Invitrogen). These cell lines were grown and maintained in a suspension culture at a density of 1 × 106 cells/ml, under humid conditions in a 37 °C incubator supplemented with 5% CO2.Human acute myelogenous leukemia (KG1a) cell line was used as the model for the CD346 cells/ml) were stained with 10 μg/ml of each of the E-selectins, E-S6-IgG, E-S6, E-S2, E-S2-A28H, or E-S0, in 100 μl HBSS with 5% FBS, in the presence of either 2 mm CaCl2 or 20 mm EDTA, at 4 °C for 20 min. Next, the stained cell samples were washed with the same HBSS buffer (containing either 2 mm CaCl2 or 20 mm EDTA) one time, and further stained with 10 μg/ml of either Strep-tag II mAb (EMD Millipore), mouse IgG1 PE conjugated isotype control antibody (R&D Systems), or PE rat anti-mouse IgG1 (Clone A85–1) (BD Biosciences) as the secondary control, and then were incubated at 4 °C for 20 min. The samples were then washed two times with the same washing buffer and incubated with 10 μg/ml of PE rat anti-mouse IgG1 (Clone A85–1) for another 20 min at 4 °C. Finally, the stained cells were again washed two times and analyzed using BD FACSCanto (BD Biosciences) and FlowJo software (version 7.6.1).The KG1a cells  (Fisher Scientific), 1× of protease inhibitor (Roche), 1 mm of PMSF (Sigma Aldrich) and 1% of Triton X-100 (Thermo Fisher Scientific) at a density of 1 × 106 cells/16 μl of lysis buffer, and were rotationally mixed for 1 h at 4 °C. The soluble fraction was collected by centrifugation at 16,000 relative centrifugal force for 30 min at 4 °C. PSGL-1 and CD44 were immunoprecipitated separately from the heterogeneous mixture of proteins by incubation with 50 μl of prewashed Protein G Dynabeads (Thermo Fisher Scientific) complexed with either 3 μg of mouse anti-human CD162 (clone KPL-1) (BioLegend) or 3 μg of mouse anti-human CD44 (clone 515) (BD Biosciences) and mixed rotationally overnight at 4 °C. Next, the supernatants were removed, and the beads were washed three times with lysis buffer chilled to 4 °C.The KG1a cells were lysed using the membrane disruption method with Triton X-100–based lysis buffer (150 m7 KG1a cells/well and transferred to an immunoblot PVDF membrane (Bio-Rad) in 1× Tris-Glycine buffer (Sigma). The resulting membrane was blocked with 5% of nonfat milk in 1× TBST buffer  containing 137 mm sodium chloride, 20 mm Tris, and 0.1% Tween 20 at pH 7.6. The prepared blots were stained individually with 1 μg/ml of E-S6-IgG (either homemade from silkworm or produced from NS0 cells) (R&D Systems), E-S6, E-S2, E-S2-A28H, or E-S0 in 1× TBST buffers containing 2 mm CaCl2 or 20 mm EDTA and were subsequently stained again with 1 μg/ml Strep-tag II mAb, followed by incubation with 0.057 μg/ml of HRP-conjugated goat anti-mouse IgG (Thermo Scientific) or, for staining the dimeric E-S6-IgG proteins from both silkworm and mammalian cells, directly with HRP-goat anti-human IgG (SouthernBiotech) at 1:10,000 . Lastly, the membranes were exposed to SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for visualization via the chemiluminescence reactions and imaged using ImageQuant LAS 4000 .Protein G Dynabeads with immunoprecipitated PSGL-1 and CD44 were prepared for PAGE by resuspending the beads in 2× NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) in PBS (Gibco) and 10% β-mercaptoethanol (Sigma-Aldrich). The sample was then heated at 95 °C for 5 min to assess the denaturation process by releasing the capturing antibody with the immunoprecipitated PSGL-1 or CD44 from the protein G beads and into the solution. For the Western blot analysis, the prepared samples were loaded into a 4–20% SDS-polyacrylamide gradient gel (Bio-Rad) in 1× Tris-Glycine SDS buffer (Sigma) as lysates of 1 × 10To characterize the possible aggregation properties of our E-selectins, protein samples were also prepared for native gel electrophoresis at a final volume of 10 μl in 1× of 4× native PAGE sample buffer (Invitrogen). The prepared samples were loaded into a 10% 10 wells TBE gel (Novex), and electrophoresis was conducted in 1× Tris-glycine buffer for 3 h at 200 voltage. Subsequently, the resulting gel was stained using G-250 Coomassie Brilliant Blue stain (Millipore), then destained in water.m CaCl2. 400 μl of each construct was then added to one glycan array. Samples were incubated for 3 h at 4 °C with continuous mixing (low shaking speed ∼60 revolutions per minute (rpm); Thermo Fisher). Each array was washed four times with 800 μl of 1× wash buffer I and two times with 800 μl of 1× wash buffer II; each washing step was performed for 5 min at low shaking speed (∼60 rpm). Next, each array was incubated overnight (4 °C) with 400 μl of biotin anti-His6 tag antibody (BioLegend)  with low shaking speed (∼60 rpm); the slide was covered with adhesive film to protect it from environment and contamination. The following day, the antibody solution was removed, and each array was washed, as stated above. Next, 400 μl of Cy3 equivalent dye-steptavidin (prepared as 1× in sample diluent buffer) was added to each array and incubated for 1 h at room temperature at low shaking speed (∼60 rpm). Cy3 equivalent dye-steptavidin was then removed, and each array was washed as described above. Subsequently, the gasket defining each array as a separate well was removed, and the slide was soaked consecutively in the following solutions in a slide washer/dryer (RayBiotech): 30 ml 1× wash buffer I, 30 ml 1× wash buffer II, and 30–40 ml milliQ; each wash was performed for 5 min at room temperature. Finally, the residual solution was removed from the surface of the chip and the chip was imaged using the Typhoon Trio variable mode imager . Finally, the resulted intensities were measured for each spot using ImageJ software.Sandwich-based protocol was followed in accordance with the manufacturer's instructions using a 4-array glycan chip . First, each array was blocked using 400 μl of sample diluent at room temperature for 30 min. Next, the sample diluent was removed and a 135 μg/ml solution of each construct  was prepared in sample diluent containing 2 mm filter) and degassed 1× HBS-EP buffer  . Mouse anti-human CD44 Hermes-3 (Abgent) was immobilized on each flow cell of the CM5 surface using an amine-coupling procedure. In this method, each flow cell was activated by injecting a 1:1 ratio of N-hydroxysuccinimide (0.1 m) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.4 m) for 8 min at a flow rate of 5 μl/min. Then, 20 μg/ml of the antibody was injected in 10 mm sodium acetate phase (pH 5.0) at a 10 μl/min flow rate, to allow amine-mediated immobilization of the antibody on the CM5 flow cell surface; the resulting amount of immobilized antibody was monitored as indicated in m ethanolamine hydrochloride at a flow rate of 5 μl/min over a total period of 8 min. Using the same procedure, mouse IgG2a κ isotype control (BioLegend) was immobilized on a control flow cell. To prepare for CD44 IP, KG1a cells were lysed at a density of 3 × 107 cells in 300 μl of a lysis buffer containing 1% Triton X-100, 250 mm NaCl, 50 mm Tris base (pH 8.0), and 1 mm CaCl2 mixed on a rotator for 1 h at 4 °C. Subsequently, the clear phase was collected by centrifugation at 16,000 relative centrifugal force for 30 min. The system was primed twice using a filtered and degassed running buffer  to exchange the buffer on the flowing cells. CD44 real-time IP was initiated by injecting the lysate at a 20 μl/min flowrate for 13 min, followed by a 15-min washing step. The E-selectins were titrated in running buffer and injected at a sequence of concentrations, as indicated in Affinity and kinetics assessments of the E-selectins' binding were performed using the Biacore T-100 system on a carboxymethylated (CM5) dextran sensor chip  at 25 °C, as described previously . First, 0.1 microscopy chamber (ibidi) were coated with protein A (Thermo Fisher) at 10 μg/ml in HBSS (Gibco) for 16 h. Then each channel was washed in one step. After the channels were completely dry, mouse anti-histidine (AbD Serotec) was added as a second coating at a concentration of 10 μg/ml in HBSS for 1 h at 4 °C, then each channel was washed as described above. The E-selectins  were added to the channels at 5 μg/ml (diluted in HBSS) and incubated at 4 °C for 16 h to uniformly immobilize them by utilizing the histidine tag located at the C terminus of each E-selectin (A). Next, the channels of the chamber were washed and blocked using HBSS containing 1% BSA for 1 h at 4 °C, then washed again. The prepared chamber was placed on the stage of an inverted microscope (Olympus) connected to a CCD camera to visualize and record the rolling events using Cell Sens software. Shear stress within a rectangular channel was calculated as described previously (2), μ is the viscosity of the fluid (0.00076 Pa.s), Q is flow volume (ml/s), a and b are the height and width of the channel (0.1 mm and 1 mm), respectively.The cell rolling assay permitted us to evaluate the incorporated shear stress conditions on the physiological interactions of the E-selectins with their ligands. In this assay, the six channels of an uncoated μ-slide VIselectin A. Next, eviously  using Eq6 cells in 1 ml of HBSS containing 1% BSA and 0.5 mm CaCl2 or 5 mm EDTA), whereas the outlet was connected to a highly programmable Harvard syringe pump. The suspended KG1a cells were gradually drawn through the channel first at 90 dyne/cm2 for 1 min, and then allowed to attach to the immobilized constructs by decreasing the shear stress rate to 0.3 dyne/cm2 for 5 min. Next, various shear stress rates were introduced sequentially, corresponding to a sequence of 1 dyne/cm2, 2 dyne/cm2, 3 dyne/cm2, 4 dyne/cm2, 5 dyne/cm2, and 6 dyne/cm2 for 30 s each. The recorded rolling incidents of the KG1a cells on the immobilized E-selectins were analyzed using Imaris software to evaluate the number of rolling cells and their rolling velocities on each E-selectin.The inlet of each channel was connected by a tube (0.8 mm silicone tubing from ibidi) to a cell-containing buffer (1.2 × 10t test. Statistical significance was defined as p ≤ 0.05.Data are reported as the means ± S.E.M. Statistically significant differences between the means of each E-selectin were resolved using an unpaired Student's F. A. A., K. S., and J. S. M. conceptualization; F. A. A., K. S., J. M. L., D. B. A., B. A., S. N., M. T., and J. S. M. data curation; F. A. A., K. S., J. M. L., D. B. A., B. A., S. M. H., S. H., T. K., and J. S. M. formal analysis; F. A. A., K. S., J. M. L., D. B. A., and J. S. M. investigation; F. A. A., K. S., J. M. L., D. B. A., B. A., and J. S. M. methodology; F. A. A., K. S., J. M. L., D. B. A., and J. S. M. writing-original draft; F. A. A. and J. S. M. writing-review and editing; J. M. L., T. K., and J. S. M. resources; T. K. and J. S. M. project administration; S. M. H., S. H., T. K., and J. S. M. supervision; S. M. H., S. H., and J. S. M. validation; T. K. and J. S. M. funding acquisition.


Thus, the m.1382A>C polymorphism is associated with susceptibility to T2D in men, possibly interacting with exercise, and contributing to the risk of T2D in sedentary males by reducing the activity of MOTS-c.Type 2 Diabetes (T2D) is an emerging public health problem in Asia. Although ethnic specific mtDNA polymorphisms have been shown to contribute to T2D risk, the functional effects of the mtDNA polymorphisms and the therapeutic potential of mitochondrial-derived peptides at the mtDNA polymorphisms are underexplored. Here, we showed an Asian-specific mitochondrial DNA variation m.1382A>C (rs111033358) leads to a K14Q amino acid replacement in MOTS-c, an insulin sensitizing mitochondrial-derived peptide. Meta-analysis of three cohorts  show that males but not females with the C-allele exhibit a higher prevalence of T2D. In J-MICC, only males with the C-allele in the lowest tertile of physical activity increased their prevalence of T2D, demonstrating a kinesio-genomic interaction. High-fat fed, male mice injected with MOTS-c showed reduced weight and improved glucose tolerance, but not K14Q-MOTS-c treated mice. Like the human data, female mice were unaffected. Mechanistically, K14Q-MOTS-c leads to diminished insulin-sensitization  The prevalence of type 2 diabetes mellitus (T2D) is growing dramatically. Over 400 million individuals were diagnosed with T2D worldwide in 2015, and the International Diabetes Federation projects that at least 600 million people will need to be treated for T2D by 2040 . HoweverWhile diabetes syndromes directly caused by mutations in mtDNA are extremely rare , severalMitochondrial-derived peptides (MDPs) are biologically active peptides derived from small open reading frames (ORF) in the mitochondrial genome –16. Emer1c  – [remaining glucose amount in the media].WT MOTS-c levels were measured as described previously . For K14® osmotic pumps carrying WT MOTS-c and K14Q MOTS-c synthetic peptides into C57BL/6 mouse . The mice were sacrificed after 24 hours implantation and plasma samples were collected to measure steady-state WT MOTS and K14Q MOTS-c levels by MOTS-c ELISA. The peptide concentrations of the WT and K14Q MOTS-c remaining in the mini pumps after implantation were also measured. All experiments with mice were performed in accordance with the appropriate guidelines and regulations and approved by the Juntendo University and University of Southern California Institutional Animal Care and Use Committee.Male and female CD-1 mice at 14 weeks (n = 8-12) and 12 weeks (n = 8-11) of age, respectively, were obtained from Jackson Laboratory  and treated with K14Q MOTS-c and WT MOTS-c peptides. Mice were singly housed under standard 12-hr light-dark cycle with access to water and rodent food a high-fat diet . Mice were randomly assigned to one of three experimental groups: 1) a control group receiving BID (twice a day) intraperitoneal (IP) injection of vehicle (sterilized water); 2) a WT MOTS-c-treated group receiving BID IP injection of 7.5-mg per kg body weight; 3) a K14Q MOTS-c-treated group receiving BID IP injection of 7.5-mg per kg body weight. Mice were anesthetized with isoflurane and sacrificed after two weeks of treatment. Tissues were collected from the mice, flash-frozen in liquid nitrogen and stored at -80° C. To examine direct effect of K14Q MOTS-c polymorphism on T2DM in human studies, a glucose tolerance test was performed. 7-week old male (n = 8-10 for each group) and female (n = 8-9 for each group) CD-1 mice were obtained from Charles River, Japan. After a week (8-week of age), mice were singly housed under standard 12-hr light-dark cycle with access to water and rodent food a HFD . Mice were randomly assigned to one of three experimental groups 1) a control group receiving IP injection of sterilized water; 2) a WT MOTS-c-treated group receiving IP injection of 0.5 mg per kg body weight; 3) a K14Q MOTS-c-treated group receiving IP injection of 0.5mg per kg body weight, daily for 21 days. After four hours fasting from the last injection of water, WT MOTS-c, or K14Q MOTS-c (11-week of age), an intraperitoneal glucose tolerance test (GTT) was performed under isoflurane anesthesia and blood glucose was measured using a glucometer . GTT consisted of a D-glucose injection  and blood was sampled from the tail at 0, 15, 30, 45, 60, 90, and 120-min post-glucose injection. To compare WT MOTS-c and K14Q MOTS-c clearance rate in mice, we implanted ALZET2 tests. The relation of the m.1382A>C genotype distribution and prevalence of T2D was evaluated using the Pearson’s χ2 test. Logistic regression was also used to predict the prevalence of T2D. All logistic regression models included the following covariates: age, BMI, smoking, and moderate-to-vigorous intensity physical activity (MVPA). The statistical significance for linearity between the prevalence of T2D and MVPA was evaluated using Cochran-Armitage trend tests. In animal and cell culture studies, group comparison was performed by t-test or one-way analysis of variance (ANOVA). The Bonferroni post hoc test was performed when the ANOVA indicated a significant difference. Delta body weight, fat mass, lean mass and glucose tolerance test were analyzed with two-way (group × time) ANOVA with replications. Values expressed as mean ± SD; p < 0.05 are considered to be statistically significant. Cell and mouse data are presented as mean ± S.E.M. Significant differences were determined by Student’s t-tests or one-way ANOVA and Turkey’s post hoc test by use of GraphPad Prism 5 software. P values of *< 0.05, **< 0.01, or ***< 0.001 were considered statistically significant.Statistical analyses were performed using PASW Statistics 18 (series of SPSS Statistics) for Windows. Differences in physical characteristics by sex were determined using unpaired t-tests and Pearson’s χSupplementary FiguresSupplementary Tables


T cells are essential mediators of the adaptive immune system, which constantly patrol the body in search for invading pathogens. During an infection, T cells that recognise the pathogen are recruited, expand and differentiate into subtypes tailored to the infection. In addition, they differentiate into subsets required for short and long-term control of the pathogen, i.e., effector or memory. T cells have a remarkable degree of plasticity and heterogeneity in their response, however, their overall response to a given infection is consistent and robust. Much research has focused on how individual T cells are activated and programmed. However, in order to achieve a critical level of population-wide reproducibility and robustness, neighbouring cells and surrounding tissues have to provide or amplify relevant signals to tune the overall response accordingly. The characteristics of the immune response—stochastic on the individual cell level, robust on the global level—necessitate coordinated responses on a system-wide level, which facilitates the control of pathogens, while maintaining self-tolerance. This global coordination can only be achieved by constant cellular communication between responding cells, and faults in this intercellular crosstalk can potentially lead to immunopathology or autoimmunity. In this review, we will discuss how T cells mount a global, collective response, by describing the modes of T cell-T cell (T-T) communication they use and highlighting their physiological relevance in programming and controlling the T cell response. In order to avoid immunologically driven tissue damage, T cells need to scale their clonal expansion and effector response according to the dose and strength of the antigenic trigger, which is accomplished by the temporal integration of T cell receptor (TCR) signalling with a number of additional regulatory signals . The prin models . Overall+ T cells are capable of proliferating and differentiating autonomously after antigen stimulation [CD8mulation ,17, suggmulation . Direct mulation ,21,22,23T cells produce and secrete soluble factors into the local environment, which enables them to translate and coordinate local interactions into tissue-wide global behaviour. Communication over long distances enables a population of immune cells to coordinate their individual behaviours and tune the global response towards an optimal outcome for the system. While few factors have been clearly established as mediating T-T cell communication, it is conceivable that others which are produced and consumed by T cells may also be implicated in T-T crosstalk. Cytokine penetration in tissues is dependent on the density of local cytokine consumers, which contributes to non-genetic cell-to-cell variability by creating variable cytokine niches in the reactive microenvironment . The cur+ T cells into their effector subsets. For CD8+ T cells, IL-2 signals have been shown to optimize both effector T cell generation and memory differentiation [+ and CD8+ T cells to become terminally differentiated effector T cells, while a low-level of IL-2 signals drive responding CD4+ T cells towards the follicular helper T cell (TFH) or central memory state, and allow CD8+ T cells to survive as long-lived memory T cells [One of the most studied cytokines involved in T-T communication is Interleukin (IL)-2 (IL-2). It has been discovered more than 30 years ago as a potent stimulator of T cell growth ,26 and ontiation . While nntiation ,32. Stro T cells .+ T cells differentiate into specific subtypes, producing a characteristic cytokine profile, depending mainly on the cytokine milieu of the microenvironment [+ T cells differentiating into T helper (Th) 1 (Th1) produce Interferon-γ (IFN-γ), IL-2, and tumour necrosis factor (TNF)-β. IFN-γ is a highly pleotropic cytokine associated with proinflammatory processes, but it also displays regulatory functions [+ T cells is sufficient to potentiate the role of IL-12 in promoting Th1 differentiation [+ T cells also skews their subsequent differentiation [+ T cells differentiating into Th2 produce IL-4, IL-5, IL-10, and IL-13. IL-4 is a signature cytokine in type II inflammatory responses and thus involved in the immune response against invading parasites and allergens. IL-4 produced by T cells [+ T cells results in the ubiquitous phosphorylation of STAT6 or STAT1 in immune cells throughout reactive lymph nodes following H. polygyrus, T. gondii and S. mansoni infection, indicating that cytokines can function in a systemic manner under certain circumstances [Multiple inflammatory cytokines are shared between T cells. Activated CD4ironment . CD4+ T unctions ,36,37,38ntiation , while intiation . IFN-γ pntiation ,35. CD4+ T cells ,41 promo T cells ,43. Bothmstances . IL-6 ismstances ,46,47, amstances , while imstances .+ T cells [+ T cells, by promoting their apoptosis during clonal expansion [+ T cells [Anti-inflammatory cytokines are also used by T cells to co-regulate their responses. Transforming growth factor β (TGF-β) is a pleiotropic cytokine that plays an essential role in T cell development, homeostasis, tolerance, and immune responses ,52,53,54 T cells  and Treg T cells . TGF-β s T cells , and conxpansion . Interesxpansion . As a coxpansion . IL-10 ixpansion ,61,62. I T cells , suggest+ T cells clustering around DCs cause CD8+ T cell accumulation through CCL3/CCL4-CCR5 binding, which enhances CD8+ T cell/DC contact formation [Chemokines are chemotactic cytokines orchestrating the migration and positioning of all immune cells and are critically involved in diverse immune cell processes, such as cell fate or activation . In vivoormation , highligormation .Immune cells are also known to communicate through the secretion of extracellular vesicles, in particular endosomally derived exosomes . ExosomeAn important feature of an effective immune response is the specific activation of appropriate responders and the subsequent directed targeting of an invading pathogen while sparing self, which necessitates excluding bystanders. In many cases this is accomplished by the formation of adhesive interfaces between the surfaces of interacting immune cells, with a synaptic space in between them. The immunological synapse (IS) formed between T cells and APCs is the best studied example of such a structure ; but othImmunological synapses between antigen-specific T cells and APCs are sites of signal initiation and integration for T cell activation and subsequent programming ,75,76. I+ T cells are organized in multifocal synapses [Antigen-specific T cells also interact with themselves, forming clusters during priming in secondary lymphoid organs (SLOs) ,78,79,80synapses . Modest synapses . T-T interactions are fairly dynamic, with dwell times of around 5−10 min and thus much shorter than the interaction times between T cells and APCs . DespiteAnother contact-mediated mode of communication between T cells which is not restricted to immediately adjacent cells is created through the formation of so called tunnelling nanotubes (TNTs) ,90. Thes5–106 T cells [+ T cells favour the priming of naive CD8+ T cells, by promoting the CCR5-dependent recruitment of polyclonal CD8+ T cells to mature dendritic cells [+ T cells and subsequent presentation of these complexes to other CD4+ T cells is used as a mechanism by T cells to inhibit the recruitment of antigen-experienced CD4+ T cells into the response, favouring the recruitment of naïve T cells [Infections with virus may generate less than 100 APCs per lymph node , and an  T cells . In orde T cells ,94,95. M T cells , suggestic cells , other sic cells ,104,105.T cells have been shown to use mechanisms similar to quorum sensing to regulate their population size. Quorum sensing is a type of cellular communication mediated by extracellular factors generally produced and detected by the same type of cells, referred to as autoinducers. The detection of autoinducers and the activation of downstream signalling pathways occurs upon reaching a sufficiently high autoinducer concentration within the local environment, which in turn requires a sufficiently high cell population density of signal producers . While m+ T cells is required to maintain critical levels of phosphorylated signal transducer and activator of transcription 5 (STAT5) in CD4+ T cell, which are necessary for T cell activation and subsequent expansion, indicating a population density-dependent regulation [+ T cells similarly rely on autocrine IL-2 production by neighbouring activating CD8+ T cells as the main source of this critical cue, rather than depending on paracrine IL-2 signals from CD4+ T cells or DCs [+ T cell expansion, based on cellular density [The cytokine IL-2 has been suggested as functioning as an autoinducer-like molecule between T cells ,32. In vgulation . Savir egulation . Their ms or DCs ,113. Rec density . ICAM-1- density , suggest+ T cells do not express IL-2 and IL-2Rα (CD25) at significant levels, but production of both proteins is strongly induced upon activation by antigen. Because IL-2Rα levels remain low at the beginning of the immune response, and increase around 2 days after priming, IL-2 can accumulate in the environment without cells immediately responding to this signal . Once the receptor levels reach a critical threshold, IL-2 signalling in CD4+ T cells is induced, leading to the activation of two feedback loops—a positive feedback loop driving further IL-2Rα expression and eventually a negative feedback loop inhibiting the production of more IL-2 [Both IL-2 receptor and cytokine expression are controlled by a number of feedback mechanisms . Under sore IL-2 . T cell differentiation is a highly complex process, and varying models have been proposed to explain the fate determination of responding cells , but T c+ T cells may differentiate into one of several subtypes of T helper (Th) cells, including Th1, Th2, Th17, and iTreg [+ T cells are capable of autonomously modulating their differentiation towards Th1 fate, in response to infection through secretion of IFN-γ [+ T cells is stochastic [During TCR activation in a particular cytokine milieu tailored to the invading pathogen, naive CD4nd iTreg . CD4+ T of IFN-γ . In Th1 of IFN-γ , inducesof IFN-γ ,118. Th1of IFN-γ . Given tochastic ,121, and+ T cells in effector and memory subsets is regulated by local cell density [+ T cells into memory precursor cells. The authors identified the cytokines IL-2 and IL-6 as the main drivers of collective CD4+ T cell memory differentiation, with IL-2 regulating the proliferation rate and IL-6 promoting the survival of responding T cells. Blocking both cytokines resulted in a reduction of CD62L expression, a phenotypic marker of memory T cells. The cell surface molecule SLAMF6 was also to be involved, by causing a reduction of CD62L expression on responding T cells. The T cell response to infection has two primary goals. The first one is to fight an invading pathogen and present infection, by the generation of a large number of effector cells. The second goal is to retain a smaller subset of responders with enhanced longevity and regenerative capacity, to protect against future encounters with the same pathogen. This is achieved by the expansion of T cells upon activation and the subsequent differentiation into effector and memory subsets. A recent study by Polonsky et al. indicated that the differentiation of CD4 density . Increas+ T cells [Multiple papers also implicated the requirement of IFN-γ for effector and memory differentiation of CD8 T cells ,124, som T cells ,35,36,37+ T cells, LFA-1/ICAM-1 ligation in human T cells promotes Th1 polarization [+ T cell activation, proliferation, and differentiation [+ T cells with a regulatory phenotype, characterised by the production of IL-4, IL-10 and TGF-β [+ T cells, LFA-1/ICAM-1 dependent clustering promotes their expansion [+ T cell differentiation and function is unclear, as blocking CD8+ T cell clustering has been shown to increase effector differentiation and IFN-γ production in vitro and following Incomplete Freund Adjuvant (IFA)-OVA priming [A number of groups investigated how homotypic clusters shape the differentiation of T cells. In CD4rization . T-T intntiation , and in nd TGF-β . In CD8+xpansion ,19,23. Mxpansion . However priming ,19 and I+ memory T cells and naïve CD8+ T cells during priming promote the accelerated differentiation of naïve cells into effector memory (TEM) cells, at the expense of less differentiated stem cell memory (TSCM) and central memory (TCM) cells [+ T cell differentiation was dependent on the density of the two subsets, suggesting again a quorum sensing-like regulatory mechanism. Finally, evidences suggest that T cell contacts happen between different T cell subsets. Direct contacts between antigen experienced CD8M) cells . These eT cells are fairly cross-reactive and occasionally autoreactive, even in healthy individuals. The control of autoimmune T cells requires a number of stringent regulatory mechanisms, to avoid detrimental reactions and a breach of self-tolerance ,130. Stuil2 gene by FOXP3 [+ T cells are the main producers of IL-2 [+ T cells get constantly activated by self-peptides, which results in IL-2 production and the subsequent recruitment and suppression by Treg cells, to prevent the expansion of autoimmune CD4+ T cells [At steady state, IL-2 drives the control of auto-reactive T cells. Treg cells, which are critically involved in maintaining self-tolerance , requireby FOXP3 ,136. At  of IL-2 ,137,138, of IL-2 . A study T cells . + T cells [+ T cell responses by limiting IL-2 production and thus shaping the differentiation of the responding cells [During an immune response, communication mediated by IL-2 during priming can both lead to cooperation or competition between effector CD4 T cells ,141,142  T cells ,111. Thi T cells ,143. A t T cells . Of noteng cells ,146,147.Finally, T-T cell contacts were also suggested to enhance Treg cell-mediated suppression by potentiating IL-35 and IL-10 production , suggest+ T cells, impeding anti-tumour immunity [+ and CD8+ T cell exhaustion [+ T cells in the tumour microenvironment [Regulatory interplay between T cells has recently been observed at peripheral sites. Treg cells in the tumour microenvironment have been shown to restrain the IL-2-dependent acquisition of cytotoxic functions by CD4immunity . Interlehaustion . T cell ironment . While n+ T cells upon hepatocellular antigen recognition acts in an autocrine/paracrine fashion to increase IL-2 responsiveness and rescue CD8+ T cells from antigen-induced apoptosis [T cell clustering was also observed in other anatomical sites, such as the liver . Primingpoptosis .Intercellular T-T communication can also be mediated by the intercellular transfer of membrane components, through a process known as trogocytosis. The sharing of TCRs between cytotoxic T lymphocytes through trogocytosis has been reported . AntigenIt is becoming increasingly clear that T cell communication is central to coordinate T cell responses and tailor it to the injury while preserving tolerance . Some meIntercellular communication between T cells has been firmly established to be a crucial regulator of the T cell response, by modulating their activation, expansion and differentiation. It has also been implied to be involved avoiding autoimmunity, by balancing the conflicting demands of self-tolerance and responsiveness to a diverse range of pathogens . Given tGiven its critical involvement in controlling T cell homeostasis, immune cell activation and population size, it is easy to see how the dysregulation of quorum sensing mechanisms may result in immunopathology or autoimmunity. A prototypic example of the result of a failure of regulatory mechanisms in maintaining homeostasis is cancer, and it is thus not surprising that disruption of quorum sensing has been implicated in tumorigenesis, which highlights the general relevance of this cellular communication mode in controlling correct cellular behaviour ,156,157.


Neuropeptide Y (NPY) has been implicated in the regulation of cellular motility under various physiological and pathological conditions, including cancer dissemination. Yet, the exact signaling pathways leading to these effects remain unknown. In a pediatric malignancy, neuroblastoma (NB), high NPY release from tumor tissue associates with metastatic disease. Here, we have shown that NPY stimulates NB cell motility and invasiveness and acts as a chemotactic factor for NB cells. We have also identified the Y5 receptor (Y5R) as the main NPY receptor mediating these actions. In NB tissues and cell cultures, Y5R is highly expressed in migratory cells and accumulates in regions of high RhoA activity and dynamic cytoskeleton remodeling. Y5R stimulation activates RhoA and results in Y5R/RhoA-GTP interactions, as shown by pull-down and proximity ligation assays, respectively. This is the first demonstration of the role for the NPY/Y5R axis in RhoA activation and the subsequent cytoskeleton remodeling facilitating cell movement. These findings implicate Y5R as a target in anti-metastatic therapies for NB and other cancers expressing this receptor. Neuroblastoma (NB) is a pediatric malignancy arising due to defects in sympathetic neuron differentiation Maris, . The disDue to their sympathetic origin, NBs express neuronal markers, such as neuropeptide Y (NPY) . To this end, non-permeabilized NB cells were immunostained for Y5R using an antibody that recognizes the extracellular N-terminus of the receptor. This approach allowed us to selectively detect the cellular distribution of the Y5R fraction present on the cell membrane and thereby able to bind the ligand. Subsequently, phalloidin was used to label F-actin and thereby assess cytoskeleton remodeling. In cells with a migratory phenotype, Y5R accumulated in specific regions of the cells, exhibiting the characterizing feature of polarized distribution on the cell edges. In SK-N-AS cells, the most profound Y5R expression was observed at the leading edge of single migratory cells, with a detectable co-localization between Y5R and phalloidin  NB cell lines, which were derived from patients before therapy or after treatment with cyclophosphamide, respectively , or with EGFP alone as a control (CHO-K1/EGFP) , and cells were tracked to compare the directionality of their movement (−9 to 10−7 M (−8 M (center of mass at the end of the experiment at 140.65 μm), while NPY at a concentration of 10−7 M had no chemotactic activity   .To further investigate the involvement of Y5R in cellular motility, we assessed its subcellular localization during cell migration. To this end, CHO-K1/Y5R-EGFP cells without prior permeabilization were immunostained with anti-Y5R antibody recognizing the extracellular domain of the receptor, as described above. In cells with a migratory phenotype, a polar distribution of the receptor was frequently observed . Detaile−8 M NPY  with anti-Y5R and anti-RhoA-GFP antibodies was employed to assess their direct interactions. Treatment with NPY at a concentration of 10stration . Initialstration . The substration . No incrstration . The memstration . High mastration . Howeverstration . Altoget−7 M. The peptide stimulated both spontaneous invasiveness when no NPY gradient was present and directional cell invasion when it served as a chemotactic agent . Y5R antagonist, CGP71683, and Y2R antagonist, BIIE0246, were obtained from Tocris .Tissue sections from 87 pediatric patients with neuroblastic tumors at diagnosis, collected between the years 2004 and 2009, were obtained from Children's Oncology Group (COG). These samples were collected by COG institutions upon obtaining appropriate consents, and their use was approved by the Georgetown University Institutional Review Board. Immunohistochemistry on the above samples was performed using rabbit polyclonal anti-Y5R antibody (1:300) .Human NB cell lines, SK-N-AS and SK-N-BE(2), were obtained from American Type Culture Collection  and cultured in DMEM media or EMEM:F12K (1:1) media with 10% FBS, respectively. CHO-K1 cells were obtained from ATCC and cultured in F12K media supplemented with 10% FBS. Stable transfectants expressing NPY receptors fused to EGFP or EGFP alone were developed as previously described  cells, as previously described . The fluorescence was measured from the bottom of the migration plates using EnSpire Multimode Plate Reader .The BD FluoroBlok™ 96-well Transwell plate or BD BioCoat FluoroBlok™ tumor invasion systems  were used to evaluate NB cell migration and invasion, respectively. NB cells, SK-N-AS, and SK-N-BE(2), were suspended in their respective media supplemented with 5% FBS and seeded in the upper chambers at a density of 2.5 × 105 cells per well. Eighteen hours after seeding, a scratch was made in the confluent monolayer using the 96-well Wound Maker™. Cells were then washed with serum-free medium to clear any floating cells within the scratch prior to treatment. The subsequent migration monitoring was performed in media with three different serum concentrations, 10%, 1%, or 0.1% FBS, depending on the experimental design. For the low-serum conditions, cells were primed in serum-free media for 6 h before creating the scratch and treating with media supplemented with 1% or 0.1% FBS and NPY or its receptor antagonists, when desired. Subsequently, the 96-well-plates were placed in the IncuCyte live cell imaging system  and the images collected every 2 h during the incubation time. The IncuCyte ZOOM software generated a wound width (WW) migration metric, which was used to calculate the migration distance (MD) according to the following formula: MD = (WWT0 – WWTn)/2.CHO-K1 cells transfected with EGFP, Y2R-EGFP, and Y5R-EGFP were seeded in IncuCyte® ImageLock 96-well plates at a density of 2–2.5 × 104 cells per well. Then, the proliferation was assessed under conditions mimicking these used in the migration assays: 10, 1, or 0.1% FBS. For the low-serum conditions, cells were primed in serum-free medium for 6 h before treatment with media supplemented with 1 or 0.1% FBS and NPY or its receptor antagonists, when desired. The cells were then monitored using the IncuCyte® Live-Cell Analysis System providing images every 2 h during incubation time. Phase object confluence metric measured as a percentage of surface coverage was used to evaluate cellular proliferation.CHO-K1 cells stably transfected with EGFP, Y2R-EGFP, and Y5R-EGFP were cultured in 10% FBS for 24 h in 96-well-plates at a density of 2–2.5 × 10–7 M NPY for 20 min or 10–6 M Y5R antagonist for 30 min. Cell lysates were collected, and the levels of active-RhoA were measured using RhoA Pull-down Activation Assay Biochem Kit  according to the manufacturer's protocol. The band intensities were quantified by Image J software . The levels of active RhoA-GTP were normalized to total RhoA expression.CHO-K1 cells stably transfected with Y5R-EGFP or SK-N-AS NB cells were incubated in serum-free media for 24 or 72 h, respectively, and then stimulated with 105 cells in a 35-mm plate) and cultured for 24 h. Then, the cells were fixed for 10 min using 4% paraformaldehyde and blocked in 1% bovine serum albumin (BSA) for 1 h at RT. The subsequent staining with rabbit monoclonal anti-Y5R antibody  was performed without permeabilization to detect the fraction of Y5R present on the cell membrane. Subsequently, the samples were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody  for 1 h at RT. Then, the cells were permeabilized and stained with mouse monoclonal anti-RhoA-GTP antibody  overnight at 4°C, followed by incubation with AlexaFluor 594-conjugated donkey anti-mouse antibody  for 1 h at RT. Alternatively, NB cells were co-stained with rabbit monoclonal anti-Y5R  and mouse monoclonal anti-ganglioside GD2  antibodies. For actin filament detection, cells were permeabilized and stained with Texas Red®-X phalloidin  for 20 min, while DAPI at a concentration of 0.5 μg/ml in PBS was used to detect DNA.Cells were seeded on 12-mm glass coverslips (2.5 × 105 cells) that were placed in a 24-well-plate. On the second day after seeding, cells were incubated in serum-free media for 24 h before stimulation with 10−8 M NPY for 20 min, with or without a 30-min pre-incubation with 10−6 M Y5R antagonist. The cells were fixed for 10 min using 4% paraformaldehyde followed by permeabilization for 10 min. To test the interaction between Y5R and RhoA-GTP, Duolink® In situ Red starter kit Mouse/Rabbit (Sigma-Aldrich) was used according to the manufacture's protocol with the following antibodies: rabbit monoclonal anti-Y5R , mouse monoclonal anti-RhoA-GTP , and mouse monoclonal anti-HDAC1 as a negative control . For actin filament detection, Alexa Fluor 488-Phalloidin  was used for 20 min before mounting. Intensities of fluorescent staining in the entire cell colonies, their outer plasma membranes, and inner areas were quantified using Image J software.NB cells were seeded on 12-mm glass coverslips (2.5 × 10−9-10−7 M) was added to the right side chamber, while 10% FBS media alone was inserted to the left side chamber. Images were captured every 20 min for a period of 24 h using live cell microscopy at a magnification of 10×. Manual cell tracking for at least 40 cells per condition was performed using ImageJ, and the data were imported into the Chemotaxis software plugin for ImageJ (Ibidi). The software calculated individual cell velocity, euclidean, and accumulated distances, directionality (euclidean distance/accumulated distance), forward migration index , center of mass , and generated trajectory plots of cell migration. Values for center of mass and forward migration index can be positive or negative, depending on whether migration was toward or away from the chemoattractant, respectively. Rayleigh tests for vector data, which account for cell endpoints and the distance from origin, were used to establish whether cellular migration was random (p > 0.05) or directed toward the chemoattractant (p < 0.05).CHO-K1 transfectants were seeded in the center of chemotaxis chambers . After cell attachment, 10% FBS media supplemented with NPY . SK-N-AS cells were fixed, immunostained for Y5R, and counterstained with Texas Red®-X phalloidin and DAPI, as described above. Twenty-five cells per treatment in all cell lines were randomly selected for quantification of filopodia. Numbers of filopodia per cell were quantified using Image J. The percentages of Y5R-positive filopodia were quantified in SK-N-AS cells.CHO-K1/Y5R-EGFP, CHO-K1/Y2R-EGFP, CHO-K1/EGFP, and SK-N-AS cells were seeded in two-well inserts (Ibidi). After removal of the insert, cells were cultured in 10% FBS media supplemented with 10post-hoc t-test, independent-samples t-tests, or paired-samples t-tests. For count data, Chi-square tests were used. All experiments were performed at least two times, and the combined data are presented as mean ± standard errors.Statistical analyses were performed using GraphPad Prism 6 software. Between-group comparisons were assessed using one-way repeated measures ANOVA with The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Georgetown University Institutional Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.NA, LC, GIG, and JK designed experiments. NA, LC, SG, EK, LA, and SZ performed experiments. SG performed histopathological analysis. NA, LC, GIG, and JK analyzed data. NA, LC, GIG, SG, EK, and JK prepared figures and wrote manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.