Understanding the genetic basis of heat and drought stress tolerance in wheat is prerequisite for wheat breeding program. In the present study, a wheat panel comprising of 192 elite bread wheat genotypes was phenotyped in eight environments for yield and related traits in field conditions. Four stress environments were created by implying four different treatments differing in sowing date and water availability, panel was evaluated for two years in field conditions. The panel was genotyped with 15K Illumina chip and 9236 polymorphic markers concentrated on B genome were employed in GWAS analysis. Consistent, fast LD decay was observed on D genome and structure analysis germplasm divided panel into three major populations. GWAS was performed using BLUEs values of combined environment data in R package GAPIT using log10(P) = 3.96 as significance threshold. The significance of association was further checked using FDR<0.05 threshold. The GWAS identified 487 loci associated with the traits and were significant at log10(p) threshold out of these 350 loci were significant at FDR threshold. For two stress indices 108 associations were significant at FDR threshold. Nine genomic regions were shared among all treatment, while multiple pleiotropic regions were present on chromosome 7D followed by unmapped chromosome. The present study validated many marker trait associations for yield and other traits, MTAs significant under combined drought and heat stress were novel. These regions are important and can be used for fine mapping and marker assisted selection to discover new genes responsible for heat and drought tolerance in wheat. These results showed that independent heat had more pronounced effects on grain yield than independent drought stress while combination of both stresses had more severe effects than independent drought or heat stress. The same trend was observed for other traits and FLL, FLW HI, LA and TILL were more effected by drought stress than heat and regarded as drought sensitive traits while DW, DTM, PH, SL and SPLS were more effected by heat stress and regarded as heat sensitive traits. The heritability values were higher under all stress treatment with DW and GY having highest H2 value under [C] treatment, FLL and LA had highest H2 value under [D] treatment, FLL had highest H2 value under [H] treatment and DTM had highest H2 value under [HD] treatment. The range, mean and heritability values for yield and related traits in the normal environment [C], drought [D], heat [H] and combined heat and drought [HD] stressed environments are summarized in Grain yield varied greatly across the environments, high grain yield was recorded under non stress treatment [C]  while under drought stress [D] it had highest positive correlation with Spikelets per spike (r = 0.88**), under heat stress it had highest correlation with harvest index (r = 0.92**) and under combined heat and drought stress it had highest correlation with plant above ground dry weight (r = 0.83**). Stress tolerance index had positive correlation with grain yield under all stress treatments while tolerance index had positive correlation under individual drought (r = 0.14*) and individual heat stress (r = 0.18*) but it had negative correlation with grain yield under combined drought and heat stress (r = -0.33**). The detailed information about correlations among all studied traits is given in 2 model highest LD decay (r2 = 0.64) was seen on chromosome 4D while least LD (r2 = 0.38) was seen on chromosome 5A with an average Pearson's correlation (r2) value of 0.45. The LD decay was faster for A and B genome resulting smaller genetic distance when compared to D genome. The rate of LD decay was ~8 cM; ~5 cM ~10 cM for A, B and D genome, respectively with mean genetic length of 8.1 cM for all groups showing that one marker is enough within ~8.1 cM region for each chromosome , 9236 polymorphic markers were employed in GWAS analysis. Most of these polymorphic SNPs were concentrated on B genome (51%) followed by A (37%) and D (12%) genome . Chromosromosome .Population structure analysis divided the whole population in three sub-populations, population_01 containing 36 genotypes while population_02 and population_03 contained 51 and 105 genotypes respectively. The members of population _01 contained genotypes with VORB, PASTOR and PWB65 as one of their parent while genotypes of population_02 contained CHIBIA, KACHU #, ND643 and PRL as one of their parents. The population_03 contained maximum 54% genotypes of whole studied panel and most of these genotypes contained ATTILA, KIRITATI, PASTOR, SERI and WAXWING as one of their parents .Genome wide association mapping was performed for independent drought and independent heat stress and for combination of both heat and drought stress using R package GAPIT with a significance threshold of log 10(P)>3.98 and FDR<0.05. Total 148, 95, 151 and 93 significant associations were detected for studied traits under non stress [C], drought [D], Heat [H] and for combined heat and drought [HD] treatment respectively. Out of these, 105, 63, 122 and 60 were significant at FDR threshold under non stress [C], drought [D], Heat [H] and for combined heat and drought [HD] treatment respectively . ManhattUnder optimum growing conditions 148 marker trait associations (MTAs) were significant at Log(P) value threshold while 105 were significant at FDR threshold. Maximum number of significant associations (36) were reported for plant above ground biomass (DW) followed by 23 significant associations for grain yield (GY). Only one marker significant at Log(P) threshold was associated with leaf area under [C] treatment while no significant association was recorded at FDR threshold for FLL, HI, LA and PH. On average all significant associations under [C] treatment explained 20.25% phenotypic variation. Maximum number of associations (18) were recorded on chromosome 3D followed by 17 associations on 5B and 6A while chromosome 2D and 3A had only one significant association. Many genomic regions on chromosome 3D were pleiotropic and were associated with more than one trait. The most potent MTAs for grain yield under optimum conditions [C] were recorded on chromosome 3D (GENE-1752_162 at 101.24 and RFL_Contig2471_119 at 23.41 cM) and explained 28.65 and 23.61% phenotypic variation. Grain yield had common QTL with DW, DTM, FLL, FLW, HI, SPLS and TILL and most consistent regions controlling these traits were present on 3D, 5B and 6A .Under Drought stress treatment [D], 95 associations were significant at Log(P) threshold while 63 associations were significant at FDR threshold. The maximum number of associations (22) were recorded for TILL followed by STI (19) and only a single marker was significantly associated with leaf area. Tillers per plant and stress tolerance index had also highest number of significant associations at FDR threshold each with 10 and 19 associations respectively. Harvest index, leaf area and spike length had no significant association beyond FDR threshold. Under [D] treatment, all the significant associations explained 19.33% phenotypic variation with maximum phenotypic variation (28.93% each) explained by markers IACX203 (67.24 cM) and wsnp_Ex_c18372_27196625 (71.76 cM) on chromosome 5D and associated with STI. Chromosome wise highest number of significant associations (24) were detected on chromosome 7B followed by 17 associations on chromosome 2B.Significant associations for GY under [D] stress were detected on chromosome 3D, 6A, 6D, 7B and on unmapped chromosome, CAP7_c1274_206 on unmapped chromosome was most significantly associated with GY and explained 23.49% phenotypic variance. Pleiotropic regions under [D] stress were concentrated on chromosome 7B followed by chromosome 4B, chromosomal region significant for GY on 7B was ~15 cM apart from region associated with DW, FLW, HI, LA, PH and TILL. The chromosomal region on 3D, 6A and 6D were specifically associated with grain yield .Under [H], 151 associations were significant at Log(P) threshold while 122 of these were significant at FDR. TOL had maximum number (21) of significant associations followed by STI (20) and FLL (12) and all these MTAs were also significant at FDR. Only DW had no significant association at FDR threshold among all the studied traits. Under [H] stress the potent associations for grain yield were present on two chromosomes viz. 7B and unmapped chromosome and IACX5767 on 7B explained 21.62% phenotypic variation. All significant associations under [H] stress explained 20% phenotypic variation with maximum value of phenotypic variation of 24.17% for leaf area. Grain yield under [H] stress shared genomic region with all studied traits except FLL, PH, SL and TILL and these common genomic regions were present between 134.06–150.6 cM on chromosome 7B .Under [HD] treatment, 93 associations were significant at Log(P) threshold while 60 of these were significant at FDR. Maximum numbers of significant associations were recorded for STI (29) and TOL (13) index and both these traits have higher number of significant associations on FDR threshold. DTM, FLW, GY and TILL were only agronomic traits which had association significant at FDR threshold. Chromosome wise, highest associations were recorded on chromosome 4B (12) followed by 2B, 7B and 7D each carrying 10 significant MTAs. MTAs for grain yield were present on 1A, 2D, 2B, 4A, 7B, 7D and on unmapped chromosome, the potent marker Kukri_rep_c68068_95 was present on chromosome 2D and explained 22.55% phenotypic variation. All significant associations significant for all studied traits on average explained 19% phenotypic variation with maximum contribution (23.67%) by RFL_Contig854_2253 present on 3A significantly associated with TOL. Grain yield share many pleotropic regions with other traits and most of these pleiotropic regions were concentrated on chromosome 7B and 7D where shares the chromosomal region with FLL, FLW, HI, PH, SL, SPLS and TILL .Marker trait associations for STI under [D] stress were recorded on chromosome 1A, 1B, 1D, 2B, 3A, 3D and 5D. BS00077789_51 on chromosome 2B at 37.03 was most potent loci associated with STI under drought stress and explained 29.08% phenotypic variation. Many genomic regions were shared between STI and other stress indices for example, genomic region on chromosome 2B and 5D were shared by STI and TOL under [D] treatment. Genomic regions significant for TOL under [D] stress were detected on chromosome 2B, 4D and 5D with the highest number of associations on chromosome 5D. The genomic region significantly associated for TOL under drought stress was common for both STI and TOL under all stress treatments. Locus BS00077789_51 on 2B at 37.03 cM was most significantly associated with TOL and explained 26.69% phenotypic variation .Twenty significant MTAs were recorded for STI under [H] stress and were concentrated on chromosome 3D. The most significant association was present on 3D at 11.23 cM and explained 22.56% phenotypic variation. The region on 3D at 176.18 cM was also significant for TOL index. The TOL index had 21 significant associations under [H] stress most of which were concentrated on chromosome 2B and on average explained 21% phenotypic variation. The most potent association on 2B was present at 37.03 cM and explained 28.8% phenotypic variation. TOL index shared regions on chromosome 2B with stress indices under studied [D], [H] and [HD], similarly genomic region significant at chromosome 5D was also shared among all stress indices in all three treatments .Twenty-nine significant associations were detected for STI under [HD] treatment concentrated on chromosome 3D and explaining average 19.62% phenotypic variation. IACX203 (67.24 cM) and wsnp_Ex_c18372_27196625 (71.76 cM) were most potent loci associated with STI under [HD] treatment and explained 21.22% phenotypic variation. Thirteen significant associations were detected for TOL under [HD] treatment concentrated on 2B and average phenotypic variation value of 20.05%. RFL_Contig854_2253 on 3A at 81.91 cM was most significant loci for TOL and explained 23.67% phenotypic variation .Many significant associations were shared between two or more stress treatments. [C] and [H] stress share four loci, [D] and [H] share 13 loci, [C] and [HD] share 3 loci, [H] and [HD] had two common loci while no common locus was recorded between [C] and [D] treatment. Some loci were common among the three treatments were also recorded [C], [D] and [H] had six loci in common [C], [H] and [HD] had three loci common among them [C], [D] and [HD] had two loci in common and [D], [H] and [HD] had three loci in common. Nine loci were common among all four treatments and were recorded as consistent loci .A total 69 pleiotropic SNPs were associated with multiple traits under all stress treatments, [H] with maximum (31) of these followed by [C], [HD] and [D] each having 20, 10 and 8 associations respectively . The maxIn last few decades lots of efforts had been made to study the effects of a single stress on plants under control and field conditions . HoweverA diverse panel of 192 advance wheat lines was planted at National Agriculture Research Center Islamabad for two cropping seasons in four treatments, i.e. [C], [D], [H] and [HD]. The GY was severely reduced by [HD] stress (51.67% reduction) followed by [H] (27.33% reduction) and [D] treatment (24.9% reduction). Similar results were reported by ,39,40 foAssociation mapping is a powerful approach to elucidate genetic basis of complex traits including grain yield ,46–48, iGermplasm evaluation in multiple environmental conditions helps us to detect and compare QTLs for desired traits common among more than one condition. Genotype × environment interaction is the main problem in breeding for diverse adaptation in bread wheat. Occurrence of common genomic region among more than one stress shows common adaptation and tolerance mechanism of accessions under these stress treatments. In our study nine common regions were identified which were associated with traits under all four treatments i.e. [C], [D], [H] and [HD] treatment. In addition, two different stress indices were also used to find out stable regions across the environments. Multiple consistent regions for both stress indices were recorded on chromosome 2B and 5D. Many regions were shared among grain yield and stress indices for instance the genomic region on 5D at 67.24–71.76 cM was shared by DTM, GY, STI and TOL. A set of MTAs on chromosome 2B at 85.07 cM was near to IWB2285 locus at 70.4 cM significant for grain yield and grains per spike in collection of tetraploid wheat . The genPhytoene synthase 1 (PSY-1) gene, presence of this gene might be responsible for pleiotropic associations on 7B [Makers shared among more than one trait are very useful in marker-assisted selection as they may play a vital role in increasing QTL pyramiding efficiency. Multi trait markers were detected on all chromosomes in present study except 1D, 4D, 5A and 6B. These markers share two or more than two traits and maximum number of these multi trait markers was detected on 7B. The locus RAC875_rep_c110526_324 on 7B at 163.3 cM and chromosomal region on chromosome 7D at 208.1 cM were shared among 8 studied traits including GY. This implies that there are genetic bases for the high and consistent phenotypic correlation recorded among grain yield and these traits. The region on chromosome 7B was approximately 39 cM and 52 cM apart from two genomic regions M1441 and M7175 respectively identified by  for GY ans on 7B . Pleiotrns on 7B . In addins on 7B .The identification and introgression of major-effect QTLs are one of the best and proven approaches to improving the stress tolerance of wheat varieties. The accuracy and consistency of QTLs have clearly shown that structured association mapping with genome-wide molecular markers is an attractive option to identify major-effect QTLs for GY under different stress treatments. Association mapping results revealed many novel regions associated with combined drought and heat stress tolerance, along with nine consistent regions common among all four treatments and many pleiotropic regions associated with more than one traits. Further exploration of common regions among treatments through marker assisted selection can help in understanding complex mechanisms of abiotic stress tolerance and fine mapping of these regions can lead to new gene discoveries. Intensity of climate change will increase in next few years and will decrease wheat production, thus it is need of time to improve germplasm by arranging crosses between diverse parents and improve genomic technology and couple these technologies with marker assisted selection. The present study will help in understanding irrigation use efficiency and tolerance of exotic lines to rain fed conditions. It will help in identification and inclusion of tolerant genotypes in breeding program for further strengthening.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.


Using bone marrow chimeric mice, we showed that PTPN22 influenced development of both regulatory and effector T cell functions in a cell-intrinsic manner. Overall the expansion of Tregs is likely to keep the expanded T effector populations in check and sparing Treg during therapeutic mAb depletion may be a useful strategy to prevent occurrence of secondary autoimmunity.Lymphopenic insult has been shown to precipitate the initiation of autoimmune disease in murine models such as the Non-obese diabetic mouse. Similarly, in man lymphopenia induced by mAb therapy, for instance Alemtuzumab as treatment for Multiple Sclerosis, can precipitate development of secondary autoimmune disease in up to 30 % of patients. We asked whether an identified autoimmune susceptibility locus might increase the risk of developing autoimmunity in the context of mAb-induced lymphopenia in a mouse model. A single nucleotide polymorphism (SNP) in the gene encoding the tyrosine phosphatase PTPN22 (R620W) is associated with multiple human autoimmune diseases, and PTPN22 has been shown to modulate T cell responses, particularly to weak antigens. In keeping with this, PTPN22-deficient or PTPN22 R619W mutant murine T cells adoptively transferred into immunodeficient lymphopenic hosts showed a higher lymphopenia-induced proliferation rate than WT cells. We induced lymphopenia by treating wild-type or PTPN22 knock-out mice with T cell depleting antibodies and monitored reconstitution of the T cell pool. We found that PTPN22 deficient T cells acquired a more activated effector phenotype, with significantly more IFNγ producing cells. This resulted from expansion driven by self-peptide MHC, as it was evident when the contribution of IL-7 to lymphopenic expansion was blocked with IL-7R Ab. Interestingly, Foxp3 Lymphopenia can lead to the expansion of autoreactive T cell clones and thus may be a risk factor for autoimmune diseases. Studies in mice have shown a direct link between lymphopenia-induced proliferation (LIP) and the development of diabetes in non-obese mice and autoimmune pancreatitis after sublethal irradiation and transfer of auto-antigen specific T cells , 2. HoweLymphopenia is therapeutically induced for hematopoietic stem cell transplant (HSCT) therapies to treat a wide range of diseases , 6. OfteHSCT is also used to treat autoimmune diseases themselves, in which intense lymphodepleting condition regiments are used in order to target autoreactive cells. In a less invasive therapeutic approach to treat Multiple Sclerosis without undergoing irradiation, patients received alemtuzumab (Campath-1H), a monoclonal antibody against CD52. In this approach the immune system is reconstituted primarily by lymphopenia induced lymphocyte proliferation. Interestingly one third of the patients developed secondary autoimmune conditions after treatment with alemtuzumab, while another third presented with asymptomatic autoantibodies. Subsequently, Jones et al. showed that alemtuzumab-induced lymphopenia stimulated residual T cell expansion and a consequent shift in T cells toward a chronically activated phenotype with a loss of clonal diversity . PatientNotably, the risk of developing secondary autoimmunity following lympho-depletion regimes might be increased for these patients given that they started with an underlying autoimmune disease and therefore may have increased pre-disposition to self-reactivity. However, it is not understood why one patient develops secondary autoimmunity whereas another does not. Given that genome-wide association studies (GWAS) have identified numerous genetic mutations that act as risk factors for autoimmune disease, we asked whether genetic pre-disposition for autoimmunity increased the susceptibility to develop autoimmunity as a consequence of lymphopenia induced proliferation.PTPN22 gene is associated with a higher risk for several autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus among others and is the strongest non-HLA risk factor described to date  followed by Ukraine (14%)  . The poine (14%) . Studiesne (14%) , 12. A rne (14%) .PTPN22 variant allele, its association with autoimmunity and most importantly its role in regulating responses to weak affinity antigens, we investigated whether alterations in PTPN22 influenced the restoration of T cell homeostasis following perturbation in vivo. In keeping with clinical human lymphodepletion regimes, we induced severe lymphopenia by treating mice with monoclonal anti-CD4 and anti-CD8 depleting antibodies and monitored the reconstitution of the T cell pool. In this model upon reconstitution, PTPN22 deficient T cells acquired an enhanced activated effector phenotype, with significantly higher production of IFNγ compared to wildtype T cells. Interestingly we found that Foxp3+ Tregs are expanded and CD25+ as well as CD25− CD4 T cells expressed higher levels of IL-10, which may keep the expanded effector population in check. Our data indicate that maintaining a high Treg:T effector cell ratio in mAb depletion therapies may be beneficial in preventing secondary autoimmunity.Given the relative high penetrance of the Introduction of the R619W mutation was carried out by microinjection of a mix of Cas9 protein, guide RNA and ssDNA repair oligo (Integrated DNA Technologies) into single cell embryos generated by mating superovulated PEP-OST OT1 Rag1KO female mice, which contain a One-STrEP-Tag sequence knocked into the 5′ end of the PTPN22 gene (details below) with OT1 Rag1KO studs. Briefly, a 20 nt crRNA with sequence: AAGACTCGGGTGTCCGTTCA was designed to span the intended mutation site in exon 14 of PTPN22. This was annealed to TRACR RNA by heating to 95°C for 5 min and allowed to cool to room temperature before incubation with Cas9 protein at 37°C for 10 min in 0.1 TE buffer. The repair oligo was subsequently added to a final concentration of 100 ng/μL. The repair oligo was a 130bp ssDNA ultramer with sequence: CTTTATTCTTATAAAACATTGCGATTACATTTCTATTACTTGGTTAGCTCCTTCTCGGAGGACAGATGATGAAATCCCCCCGCCACTGCCTGAATGGACACCCGAGTCTTTTATTGTGGTTGAGGAAGCC which differed from the genomic DNA by three bases. The intended C to T change introduced a new XcmI restriction digest site and the arginine to tryptophan substitution. A further two silent mutations in the PAM region for the guide RNA were introduced to halt any subsequent Cas9 activity at this site. Introduction of the intended mutations were confirmed by restriction digest with XcmI and Sanger sequencing.Ptpn22 gene comprising at its 5′ end a Twin-Strep-tag-coding sequence which encodes for the peptide tag OST (One-STrEP-Tag) and a Gly-Ser-Gly spacer sequence as well as two loxP sites bracketing the modified exon 1  and 50 μg of anti CD8 (clone YTS 169.4), both BioXcell were given intraperitoneally. Where stated, groups of mice were given 300 μg mAb to IL-7R  intraperitoneally every second day over the 14 days experimental time-course. The anti CD25 antibody (clone PC61) was purchased from BioXcell and a single dose of 500 μg per mouse was injected intraperitoneally.+CD62L+CD44−CD25− T cells mixed at a ratio of 1:1. Naïve CD4 T cells were obtained by enrichment of CD4+ T cells using a mouse CD4+ T cell Isolation Kit (Miltenyi), followed by sorting for naive CD4+CD62L+CD44−CD25− T cells with a FACSAria sorter (BD).For cell transfer into Rag-1 KO recipient mice, CD45.1 PTPN22 WT, CD45.1/2 PTPN22 R169W, and CD45.2 PTPN22 KO OT-I T cells were mixed at a ratio of 1:1:1 and a total of 5x10e5 cells were injected intravenously. In some experiments, recipient mice were lethally irradiated before cell transfer. Mixed bone marrow chimeras were generated by reconstituting CD45.1/2 PTPN22 WT recipients with T cell depleted bone marrow cells from CD45.1 PTPN22 WT and CD45.2 PTPN22 KO mice, mixed at a ratio of 1:1. In other experiments CD45.1/2 recipients were reconstituted with Rag1 KO bone marrow and received in addition 2.5x10e5 CD45.1 WT and CD45.2 PTPN22 KO naïve CD4The following specific antibodies were used: a(anti)CD4 fluorescin isothiocyanate (Fitc), allophycocyanin (APC) or Pacific Blue (clone RM4-5), aCD4 phycoerythrin (Pe) cyanine (Cy)7 or Pacific Blue (clone RM4-4), aCD8 Fitc or peridinin chlorophyll protein (PerCP)/Cy5.5 (clone YTS156.7.7), aCD25 PeCy7 (clone 3C7), aCD45 PerCP/Cy5.5 (clone 30-F11), aCD45.1 Fitc or APC (clone A20), aCD45.2 PcP, PeCy7 or Pacific Blue (clone 104), aCD62L Pe, APC or Pacific Blue (clone MEL-14), aIFNγ Fitc, PeCy7 or APC (clone XMG1.2), aIL-10 Pe or APC (clone JES5-16E3) and aIL-10R (aCD210) Pe (clone 1B1.3a) were purchased from BioLegend. aCD8 PeCy7 (clone eBioH35-17.2), aCD25-APC or PeCy7 (clone PC61.5), aCD44 Alexa eFlour780 (clone IM7), aFoxp3 eFlour 450, Pe or APC (clone FJK-16s) and aT-bet Pe (clone 4B10) were purchased from eBiosciences. aKi67-PE or Fitc (clone B56) and aCD4-Fitc (RM4-5) were purchased from BD. Live/Dead Aqua or Far Red (both Life Technologies) were used to identify live cells. Intracellular cytokine staining was performed using Fixation buffer (BioLegend) to permeabilise and fix cells, for subsequent antibody staining the Intracellular Perm Wash Buffer (BioLegend) was used. For staining of transcription factors, cells were permeabilized and fixed in FoxP3 Fix/Permeabilization Buffer (eBioscience) before staining with antibodies in Permeabilization/Wash buffer (eBioscience). Samples were acquired with a Fortessa (BD) or a MACSQuant flow cytometer (Miltenyi). Data was analyzed with FlowJo software (Treestar).in vitro cell proliferation assays cells were labeled with 500 nM CellTracer Violet (Life Technologies). N4, T4, and G4 peptides (Peptide Synthetics) were added to culture medium at indicated concentrations. Recombinant IL-7 (Peprotech) was used at 20 ng/mL, recombinant IL-10 (Peprotech) was used at indicated concentrations. For cytokine recall responses cells were restimulated with 20 ng/mL phorbol 12,13-dibutyrate (PdBU) and 0.5 μg/mL Ionomycin  in presence of 1 μg/ml Brefeldin A for 4 h before intracellular staining (antibodies identified above). Transgenic OT-1 T cells were restimulated with T4 peptide (SIITFEKL) in presence of 1 μg/ml brefeldin A for 4 h. Where stated cells were stimulated in media with 0.5 μg/mL anti CD3ε antibody and 2 μg/mL anti CD28 antibody (both BioLegend) for 4 h.Cells from lymph nodes or spleens were cultured in IMDM medium supplemented with heat-inactivated 10% FBS, 2 mM L-glutamine, 100 U/mL Penicillin, 100 mg/mL Streptomycin and 50 μM 2-mercaptoethanol . For t-test or a 1way ANOVA with a Sidak's multiple comparison test was performed. For experiments with two factors a 2way ANOVA test was used followed by Sidak's multiple comparison test. In experiments in which cells of different genotypes were adoptively transferred into one host a paired analysis was used. When a dataset failed to meet the requirements for a parametric test, a non-parametric unpaired Kruskal Wallis test with a Dunn's multiple comparison test was used. All experiments were repeated at least twice. The figure legends are stating the number of repeats for each experiment and how many animals were used.Prism software Version 7 was used for statistical significance analysis. All data was tested for normality and depending on the experimental set up a Student's Ptpn22 regulated homeostatic T cell proliferation in response to transient lymphopenia in mice we chose an Ab-mediated lymphopenia model, as this was similar to the mode of action of drugs such as Alemtuzumab which is used for lymphodepletion therapy in man. A single injection of 100 μg CD4 and 50 μg CD8 mAbs per mouse mediates severe T cell depletion with high specificity (Ptpn22 wildtype (WT) and Ptpn22 knockout (KO) mice (p = 0.05) (To test whether cificity . At day cificity . The depKO) mice . After dKO) mice . In contKO) mice . CD4 T cKO) mice , and wer = 0.05) , suggest+ cells was slightly elevated in CD4 T cells in comparison to untreated mice where there was no difference between the genotypes. However, IFNγ expression was significantly increased in PTPN22 KO CD4 T cells indicating that PTPN22 influences the inflammatory potential of T helper cells and the difference between WT and KO was particularly pronounced in T cells of mice that underwent lymphopenia induced proliferation  and 60%  of Tregs were depleted 3 days after antibody injection ; the ove+ Treg cells in steady state  than the co-generated WT Treg, indicating that their acquisition of regulatory function and state of activation is also regulated by PTPN22 , lethally irradiated CD45.1/2 PTPN22 WT mice were reconstituted with mixed BM from CD45.1 PTPN22 WT and CD45.2 PTPN22 KO mice in a 1:1 ratio. In this scenario, newly formed T cells exit the thymus into a lymphopenic adult host and undergo homeostatic expansion and we should expect to observe similarly expanded Treg populations in both WT and PTPN22 KO T cells if this is driven by the presence of PTPN22 KO hematopoietic cells. Fifteen weeks after BM transplant we found higher Foxp3neration . Furthery PTPN22 .+ CD4 T cells may be restraining lymphopenia-induced proliferation in the context of the PTPN22 KO and attempted to address this point by specific depletion of Treg using an anti-CD25 antibody, but were unsuccessful due to the poor depletion efficacy of this antibody  behaved more similarly to either PTPN22 WT or KO cells in the context of lymphopenia. First, in order to exclude extrinsic influences of other hematopoietic populations and in particular Treg on lymphopenia-induced expansion, we used TCR transgenic OT-1 Rag-1 KO CD8 T cells, and adoptively transferred these cells into Rag-1 KO lymphopenic hosts. The availability of transgenic OT-1 CD8 T cells expressing CD45 congenic markers on the three genotypes, PTPN22 WT, KO, and R619W, allowed us to compare all genotypes in the same environment. The use of transgenic OT-1 CD8 cells, which express a T cell receptor that recognizes chicken ovalbumin, precludes responses to bacterial components from gut which can drive a much faster homeostatic expansion than the slow proliferation seen in response to self-peptide:MHC  were recovered with the R619W compared with the WT genotype .R619W is also defective in regulating lymphopenia-induced proliferation and CD8 T cells expressing this variant also have increased production of inflammatory cytokines indicating that this mutation could pose a risk factor for the development of secondary autoimmunity in man as a sequela to Ab-mediated lymphodepletion that failed to spare Tregs.Together these data show that proliferation induced by lymphopenia is intrinsically regulated by PTPN22 protein in both CD4 and CD8 T cells. The variant allele PTPN22PTPN22, identified as a risk factor for autoimmunity might increase susceptibility to development of autoimmune conditions following lymphodepletion. Using anti-CD4 and anti-CD8 antibodies in combination we found that severe T cell depletion was followed by transient proliferation and activation of T cells until T cell homeostasis was restored. PTPN22 deficient T cells showed higher expression levels of activation markers, such as CD44, and produced more pro-inflammatory cytokines compared to WT T cells. This activated phenotype persisted amongst PTPN22 KO T cells after restoration of homeostasis but did not lead to weight loss nor the induction of colitis. Unexpectedly PTPN22 deficiency had no impact on the rate of proliferation nor on absolute lymphocyte numbers recovered after Ab-mediated depletion, provided IL-7 cytokine was available. IL-7R expression is down-regulated in response of TCR mediated signaling (In this study we tested the hypothesis that a gene, ignaling . Thus, Iignaling , 21. In ignaling .in vitro and in vivo 45 days after transfer into Rag-1 KO recipient mice (de novo thymic output or enhanced iTreg generation.The most striking differences we found between the two genotypes were increased levels of Tregs and IL-10 producing T cells in PTPN22 KO compared to WT mice from 2 weeks post depletion. IL-10 was shown previously to be critical for Treg control of LIP . Tregs went mice . TogetheRegulatory T cells have been shown to be important to keep conventional T cells in check and prevent further autoimmunological complications in lymphopenic conditions. Suppression of antigen mediated T cell proliferation by Tregs through CTLA-4 mediated downregulation of costimulatory CD80/CD86 expression on APCs and thus modulation of CD28 signaling is well-described , 26. ThePtpn22 gene resembles the full knock-out suggesting that R619W is a loss of function mutation (R619W knock-in mice (Given that PTPN22 is a major risk allele for autoimmunity and its role in the negative regulation of T cell activation in response to antigens with a weak TCR affinity, such as self-antigens, we hypothesized that functional PTPN22 might be critical for “safe” T cell restoration after a lymphopenic insult. The association of the PTPN22 R620W SNP with various autoimmune diseases suggests a regulatory role of functional PTPN22 for T cell activation in human. In mouse the R619W point mutation in the mutation , 33. Intmutation , 32. ThiHigher TCR affinity might lead to chronic signaling in PTPN22 KO T cells, which could cause the upregulation of co-inhibitory receptors and thereby limit excessive proliferation in absence of PTPN22. The co-inhibitory receptor PD-1 is known to be upregulated in response to T cell activation and negatively regulates T cell function. Transgenic male antigen specific PD-1 KO CD4 T cells outnumbered WT cells when adoptively transferred into female Rag1 KO, suggesting that PD-1 regulates tonic pMHC signaling and LIP . In contR619W mice had a similar expansion of Tregs to the PTPN22-KO mice, raises the intriguing possibility that in man, carrying the PTPN22R620W allele might even be protective against secondary autoimmunity following therapeutic Ab depletion, rather than being an increased risk factor as we originally hypothesized. Conversely, enhanced T cell activation mediated by LIP in absence of PTPN22 could promote tumor immunity and be attractive for cancer therapies.Based on our findings we conclude that antibody mediated T cell lymphopenia does not trigger overt auto-aggression in PTPN22 KO mice although PTPN22 deficient T cells showed a pronounced effector phenotype. High abundance of Tregs and IL-10 producing cells in PTPN22 KO mice have been previously associated with increased tolerance in a model of pancreatic islet transplantation and might suppress excessive T cell proliferation and keep activated T cell clones in check . T cell The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by UK Home Office and performed in accordance with the institutional and ethical guidelines of the University of Edinburgh.Ptpn22 gene construct and provided OST-tagged PTPN22 mouse. RZ led the overall project, designed experiments, and wrote the manuscript.JK designed and performed experiments, analyzed data, and wrote the manuscript. DW designed the PTPN22 R619W mutant mouse strain. AC provided intellectual input into the project and design of the PTPN22 R619W mouse. BM designed the OST-tagged 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.


BAX and CDKN1A. Interestingly, microgravity increased the PTEN, DRAM1, and PRKAA1 mRNA levels. However, microgravity decreased the levels of mTOR and increased the LC3-II/I ratio, suggesting the activation of autophagy. The CDDP-induced cleavage of caspase-3 was increased during the early phase in Group MG (+), and cleaved caspase-3 was detected even in Group MG (+) with constitutive expression of a mutant type of p53 . These results interestingly indicate that microgravity altered CDDP sensitivity through activation of caspase-3 by p53-independent mechanism.Although the biological systems in the human body are affected by the earth’s gravity, information about the underlying molecular mechanisms is limited. For example, apoptotic signaling is enhanced in cancer cells subjected to microgravity. We reasoned that signaling regulated by p53 may be involved because of its role in apoptosis. Therefore, we aimed to clarify the molecular mechanisms of modified cis-diamminedichloroplatinum (CDDP)-sensitivity under simulated microgravity by focusing on p53-related cell death mechanisms. Immunoblotting analyses indicated that, under microgravity, CDDP-induced ATM/p53 signaling increased and caspase-3 was cleaved earlier. However, microgravity decreased the levels of expression of p53 targets  Our biological systems work properly in 1G gravity, indicating that those are governed by gravitational force on the earth –5. Under−3 G). Using the 3D-clinostat, we demonstrated that simulated microgravity affects biological processes such as embryogenesis, stemness of embryonic stem cells, and the differentiation of skeletal muscle [For these reasons, our group developed the 3D-clinostat to conduct cell culture under simulated microgravity  according to a published method [TP53 (R248W) was constructed using site-directed mutagenesis of pCMX-p53 (pCMX-p53-R248W). The integrities of the vectors were verified using nucleotide sequence analyses.Human d method . Mutant 2. After reaching 80% confluence, the cells were detached using 0.25% trypsin, seeded into a 12.5 cm2 flask (2.0 × 105 cells), and cultured for one day. The flasks were subsequently divided into the groups as follows , 100 μg/mL kanamycin (Sigma-Aldrich) at 37°C in an atmosphere containing 5% CO follows : HepG2 c−3G in 8 min, and the gravity condition was measured by a gravity acceleration sensor. Cells were harvested and counted at the indicated times  cancels the effect of 1G gravity through controlled rotation of two axes to generate an average of 10ed times . For expACTB  was used as an internal control. ACTB mRNA.Total RNA was prepared from frozen cell pellets using NucleoSpin RNA (Macherey-Nagel) according to the manufacturer’s protocol. A ReverTra Ace-α Kit (Toyobo) was used to synthesize cDNA. Real-time RT-PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems) and FastStart Universal Probe Master (Roche), following the TaqMan probe method described in manufacturer’s protocol. Standard curves were generated from serial dilutions of cDNA from various cell lines. Gene expression levels were then quantified using 7500 System SDS software version 1.5.1 (Applied Biosystems). Whole-cell extracts were prepared from frozen cells as described previously . Each prt-test or the Mann–Whitney test was performed as appropriate. P < 0.05 was considered statistically significant.SPSS Statistics version 17.0 (IBM) was used to perform statistical analyses. When compared with set 0, statistical analyses were performed using one-way ANOVA, and Dunnett’s post-hoc test was used to compare the groups. For comparisons of each group analyzed at the same time, the Student’s 50 value of CDDP for HepG2 was determined to be 419.6 ng/mL  and Group MG (+) . The levBAX increased after 48 h in Group 1G (+). In contrast, there was no significant change in Group MG (+)  but did not in Group 1G (+)  . Furtherp MG (+) . The levp MG (+) . When wep 1G (+) .BAX and CDKN1A mRNAs in HepG2 mt cells were significantly lower compared with those of HepG2 mock cells , we used HepG2 cells stably transfected with a vector expressing a mutant p53 cDNA, pCMX-p53-R248W (HepG2 mt cells), or cells transfected with the empty pCMX vector (HepG2 mock cells). The dominant-negative effect of enforced expression of the p53 mutant was evaluated using a reporter assay. The p53 target promoter activity was inhibited in pCMX-p53-R248W dose-dependent manner  and Group MG (+) . The levp53-mediated apoptosis and autophagy-related signaling in HepG2 mock cells was not significantly different from that of native HepG2 cells Figs .Then, we evaluated p53-mediated signaling in HepG2 mt cells cultured under normal gravity or microgravity. The kinetics of phosphorylation and total ATM were identical in both cell types under both conditions . The levBAX mRNA in HepG2 mock cells increased with CDDP treatment in a time-dependent manner under both gravitational conditions. BAX levels were higher in Group MG (+) after 24 h but decreased by 48 h (BCL2 mRNA increased only in Group 1G (+)  . The levnditions . Any spere found .BAX and CDKN1A mRNAs in HepG2 mt cells were significantly lower compared with those of HepG2 mock cells (BAX and CDKN1A mRNAs in HepG2 mt cells increased after 72 h in Group 1G (+), while those in Group MG (+) increased by 48 h  increased after 24–48 hours; however, in Group MG (+), they increased after 72 h  HepG2 cells were treated with 500 ng/mL CDDP for 0, 24, 48, or 72 h. Immunoblotting was conducted using whole-cell extracts prepared from HepG2 cells at each time point. Representative images from three independent experiments are shown. (A) The line graphs of percent cell viability relative to control are shown as the mean ± SD; (B) Expression levels for each immunoblot were quantified; relative protein levels were calculated as the ratio to β-actin level. Values are expressed as mean ± SE.(A) HepG2 cells were treated with various concentrations of CDDP from 0 to 10000 ng/mL for 72 h. The MTT assay was then performed, and the drug concentration of 50% absorbance of the control was calculated as the IC(EPS)Click here for additional data file.S2 FigACTB mRNA levels for each experiment, and the values are expressed as the mean ± SD (n = 3). *p < 0.05; **p < 0.01, (C) Value are represented as the mean ± SD (n = 5); **p < 0.01.(A) HepG2 cells were transiently transfected with different amounts of the p53 luciferase reporter vectors pCMX-p53 (p53wt) and pCMX-p53-R248W (p53 mt). Promoter activity was calculated as the ratio of firefly to renilla luciferase activities. (B) Real-time RT-PCR analysis of the expression of p53 target genes in HepG2 mock cells and HepG2 mt cells. (C) The number of HepG2 mock cells and HepG2 mt cells cultured for 72 h under normal gravity. (A) Values are expressed as the mean ± SD (n = 5); **p < 0.01, (B) Relative mRNA levels were calculated as the ratio to (EPS)Click here for additional data file.S3 Fig(A) The immunoblots in (EPS)Click here for additional data file.S4 Fig(A) The effects of p53-related signaling in Group 1G (+) and Group MG (+). (B) HepG2 mt cells. The effects of p53-mediated signals in Group 1G (+) and Group MG (+).(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P[8] VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006–2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers. Rotavirus A (RVA), a member of the genus Rotavirus and family Reoviridae , G2P[4], G3P[8], G4P[8] and G9P[8] ,d G9P[8] –19.P8] and P[4] RVAs have been identified as the first and second predominant P- genotypes, respectively, causing more than 90% of rotavirus-associated diarrhoea cases in many countries , 21. Of  and P[4]Phylogenetically, four distinct lineages of the P[8] genotype namely P[8]-Lineage I, P[8]-Lineage II, P[8]-Lineage III and P[8]-Lineage IV have been described , 23. CurWidespread use of rotavirus vaccines could result in strain selection due to vaccine-induced selective pressure as demonstrated by Roczo-Farkas et al.  who noteThere is an abundance of molecular epidemiological data on the prevalence of both common and unusual G/P types determined by RT-PCR in Ghana. These studies described RVA strains with unusual G and P combinations and revealed the G9 genotype as an important emerging RVA in Ghana. In addition, it was also shown that G1P[8] RVA was replaced as the major circulating strain during the pre-vaccination era with G12P[8] as the most predominant strain post vaccine introduction. –29. HoweThe study protocol was approved by the Institutional Review Board, Noguchi Memorial Institute for Medical Research, Legon, Accra, Ghana. A total of 57 RVA positive samples analysed in this study were collected from children <5years who were hospitalized with a primary diagnosis of acute gastroenteritis between 2007 and 2011. The G-genotypes of these 57 samples were previously reported in studies carried out by Enweronu-Laryea et al. , DamankaTotal RNA was extracted from 10% stool suspension by the phenol/chloroform method as described by Steele and Alexander  and the The amplicons were purified with the QIAquick PCR purification kit (Qiagen/Westburg) according to the manufacturer’s protocol and sequenced in the forward and reverse directions using the respective amplification primers and the BigDye Terminator cycle sequencing kit v.3.1 (Applied Biosystems). The cycle sequenced products were purified, using ethanol-sodium acetate precipitation method, dried under vacuum and reconstituted in Hi-Di formamide. DNA sequences were determined using ABI 3130xl genetic analyser .Near-full-length VP7 and VP8* portion of the VP4 gene were obtained by combining the sequences obtained from the forward and reverse reactions. The genotypes were determined for the VP8* portion and confirmed for the G-types using the online RotaC v2.0 RVA genotyping tool . Using tBased on the best fit nucleotide substitution models with the lowest Bayesian Information Criterion scores , i.e. TaAll 57 rotavirus-positive samples had enough bulk stool specimens for further analysis in this study. The VP8* fragment of the VP4 genes of these samples were successfully sequenced and genotypes were determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare P[8]b (OP354-like) subtype. The P-genotypes of the remaining 9/57 samples were P[6]: 7(12.3%), P[4]: 1(1.75%), and P[14]: 1(1.75%). Out of the 43 P[8]a sequences, 39  were of The nucleotide and amino acid sequence diversity of the VP8* fragment of the VP4 gene among the Ghanaian P[8]a strains was relatively high even between strains detected from the same place and year, ranging from 0% - 5% and 0% - 9.4%, respectively. Specifically, strains from DC/2008 varied from 0%– 2.9%, those from DC/2010 varied from 0.2% - 3.2%, those from PML/2010 varied from 0.7% - 3.9%, those from EB/M/P/2010 varied from 0% - 2.9%  Tables. Phylogenetic analysis of the VP8* fragment of the VP4 gene using sequences available in the DNA databases maintained the previously established four distinct lineages designated lineage I–lineage IV . The Pa when the ambiguous codes N, Y, and Y were respectively substituted with C, C, and T (Twenty-nine of the P[8]a sequences consistently showed three mismatches at nucleotides 7 (T→C), 10 (C→T) and 12 (A→C)  upon aliC, and T . The newCAN GTY AAY CCA GTA GA), a single nucleotide mismatch at nt 8 (A→G) was detected.Additionally, nine of the strains showed five consistent mismatches at nucleotide (nt) 7 (T→C), 8 (A→G), 9 (T→A), 10 (C→T) and 12 (A→C) when aligned with 1T-1 primer . These nReverse transcription polymerase chain reaction genotyping methods used in rotavirus surveillance studies, have been useful in monitoring circulating strains and identifying rare and emerging strains worldwide. Advances in rotavirus molecular assays such as sequence analysis of rotavirus genes provide us the opportunity to i) genotype non-typeable rotavirus strains that have mutated hence might be recognized by the routine genotyping primers and ii) understand their evolutionary histories using molecular phylogenetic inferences. Our previous study on Ghanaian rotavirus strains revealed that a considerable proportion of the RVAs could not be G and/or P genotyped by the routine multiplex RT-PCR strategy by Gouvea et al.  and Gentth quarter of 1999  strains were rarely reported in Ghana while the G9P[8] strains emerged and predominated in the 4 of 1999 , 44. Fro of 1999 . The G1  of 1999 , 46. We  of 1999  and Heyl of 1999  from Gha8 x10-3)  and G9 V7 x10-3) .Phylogenetic analysis of P[8]—VP4 sequences from the global rotavirus database maintained that P[8] rotaviruses could be divided into the four previously established distinct phylogenetic lineages: P[8]-lineage I, P[8]-lineage II, P[8]-lineage III and P[8]-lineage IV. All the Ghanaian strains were grouped into lineage III and seemed to be related to African, Australian and European strains detected between 2006 and 2014, indicating that they shared common ancestral strains . In the http://apps.who.int/iris/bitstream/handle/10665/70122/WHO_IVB_08.17_eng.pdf;jsessionid=8C5703F323752AD2934C58EDEAE338AA?sequence=1). The observed difficulty in P-typing was solved by including a new primer 1T-1GHA—designed based on the 1T-1D degenerate typing primer in our PCR reactions and this additional primer helped genotype the non-typeable strains reported previously in the reference laboratory in Ghana.Sequence comparison of the VP4 genes revealed mismatches at the P[8] primer (1T-1) binding site which we considered to be responsible for the genotyping failure. The substantial numbers of incompletely genotyped strains could be attributed to genetic drift due to the accumulation of point mutations or the lack of specific oligonucleotide primers for amplifying novel strains . MajoritOur analysis provided information on how Ghanaian pre-vaccine introduction period G1P[8] and G9P[8] strains relate to a global collection of G1 and G9 strains at the lineage level. The study also resolved the methodological limitations of the standard genotyping techniques employed in the Rotavirus Regional Reference laboratory in Ghana. Genotyping failure attributable to natural variation in primer binding sites could be cited among problems encountered in large surveillance studies. The study highlights the importance of close monitoring of rotavirus genotyping methods as well as regularly updating the primer sequences employed for molecular typing of rotaviruses. The findings also suggest that regions should start to use sequencing data from local rotavirus strains to design and update genotyping primers.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Multifocal lung cancers (MLCs) are common in patients newly diagnosed with lung cancer, and histological results of most synchronous MLCs are similar. Few cases with different histology findings have been reported, and no genomic or transcriptomic profiling of this kind of cases were done before. Here, we analyzed genomic and transcriptomic profiles of all lung tumors from 2 patients with synchronous adenocarcinoma and squamous cell carcinoma in the same lung lobe.TP53 and KEAP1, were also detected. Mutation signature analysis demonstrated that the tumor initiation might be related to the transcription coupled nucleotide excision repair process. Two tumors for these 2 patients had loss of heterogeneity (LOH) in HLA genes, showing tumor escaping mechanism. Furthermore, tumor microenvironments showed different patterns in 2 tumors from the same patient. The tumor with more neoantigens and no HLA LOH showed more infiltrating CD8+ T cells and more clonal TCRs, indicating a more active microenvironment.Two patients were diagnosed as synchronous adenocarcinoma and squamous cell carcinoma and underwent surgical resection. All 4 tumors showed distinct genomic profiles, therefore were independent primary tumors. Several cancer-associated pathways, such as RTK-RAS pathway and Notch pathway, exhibited different mutated genes in different tumors from the same patient. Several known cancer genes with different mutations, including The lung squamous cell carcinoma and lung adenocarcinoma form the same patient are from independent origins. The genetic profiles and transcriptomic microenvironments are quite different for these 2 tumors. With the same genetic background, the 2 tumors in one patient exhibited different tumor escape mechanisms and immune responses, including HLA LOH and T cell infiltrating and expansion. Lung cancer is the leading cause of cancer incidence and mortality in China and all over the world . Among aThe synchronous MLCs represent complex evolutionary process. Some synchronous MLCs occur independently, but some others are originated form a single site and then spread to other sites , 7, 8. TIn this study, we analyzed 4 tumors from 2 patients with synchronous lung adenocarcinoma and lung squamous cell carcinoma to evaluate genetic profiles and transcriptomic microenvironments. Our results demonstrated that lung squamous cell carcinoma and lung adenocarcinoma form the same patient were from independent origins, showing quite different genetic profiles and transcriptomic microenvironments.Two patients were diagnosed with pathologically confirmed synchronous MLCs and were free of extrathoracic metastases. The two tumors from the same patients were all on the same lung lobe. The patients underwent surgical resection, and the tumor sizes ranged from 1.3 to 1.6 cm according to pathology reports   of these pathways were detected. Of these genes, 73% genes existed SNVs or CNVs in only one sample. Tumors of P1 showed amplifications of oncogenes in ADC and SQCC, while tumors of P2 occurred deletions of tumor suppressor genes in ADC and SQCC  of human leukocyte antigen (HLA) in P1S and P2A Fig. c. It’s kTumor microenvironment (TME) evolved by tumor surrounding cells, including immune cells, fibroblasts, endothelial cells and other kinds of cells. We performed RNA-sequencing to investigate on the tumor microenvironments between P1A and P1S , indicating more T cell expansion events in P1A .Additional file 2: Figure S2. Immunohistology of CD8+ and CD4+ T cells in P1 (X400). (A) CD8+ T cells of P1A. (B) CD8+ T cells of P1S. (C) CD4+ T cells of P1A. (D) CD4+ T cells of P1S.Additional file 3: Table S1. Non-synonymous mutations of P1 and P2. Table S2. Gene expressions (FPKM) of P1.


Both inactivated influenza vaccines (IIV) and live-attenuated influenza vaccines (LAIV) have been recommended for administration to children. Children are a high-risk group for severe influenza, and a major source of transmission. Therefore, prevention of infection by vaccination is particularly important. However, efficacy and immunogenicity of these vaccines are known to vary by season and geographic location. We compared the immunogenicity of the 2014–2015 Northern Hemisphere trivalent IIV and LAIV against influenza A virus in Canadian Hutterite children aged 2 to 17 using hemagglutination inhibition (HAI) assays, and enzyme-linked immunosorbent assays to measure hemagglutinin-specific serum IgA and mucosal IgA. Both vaccine formulations induced significant increases in HAI titers against H1N1 and H3N2 vaccine strains. Serum IgA titers against H3N2 were significantly boosted by both IIV and LAIV, while only IIV induced a significant increase in serum IgA specific to the H1N1 vaccine strain. While HAI titers correlated with protection conferred by IIV, mucosal IgA titers correlated with protection conferred by LAIV . IIV and LAIV were previously reported to be equally efficacious in this cohort, although the immunogenicity of IIV was generally superior.  Seasonal influenza virus epidemics continue to cause millions of hospitalizations, and hundreds of thousands of deaths globally each year . Attack ®/Fluenz™) and became available in 2003 [While inactivated influenza vaccines (IIV) formulations have been available for several decades, the first live-attenuated influenza vaccines (LAIV) widely approved for use in humans was manufactured by MedImmune  titers, serum IgA endpoint titers, and mucosal IgA titers against the influenza A virus (IAV) components (H1N1 and H3N2) of the vaccines were evaluated.In this study, we assessed 618 healthy Hutterite children and adolescents aged 3 to 15 years in the 2014–2015 flu season who participated in a cluster randomized clinical trial  that compared the direct and indirect protectiveness of trivalent IIV and trivalent LAIV, as reported previously [Blood samples were drawn from participants at baseline and between 3 and 5 weeks post-vaccination. For previously unvaccinated children under 9 years of age who received two doses of vaccine, samples were collected after the second dose. Active surveillance was conducted twice weekly during the influenza season  and those with two or more signs or symptoms compatible with influenza were tested by RT-PCR of nasal swabs. We collected nasal swabs to test for mucosal IgA antibodies at baseline and three weeks after vaccination with either IIV or LAIV.The HAI assay was performed as previously described using turkey erythrocytes and reference antigens for A/California/07/2009 (H1N1) pdm09-like virus  and A/Texas/50/2012 (H3N2)-like (Tex/50 H3N2) .ELISAs were performed on 96-well Nunc-immuno maxisorp plates . The plates were coated overnight with 2 ug/mL per well of purified recombinant HA  in bicarbonate–carbonate coating buffer . The plates were blocked with 5% skim milk for 1 h. Serum samples were serially diluted starting with 1:50 dilution in 5% skim milk and then incubated for 1 h at room temperature. Mucosal samples were undiluted and were incubated for 1 h at room temperature. Plates were washed three times with PBS-T (0.1% Tween 20). Secondary goat anti-human IgA-horseradish peroxidase (HRP)  at 1:4000 dilution was added in 5% skim milk for 1 h at room temperature, followed by 3x PBS-T wash prior to addition of HRP substrate . Reactions were stopped after 10 min by the addition of 3 M HCl, and the optical densities were read at 490 nm on a Spectramax I3 . Endpoint titers of serum IgA were defined as the lowest dilution whose optical density remained three standard deviations above the mean of negative control wells. Since mucosal IgA could only be reliably measured using undiluted samples, only normalized optical density (O.D.) values were reported. We chose to favor specificity over sensitivity, and therefore, normalization was performed by subtracting the mean O.D. plus three standard deviations of blank samples from each experimental sample.We compared pre- and post-vaccination antibody titers in terms of three types of antibody measures through paired t tests for both the IIV and LAIV groups. A Mann–Whitney U test was used to compare the changes of the three antibody titers between the IIV and LAIV groups. We used linear regression to estimate the associations between antibody changes and their related factors. A backward stepwise selection method was used to determine the variables in the final model. Finally, we evaluated the correlation of protection against influenza by calculating protective effectiveness using Cox’s proportional hazards model. The protective effectiveness was defined as (1-hazard ratio) X 100%. All analyses were performed in R version 3.4.2.The characteristics of participants in the IIV group and LAIV were similar . The meap < 0.01) (p < 0.01)  A. In com < 0.01) B. The unp < 0.01), it was substantially lower than that observed for the children vaccinated with IIV (1.64) (p < 0.01), which again was lower than that observed for participants who received IIV (p < 0.01). Furthermore, the elevated pre-vaccination titers observed in the IIV group  log transformed HAI titer against Cal/09 H1N1 was 4.17 (1.91) pre-vaccination and 4.29 (1.87) post-vaccination. While this increase of 0.11 was statistically significant  A. In LAIived IIV B. OveralIV group  likely rIV group .p < 0.01) (p < 0.01)  A. The me < 0.01) B. The unp = 0.22) (p = 0.03) (The mean (SD) log transformed serum IgA endpoint titer against Cal/09 H1N1 in the LAIV group was 2.88 (2.27) pre-vaccination and 2.96 (2.36) post-vaccination. This increase was not statistically significant  A. The me = 0.03) B. While p = 0.03) (p < 0.01) (The mean (SD) mucosal IgA titer against Cal/09 H1N1 from the IIV group was 0.08 (0.16) pre-vaccination and 0.10 (0.14) post-vaccination, representing a statistically significant increase of 0.02  A. The me < 0.01) B.p = 0.23) (p < 0.01) (The mean (SD) mucosal IgA endpoint titer against Cal/09 H1N1 in the LAIV group was 0.11 (0.25) pre-vaccination and 0.10 (0.14) post-vaccination, corresponding to a non-significant decrease of −0.01  A. The me < 0.01) B. IndiviPre-vaccination mucosal IgA titers against Tex/50 H3N2 were substantially higher in the LAIV group , which wAs shown in The change of HAI titers against Tex/50 H3N2 was significantly associated with vaccine type , pre-vaccination titer, and age. The change of serum IgA titers was significantly associated with vaccine type and pre-vaccination titer. The change of mucosal IgA titer was significantly associated with vaccine type and pre-vaccination titer .We only detected H3N2 cases in the study participants, consistent with the known dominance of H3N2 circulation during the 2014–2015 season. Therefore, the correlates of protection analysis were only performed for the H3N2 virus.For HAI titers, protective effectiveness estimates in the IIV group were statistically significant at titer values of 1:80 and 1:160. The estimates at these two titer values were similar (77% vs. 76%) . HAI titSerum IgA titers did not correlate with protection in either the IIV group or the LAIV group . For mucChildren represent an extremely important group when it comes to the prevention of influenza virus infection by vaccination due to their elevated risk for severe illness, and their role as a major source of viral transmission ,23. IndeDespite observational data from the U.S. Vaccine Effectiveness network that led ACIP to recommend against the use of LAIV during the 2016–2017 influenza season, members of our group previously published the results of a cluster-randomized control trial in which the efficacy of IIV and LAIV was equivalent across the three influenza seasons spanning October 2012 to May 2015 for all strains present in the trivalent vaccine . HoweverBoth IIV and LAIV induced significant increases in serum HAI titers against both H1N1 and H3N2 however, the magnitude of seroconversion induced by IIV was much greater—consistent with prior studies . HAI titIIV recipients experienced a small but significant boost in mucosal IgA titers against both H1N1 and H3N2 vaccine components. No increase in mucosal IgA was observed in the LAIV group. However, consistent with an earlier H1N1 challenge study of children vaccinated with LAIV, detection of any mucosal IgA correlated with protection against H3N2 infection . Given tThe lack of a detectable boost in mucosal IgA titers in the LAIV group was surprising, since LAIV has been reported to boost mucosal IgA titers in several previous studies ,26,27,28The influence of pre-existing immunity on the vaccine-induced responses during the 2014–2015 influenza season is also an important consideration in this study—since most of the participants had been enrolled in the study and vaccinated for each of the two previous seasons ,30. HighA notable limitation of this study is that peripheral blood mononuclear cells were not collected and thus, T cell responses were not measured. LAIV is known to effectively boost T cell responses, including cross-reactive T cells that may help to protect against drifted or novel strains ,33,34. GTaken together, the results of this study provide new insights into the nature of the humoral immune response elicited by IIV and LAIV in children. While vaccine efficacy against all strains of influenza virus were equivalent in this cohort, the immune responses elicited by each vaccine were distinct, as were their respective correlates of protection. By most measures, IIV demonstrated superior immunogenicity compared to LAIV. The data presented here reinforce the notion that the HAI titer suggestive of antibody-mediated protection may be greater than 1:40 in children—and that distinct correlates of protection are required for IIV and LAIV.


These co-cultures support longterm production of a novel dendritic-like cell type along with transient production of myeloid cells. They also maintain a progenitor cell population. The splenic stromal lines 5G3 and 3B5 have been identified as a supporter and a non-supporter of hematopoiesis. Based on their gene expression profile, both 5G3 and 3B5 express genes related to hematopoiesis, while 5G3 cells express several unique genes, and show upregulation of some genes over 3B5. Based on gene expression studies, specific inhibitors were tested for capacity to inhibit hematopoiesis in co-cultures. Addition of specific antibodies and small molecule inhibitors identified VCAM1, CXCL12, CSF1 and SPP1 as potential regulators of hematopoiesis, although both are expressed by 5G3 and 3B5. Through inhibition of function, SVEP1 and ALDH1 are also shown here to be deterministic of 5G3 hematopoietic support capacity, since these are uniquely expressed by 5G3 and not 3B5. The achievement of inhibition is notable given the dynamic, longterm nature of co-cultures which involve only small numbers of cells. The alternate plan, to add recombinant soluble factors produced by 5G3 back into 3B5 co-cultures in order to recover in vitro hematopoiesis, proved ineffective. Out of 6 different factors added to 3B5, only IGF2 showed any effect on cell production. The identification of differentially expressed or upregulated genes in 5G3 has provided an insight into potential pathways involved in in vitro hematopoiesis leading to production of dendritic-like cells.Cultured splenic stroma has been shown to support  A cell pellet was resuspended in 40μl of labelling buffer containing a cocktail of biotin-labelled antibodies specific for hematopoietic lineage markers CD5, CD45R, CD11b, Gr-1 (Ly-6G/C), 7–4, and Ter-119  equipped with DFC digital camera (Leica) to obtain phase contrast photomicrographs. Images were processed using Leica IM software v4.0.The morphology of stromal cells was monitored microscopically before and after addition of overlay cells. Cultures of stroma alone, or cocultures of Linμg/106 cells in 1ml to block non-specific antibody binding. Fluorochrome or biotin-conjugated antibodies for CD11b (M1/70), CD11c (N418), MHC-II (AF6-120.1), CD45RB (C363.16A), Sca-1 (D7), c-Kit 2B8), streptavidin-PE-Cy7, streptavidin-PE, streptavidin-FITC and isotype control antibodies were purchased from either Biolegend or eBiosciences . All antibodies were titrated to determine concentration giving minimal saturating binding. Cells labelled with fluorochrome-conjugated antibodies were analysed in both multicolour and single colour staining protocols. Discrimination of live cells utilized staining with 1μg/ml propidium iodide (PI) . Isotype matched control antibodies or fluorescence minus one (FMO) controls were used to set gates to delineate specific antibody binding. Flow cytometric analysis was carried out using BD FACSDiva (Becton Dickinson) and FlowJo software .For analysis of cell surface marker expression and subsequent subset delineation, cells were stained with antibodies diluted in fluorescence-activated cell sorting (FACS) buffer . Antibody specific for FcγII/IIIR (CD32/CD16)  was used at 5 - bone marrow cells, at concentrations of 0.3, 1 and 3 μg/mL. These are listed in - bone marrow cells, giving shifts in median fluorescence intensity between 300 and 13000. Anti-CXCR7 and anti-ITGA9 bound to only small numbers of cells amongst Lin- bone marrow cells and so were titrated on hematopoietic stem cells (HSC) sorted from Lin- bone marrow as the Lin- Sca-1+c-Kit+ subset.Specific antibodies used in inhibition studies were titrated before use in co-cultures through binding to control Lin24 primers and Superscript II reverse transcriptase . This was followed by second strand cDNA synthesis. Double-stranded DNA was then purified using phenol-chloroform together with phase-lock gels . In vitro transcription and biotin labelling were performed using the BioArray High Yield RNA Transcript Labelling Kit . cRNA was purified on RNeasy Spin columns , fragmented, and labelled with biotin. Labelled cRNA was then hybridized to Murine Genome 430v2 genechips (Affymetrix) following the manufacturer’s instructions. They were washed followed by staining on the fluidics station (Affymetrix), ahead of scanning and image analysis using a Gene Array Scanner (Affymetrix). Scanned images of genechips were processed using Microarray Suite 5.0 software . Data files were prepared in Microsoft Excel containing probeset numbers, signal values and p-values. Further analysis involved extraction of data according to set criteria. The preparation of label, hybridisation to genechips, scanning, data compilation and basic analysis was performed by staff in the Biomolecular Resources Facility .Total RNA was isolated from stromal cell lines, and concentration and purity determined spectrophotometrically. Double stranded cDNA was synthesized from RNA in a two-step process. The first strand of cDNA was synthesis using T7-(dT)Abcg1 (PPM03895A); Actb (PPM02945A); Aldh1 (PPM04398F); Apod (PPM05327F); Ccl8 (PPM03165A); Clca1 (PPM04051E); Csf1 (PPM03116C); Ctla2a (PPM27745A); Cxcl12 (PPM02965E); Igf2 (PPM03655A); KitL (PPM02983C); Ms4a4d (PPM24757A); Postn (PPM34333E); Rspo2 (PPM32746A); Serpinα3n (PPM05779F); Sfrp2 (PPM03616C); Spp1 (PPM03648C), Svep1 (PPM05259A); Wnt5a (PPM04748C). The cDNA/primer mixture was then added to RT2 SYBR Green Mastermix and RNase-free water in a ratio of 1:6.25:5.25, respectively. Samples were loaded on to a LightCycler 480 . The cycling conditions used were: 1 cycle for 10 mins at 95°C to activate DNA Taq Polymerase, followed by 45 cycles of 15 sec at 95°C for extension, and then 1 min at 60°C for fluorescence data collection.Total RNA was isolated from stromal cell lines using the RNeasy mini kit following the manufacturer’s protocol (Qiagen). RNA was purified using the genomic DNA elimination mix, and concentration and purity determined spectrophotometrically. Following this, Buffer BC3, Control P2, Reverse Transcriptase mix and RNase-free water were added in ratios of 4:1:2:3 for preparation of cDNA. Incubation proceeded for 15 mins at 42°C for denaturation, then for 5 mins at 95°C to convert RNA into cDNA. Subsequently, cDNA was diluted in RNase-free-water for use in Realtime PCR reactions. Equal volumes of cDNA and primer (10μM stock) were mixed. Primers were purchased from SABioscience . These included: nd derivative max) method was used to obtain a cross point value (Cp), referred to as the cycle threshold (Ct). Cp is the point of maximal increase in fluorescence emitted by a single PCR reaction within the log-linear phase. To further analyse data, Ct values for genes of interest (GOI) along with housekeeping genes (HKG) were imported into Excel . ΔCt = Ct (GOI)–Ct (HKG) was calculated, and the average ΔCt taken from quadruplicate experiments. To calculate the fold change between two samples, the calculation 2-ΔCt (Sample 1) / 2-ΔCt (Sample 2), was used. The resulting value corresponds to the relative difference in mRNA quantity between two samples. The presence of an amplified product was confirmed by gel electrophoresis.Data analysis involved LightCycler 480 software v.1.2.9.11. (Roche). The Absolute Quantification  for sample size n. The Wilcoxon Rank Sum test has been routinely used to test significance (p≤0.05).In experiments involving addition of inhibitors (or replacement factors) where very small numbers of progenitor cells were available, a titration of additives was performed in preference to replication of tests of additive at just one concentration. A significant inhibitory (or enhancement) effect was indicated by the outcome of even or decreasing (increasing) cell production over 4 dilutions of additive. The null hypothesis is that cell production is random across the titration, and the alternative hypothesis is that decreasing (or increasing) cell production occurs with increasing concentration of additive. The probability of an ordered reducing (or expanding) sequence for cell production over four different dilutions is 1/24 or 0.0417 which is significant at p≥0.05.- bone marrow progenitors. Large non-adherent cells were detectable at 7 and 14 days above 5G3 but not above 3B5 (- bone marrow cells showed no viable cells after 14 days (not shown). Cell yield (%) determined by Trypan blue exclusion of recovered non-adherent cells at 7 and 14 days showed that 5G3 produced a significantly higher yield of live cells relative to input cell number than did 3B5  cells. After gating to remove debris on a forward scatter (FSC) versus side scatter (SSC) plot, DC were identified as CD11b+CD11c+ and myeloid cells as CD11b+CD11c-. MHC-II and CD45RB were used to identify mature and any regulatory DC amongst CD11c+ cells, respectively  (24%), compared with 3B5 co-cultures (4%). This showed 5G3 to be the best supporter, and 3B5, a weak or non-supporter of myelopoiesis. These co-cultures produced few CD11b+CD11c+CD45RB+ cells, described by others as regulatory DC [Flow cytometry was used to distinguish live cells produced in co-cultures and to analyse their cell surface phenotype. Non-adherent cells produced in Linectively . CD11b+CD8α- cDC , while Cmed L-DC . From asatory DC .in vitro hematopoiesis. Affymetrix Murine Genome 430v2.0 genechips were employed and duplicate experiments conducted. Several hypotheses were investigated. Firstly that supporting stroma like 5G3 express genes which encode molecules important for cell-contact dependent signalling leading to hematopoietic differentiation. The second was that 5G3 secretes cytokines or chemokines which specifically support hematopoiesis. Comparative analysis across two experiments involved normalisation of genechips and removal of variation due to batch effect.Transcriptome analysis was performed to identify genes specifically expressed or upregulated in 5G3 stroma compared with 3B5, to identify specific regulators of Apod, Svep1, Plxdc2, Aldh1, Serpina3n and Abcg1 were selected, on the basis of high signal value and potential role in hematopoiesis, for verification of differential gene expression using Realtime PCR  effect was determined by the outcome of decreasing cell (or increasing) production over 4 dilutions of added factor.Genes of interest either upregulated or specifically expressed by 5G3 over 3B5 included soluble factors available as commercially recombinant molecules in highly purified form . The pos-CD11c- fraction containing progenitors with a reduction in myeloid cell production , metabolism (Akr1c13), signalling , cell adhesion (Svep1), and development . Genes which dictate development or cell-cell contact are of primary interest, particularly those expressed in highest levels like Apod, Svep1, and Serpina3n and the proteins encoded by Plxdc2 and Aldh1a1. The dominant expression of many of these genes in 5G3 over 3B5 was confirmed by Realtime PCR (in vitro hematopoiesis is predicted through studies showing that specific inhibitors DEAB (for ALDH1a1) and anti-ITGA9 (for SVEP1) inhibited cell production in 5G3 co-cultures , a ligand for CD44 expressed by 5G3, may play a fundamental role in L-DC development. SPP1 is known to maintain HSC in the quiescent state by promoting engraftment to the stem cell niche , 60. SPPin vivo hematopoiesis have been identified in this study. CXCL12 binds to CXCR4 and CXCR7 which are receptors on HSPC [Vcam1-/- mutant mice gave an increase in circulating CFU-C over wild type mice, with a decrease in CFU-C accumulation in bone marrow and spleen [Several known regulators of  on HSPC . While Cd spleen .in vitro hematopoiesis. CTLA2 is expressed highly in the trabecular bone region of the bone marrow, suggesting a potential role in hematopoiesis [Several other molecules identified reflect potential regulators of opoiesis . PLXDC2,opoiesis . These mCxcl12, KitL, Spp1, Vcam1 and Csf1. Hematopoiesis is responsible for the production of myeloid cells, and this is supported by evidence that antibodies or inhibitors of several of these gene products which act through blocking receptors  led to reduced cell production of in 5G3 co-cultures. Such evidence justifies the search for differentially expressed or upregulated genes in 5G3 over 3B5 as determinants of in vitro hematopoiesis, and verifies the model co-culture system as highly suitable for returning genes which determine the hematopoietic support environment of HSC.The cloned stromal cell line 5G3 is a supporter of hematopoiesis while 3B5 is a non-supporter. 5G3 supports production of dendritic-like cells as well as myeloid cells and progenitors, while 3B5 transiently supports the production of only low numbers of myeloid cells. Gene expression profiling was performed using Affymetrix genechips to compare gene expression between 5G3 and 3B5 stroma and to identify genes which regulate this process. Initially, data mining identified gene expression essential for hematopoiesis in bone marrow including S1 Table(DOCX)Click here for additional data file.


H9N2 is the most prevalent low pathogenic avian influenza virus (LPAIV) in domestic poultry in the world. Two distinct H9N2 poultry lineages, G1-like (A/quail/Hong Kong/G1/97) and Y280-like (A/Duck/Hong Kong/Y280/1997) viruses, are usually associated with binding affinity for both α 2,3 and α 2,6 sialic acid receptors (avian and human receptors), raising concern whether these viruses possess pandemic potential. To explore the impact of mouse adaptation on the transmissibility of a Y280-like virus A/Chicken/Hubei/214/2017(H9N2) (abbreviated as WT), we performed serial lung-to-lung passages of the WT virus in mice. The mouse-adapted variant (MA) exhibited enhanced pathogenicity and advantaged transmissibility after passaging in mice. Sequence analysis of the complete genomes of the MA virus revealed a total of 16 amino acid substitutions. These mutations distributed across 7 segments including PB2, PB1, PA, NP, HA, NA and NS1 genes. Furthermore, we generated a panel of recombinant or mutant H9N2 viruses using reverse genetics technology and confirmed that the PB2 gene governing the increased pathogenicity and transmissibility. The combinations of 340 K and 588 V in PB2 were important in determining the altered features. Our findings elucidate the specific mutations in PB2 contribute to the phenotype differences and emphasize the importance of monitoring the identified amino acid substitutions due to their potential threat to human health. The first case of a human infection with H9N2 was reported in 1998, in China7. Seroprevalence studies have shown that the antibodies to H9N2 were higher in humans who had direct exposure to poultry, particularly in occupational poultry workers, suggesting that infections with H9N2 commonly occur9. H9N2 AIV can also replicate in mammals without prior adaption, and its ongoing mammalian adaption poses a significant public health risk12. Notably, H9N2 AIV is presumed to be the donor of the internal genes of several prevalent reassortant AIVs, such as H5N1, H7N9 and H5N6 viruses17. Airborne transmission contributes to the dissemination of H9N2 worldwide and has enabled H9N2 to transmit efficiently among poultry, but no human-to-human transmission of H9N2 has been observed. The airborne transmissibility of H9N2 in mammals remains unknown18.H9N2 avian influenza virus (AIV) was first detected from turkeys in 1966 in the United States of America and transmitted to a variety of mammalian species including swine, dogs, weasels, mink, bats and humans19. A highly pathogenic strain of H7N1 avian influenza virus became capable of airborne transmission in mammals after 10 serial passages20. An avian H5N1 virus acquired airborne transmission in guinea pigs after receiving genes from 2009/H1N1virus21. The HA and PB2 genes are important for cross-species transmissions of AIVs22. An H5N1 virus modified by site-directed mutagenesis and subsequent serial passage in ferrets became airborne transmissible23.Several experimental methods have been used to explore the transmissibility of AIVs in mammals. In general, the methods rely upon mammalian adaptation through serial passage, reassortment between an AIV and a human influenza virus, point mutations in genes, or a combination of the above25. Human receptor binding specificity, specifically leucine (L) at position 226 in HA receptor binding site, is critical for direct transmission of avian H9N2 viruses in ferrets10. Increased viral polymerase activity mediates adaptation of AIVs to a mammalian host26. The high thermostability of HA facilitates H5 AIV transmission via respiratory droplets in mammals27.The capacity of AIVs to transmit among mammals appears to require multiple viral features, such as human receptor binding, increased polymerase activity and high thermostability of HA30. Y439 (A/Duck/Hong Kong/Y439/1997), G9 (A/Chicken/Hong Kong/G9/1997), G1 (A/quail/Hong Kong/G1/97) and Y280 (A/Duck/Hong Kong/Y280/1997) are four different H9N2 poultry lineages. The G1 and Y280 poultry lineages are usually associated with both avian and human receptor binding affinity and could potentially transmit between mammals8. Mice have been widely applied to study mammalian adaptation of AIVs. Serial passage of AIVs in mammals can result in adaptive changes that confer enhanced pathogenicity and transmissibility in mammals33. Although the pathogenicity and transmissibility of H9N2 AIVs have been characterized previously35, the molecular features that account for H9N2 airborne transmissibility in mammals are not clear. In the current study, a Y280-like H9N2 virus transmitted among guinea pigs after mouse adaption. To explore which gene-specific mutations contribute to altered phenotype, we generated recombinant and mutant viruses using reverse genetics technology.The transmission of AIVs to mammals appears to acquire human receptor binding preference36. Briefly, all animal studies were conducted in strict accordance with the Guidelines of Animal Welfare of World Organization for Animal Health and the protocols approved by the Hubei Provincial Animal Care and Use Committee .The ethics statement was described in our previous workMA, WT-PB1MA, WT-PAMA, WT-NPMA, WT-HAMA, WT-NAMA, WT-NSMA) and the mutant viruses (WT-PB2340K and WT-PB2588V) as described previously37. The recombinant reassortant viruses each contained one gene from the MA virus and the mutant viruses each was a single amino acid substitution in the PB2 of WT. The viruses were propagated in 9-day-old specific pathogen free (SPF) embryonated eggs and stored at −80 °C.The wild type H9N2 virus used in this study was isolated from chickens in 2017, in China, and named A/chicken/Hubei/214/2017 (abbreviated as WT). A single amino acid substitution in PB2 was generated by using A Quick Change XL Site-Directed Mutagenesis Kit . The WT and MA were the parental viruses. We used the WT as the backbone to generated the recombinant reassortant viruses  was isolated from the homogenized lung tissue supernatants using 9-day-old SPF embryonated eggs for subsequent use in pathogenicity and transmissibility studies.The mouse-adapted H9N2 virus was derived from series of sequential lung-to-lung passages of the WT virus in mice as described previously36. In brief, viral RNA was extracted from allantoic fluid using TRIzol reagent (Invitrogen) and reverse transcribed into cDNAs using the primer Uni12 (5′-AGC RAA AGC AGG-3′) primers, an RT reagent kit and viral genes were amplified using a PCR kit  according to the manufacturer’s protocol. The PCR products of the eight segments of the viruses were amplified by PCR using specific virus primers as described by Hoffmann et al.38. The PCR products were purified and sequenced by Sangon Biotech Company. Amino acid substitutions between the WT and MA viruses were identified. All the sequence data were analyzed with the SeqMan program . All reference sequences used in this study were obtained from the National Center for Biotechnology Information (NCBI) GenBank database.The viral gene sequences were acquired as described in our previous work36. For the HA assay, sialic acid residues were enzymatically removed from cRBCs by incubating the cells with 50 mU of Vibrio cholerae neuraminidase  at 37 °C for 1 h, followed by resialylation using either α2-,6-N-sialyltransferase or α2-,3-N-sialyltransferase  at 37 °C for 4 h. The sample was then washed two times with phosphate-buffered saline (PBS), centrifuged at 1500 rpm for 5 min each time, adjusted to a final working concentration (1%) with PBS, and stored at 4 °C. For the HA assay, viruses were serially diluted 2-fold with 50 μL of PBS and mixed with 50 μL of a 1% RBC suspension in a 96-well plate. HA titers were determined after 1 h at 4 °C.The receptor-binding specificities of the WT and MA viruses were determined by HA assays with 1% chicken red blood cells (cRBCs) as described in our previous work39. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . The growth kinetics of the WT and MA viruses were determined by inoculating MDCK cells at a multiplicity of infection (MOI) of 0.001 50% tissue culture infectious dose (TCID50) per cell. One hour after inoculation (hpi), the cells were washed twice with PBS, and fresh medium supplemented with 1 μg/mL tosyl phenylalanyl chloromethyl ketone (TPCK) and trypsin  was added. The supernatants were sampled at 12, 24, 36, and 48 hpi. The virus titers were determined by calculating the lg TCID50/mL in MDCK cells. The TCID50 values were calculated according to the method of Reed and Muench.The virus growth curve experiment was performed as described in our previous work40. Groups of five six-week-old female BALB/c mice  were anesthetized with ether and intranasally inoculated with 50 μL of 106 EID50 solution of the test virus or PBS. The weight loss and mortality of mice in these groups were monitored daily for 14 days. Mice that lost >30% of their original body weight were humanely euthanized.Mouse experiments were performed as described in our previous work36. Hartley strain female guinea pigs weighing 300 to 350 g , confirmed to be seronegative for influenza viruses prior to the experiment, were used in these studies. In the transmission studies, groups of three guinea pigs were anesthetized with ether and intranasally inoculated with 300 μL of 106.0 EID50 solution of the test virus and housed in a cage placed in an isolator. The next day, three naive guinea pigs were individually paired and cohoused with an infected guinea pig for the direct contact transmission studies, and another naive guinea pig was housed in a wire frame cage adjacent to the infected guinea pig for the aerosol transmission studies. The distance between the cages of the infected and aerosol-contact guinea pigs was 5-cm. To monitor virus shedding, nasal washes were collected from all animals at 2, 4, 6, and 8 dpi and titrated.Guinea pig experiments were performed as described in our previous workStatistically significant differences were determined using one-way analysis of variance (ANOVA) with GraphPad Prism software . All assays were run in triplicate, and the data are representative of at least 3 separate experiments. The error bars indicate the standard deviation.50 was 104.5 EID50/mL. In contrast, the WT-inoculated mice experienced no substantial body weight loss and had nonlethal infections . No virus shedding was detected in the lungs of the WT-infected mice at 5 and 7 dpi , and resialylated cRBCs contained either human  or avian  receptors. The HA titers represent 3 separate experiments. The results showed that the WT and MA viruses bind to both avian and human receptors  exhibited increased virulence, and the body weight loss and mortality of the infected mice were comparable to those of the MA-infected mice. Whereas the other recombinant viruses WT-PB1MA, WT-PAMA, WT-NPMA, WT-HAMA, WT-NAMA, WT-NSMA caused no substantial body weight loss and nonlethal infections . The two variants also displayed increased pathogenicity than the recombinant viruses, with the exception of the WT-PB2MA virus  and 228-Gly(G) (H3 numbering) in HA have been reported to prefer both avian and human receptors45. The loss of glycosylation at residue 158 in the HA was also shown to be responsible for H5N1 AIV binding to human receptors46. In this study, there was no difference in receptor binding specificity between the WT and MA viruses with the in vivo results for the WT-HAMA virus that didn’t show difference from the WT, suggesting the HA was not involved in the altered phenotype.Influenza A virus can infect a variety of animal species. The receptor binding specificity of AIV is recognized as an important factor in interspecies transmission48. The major reason is their limited airborne transmissibility among humans. Previous studies found that ferrets and guinea pigs adaptation enabled AIVs to transmit in mammals33. In our previous study, mouse adaption could not enable the H5N6 to transmit in the guinea pig model32, but it enabled airborne transmission of the H9N2 in this study. We suppose the reason for its airborne transmissibility after mouse adaption might be correlate with its avian and human receptors binding affinity.H5N1, H7N9, H9N2 and H5N6 AIVs have been reported to occasionally break the species barrier to infect humans, but they have not been able to disseminate among humans50. Passaging H9N2 in swine increased its replication and transmissibility51. The PB1-577E increased pathogenicity of H9N2 in mice52. The HA1-227P, HA2-46E and NP-434K enabled H9N2 contact transmission in guinea pigs33. The loss of glycosylation at 166 in HA and PB2-627K were also shown to increase virulence of H9N2 in mice53. Previous studies found that the PB2 gene of H9N2 played an important role in mammals54, they had identified PB2-404L, PB2-235N PB2-147L and PB2-627K enhanced pathogenicity of H9N2 in mice56. The PB2-E627K substitution was consistently found to mediate mammalian adaptation and a known determinant of pathogenicity and host specificity of AIVs58. However, the previous identified amino acid changes in H9N2 were not observed in this study. The PB2-R340K, PB2-A588V, PA-K356R, PA-S343A, NP-V239M and NS1-T216P identified in this study have been previously implicated in increasing virulence of other subtypes of AIVs63. PA-R356K was considered as a unique signature of H7N9 viruses with bird-to-human transmissibility and was also found to enhance viral polymerase activity, replication and pathogenicity in mammals61. The PA-S343A mutation was found to increase the polymerase activity and virulence of a low-pathogenic H5N1 influenza virus62. The NP-V239M and NS1-T216P mutations were defined as signature amino acids of H7N9 viruses isolated from confirmed human cases in Shenzhen of China63. PB2-R340K and PB2-A588V were previously found to increase viral polymerase activities, replication and pathogenicity of H10N8 and H7N959. In the present study, we also found the substitutions PB2-R340K and PB2-A588V in combination enabled H9N2 airborne transmission among guinea pigs. These findings further highlight the need for persistent surveillance efforts to detect the emergence of H9N2 isolates with the identified amino acid substitutions in PB2.Several previous works have studied H9N2 adaptation to chickens or mammals. The HA-363K and PA-672L enabled H9N2 airborne transmission among chickens