Traf3-deficient (M-Traf3-/-) mice exhibited MDSC hyperexpansion during chronic inflammation. Interestingly, MDSC hyperexpansion in M-Traf3-/- mice led to accelerated growth and metastasis of transplanted tumors associated with an altered phenotype of T cells and NK cells. Using mixed bone marrow chimeras, we demonstrated that TRAF3 inhibited MDSC expansion via both cell-intrinsic and cell-extrinsic mechanisms. Furthermore, we elucidated a GM-CSF-STAT3-TRAF3-PTP1B signaling axis in MDSCs and a novel TLR4-TRAF3-CCL22-CCR4-G-CSF axis acting in inflammatory macrophages and monocytes that coordinately control MDSC expansion during chronic inflammation. Taken together, our findings provide novel insights into the complex regulatory mechanisms of MDSC expansion and open up unique perspectives for the design of new therapeutic strategies that aim to target MDSCs in cancer patients.Myeloid-derived suppressor cells (MDSCs) are aberrantly expanded in cancer patients and under other pathological conditions. These cells orchestrate the immunosuppressive and inflammatory network to facilitate cancer metastasis and mediate patient resistance to therapies, and thus are recognized as a prime therapeutic target of human cancers. Here we report the identification of the adaptor protein TRAF3 as a novel immune checkpoint that critically restrains MDSC expansion. We found that myeloid cell-specific  The adaptor protein TRAF3 is an immune checkpoint of MDSC expansion.Traf3-/- mice with chronic inflammation exhibit accelerated growth and metastasis of transplanted lymphomas.Young adult M-A novel TRAF3-CCL22-CCR4-G-CSF axis acts in inflammatory macrophages and monocytes to restrain MDSC expansion during chronic inflammation.TRAF3 specifically inhibits the GM-CSF-STAT3 signaling axis in MDSCs.ex vivo expanded tumor-specific T cells or chimeric antigen receptor T cells (CAR-T), and cancer vaccines, are recognized as breakthroughs in cancer treatments . We reported that specific ablation of TRAF3 in myeloid cells does not affect the maturation or homeostasis of macrophages and neutrophils in young adult naïve mice , and cytokine receptors, among others –13. TRAFïve mice . Interesid cells , 28. ThuIn the present study, we identify TRAF3 as a critical immune checkpoint in myeloid cells that restrains MDSC expansion during chronic inflammation. Our findings provide novel insights into the complex cellular and molecular mechanisms underpinning the regulation of MDSC expansion. This knowledge will open up unique avenues for the design of new strategies that aim to target or exploit MDSCs in different clinical settings.Traf3flox/flox and Traf3flox/floxLysM+/Cre (M-Traf3-/-) mice on the C57BL/6J genetic background were generated as previously described (Traf3 gene in MDSCs and macrophages was detected by genomic PCR using primers U7 and BT6 as previously described (Traf3flox/flox mice with Traf3flox/floxLysM+/Cre mice. Traf3flox/flox littermates (LMC) were used as controls for all experiments. C57.SJL (CD45.1) mice were purchased from Jackson Laboratory . Traf3flox/flox mice (CD45.2) were bred with C57.SJL mice to generate wild type (WT) donor mice (CD45.1/CD45.2) for mixed bone marrow (BM) chimera experiments. All mice were kept in specific pathogen-free conditions in the Animal Facility at Rutgers University and were used in accordance with NIH guidelines under an animal protocol (PROTO999900371) approved by the Animal Care and Use Committee of Rutgers University. Mice were monitored daily for signs of illness or discomfort, including weight loss, enlarged lymph nodes or abdomen, labored breathing, hunched posture and paralysis. Equal numbers of male and female mice were used in this study.escribed . Mouse eescribed , (Lys-Coescribed . Deletioescribed . All expTraf3-/- mouse (mouse ID: 291-6) (Traf3-/- mouse (mouse ID: 291-7) (5 cells/well in mouse B cell media (Mouse B-cell lymphoma cell lines 291-6.1.1-8 (291-6) and 291-7.1.2 Asc C1 (291-7) were generated and cultured as described previously , 29. Bri: 291-6)  and from: 291-7) , and thell media  containiM. tuberculosis H37 Ra), and Matrigel Matrix were from BD Biosciences . Mouse Cytokine Array Kit Panel A, ELISA Ab pairs for mouse G-CSF, CXCL-13, S100A8/A9, and CCL22, as well as neutralizing Abs for mouse G-CSF and CXCL-13 were obtained from R&D . Anti-mouse HMGB1 ELISA kit was purchased from Biomatik . Rabbit Abs for HA tag and V5 tag were from Cell Signaling Technology . Mouse monoclonal Abs to SBP tag were purchased from EMD Millipore Corp . HRP-labeled secondary Abs were from Jackson ImmunoResearch Laboratories, Inc. .Fluorochrome-labeled antibodies (Abs) against mouse CD11b, Gr1, Ly6C, Ly6G, CD45R (B220), CD3, CD45.1, CD45.2, F4/80, CD11c, MHC class II, CD4, CD8, CD115, CD169, NK1.1, CD49b, CD62L, CD69, CX3CR1, CCR2, CCR4, PD-L1, CD38, KLRG1, NKp46, and Sca1 were purchased from BioLegend  and eBioscience . Anti-BrdU, anti-p-STAT3 (pY705), anti-p-STAT5 (pY694), anti-p-ERK1/2 (pT202/pY204), heat-killed Bacillus Calmette-Guérin , LPS, Stattic, Baricitinib, anti-FLAG M2 Affinity Gel, rabbit Abs for FLAG tag, FLAG peptides, and Microcon-10kDa spin columns were purchased from Sigma . Mouse MDSC purification kits and anti-phycoerythrin (PE) magnetic beads were from Miltenyi Biotec Inc. . Tissue culture supplements including stock solutions of sodium pyruvate, L-glutamine, non-essential amino acids, and HEPES (pH 7.55) were from Invitrogen . Lymphocyte Separation Medium was purchased from Lonza . GM-CSF, G-CSF, M-CSF, IL-4, and IL-6 were from PeproTech . Hyaluronidase, Collagenase type 3, and type 4 were from Worthington Biochemical Corp . DNase I, Phosphatase Inhibitor Mini Tablets, and Streptavidin-Sepharose beads were purchased from Pierce . EDTA-free Protease Inhibitor Cocktail Tablets were obtained from Roche Diagnostics Corp . SureBlue™ TMB Microwell Peroxidase Substrate Kit was purchased from KPL Inc. . DNA plasmids of STAT3, PTP1B, and JAK2 were purchased from Addgene . DNA oligonucleotide primers were obtained from Integrated DNA Technologies .’s protocols. Cells (1x106) were blocked with rat serum and FcR blocking Ab (2.4G2), and then incubated with various Abs conjugated to different fluorochromes for multiple color fluorescence surface staining. Intracellular markers were stained after fixation and permeabilization of cells using an Intracellular or Phospho-Flow Staining kit (BD Biosciences). BrdU labeling on cellular DNA was measured using the BrdU Flow Kit (BD Biosciences). FACS data were acquired on a Northern Lights spectral flow cytometer  or a FACSCalibur . The results were analyzed using the FlowJo software  , BM, peripheral blood cells (PBL), and tumor. Immunofluorescence staining and FACS analyses were performed as previously described , 16, 21.MDSCs were purified from splenocytes by first depleting B cells and T cells using anti-B220 and anti-CD90.2 beads (Miltenyi Biotec), and then collecting Gr1+ MDSCs from the B220-CD90.2- fraction using anti-Gr1-PE followed by anti-PE beads (Miltenyi Biotec). The purity of prepared MDSC populations was verified to be greater than 95% of B220-CD3-CD11b+Gr1+ as determined by FACS.Traf3-/- mice with chronic inflammation or B-cell lymphomas. Wright-Giemsa staining of cytospin slides was carried out using a Diff-Quick staining kit  following the manufacturer’s procedures. Bright field micrographs of stained slides were taken using a microscope .Morphological characterization of MDSC subtypes was performed on cytospin preparations of splenic MDSCs purified from aging M-CD8 T cells were purified from naïve LMC mice using a CD8+ T Cell Purification Kit (Miltenyi Biotec) following the manufacturer’s instructions. Purified CD8 T cells were labeled with 0.5 µM of a cell proliferation dye eFluor 670 (eBioscience) at 37°C for 10 min according to the manufacturer’s protocol . Labeleds.c. injected with heat-killed BCG (100 μg/100 μl/mouse) at 1-week intervals. The first two injections were administrated as a mixture of BCG/PBS and incomplete Freud adjuvant (IFA) at a 1:1 ratio emulsified by vortexing for 15 minutes. The third BCG injection was in PBS only. Unless stated otherwise, cells were collected on day 2 after each injection.Chronic inflammation was induced in young adult (2-4-month-old) mice by repeated injections of heat-killed BCG as previously described . BrieflyTraf3-/- mice were injected s.c. with 5x106 cells of 291-6 and 291-7 B lymphoma cell lines (mixed at a 1:1 ratio) for tumor transplantation. For mice that received 3 injections of heat-killed BCG, tumor transplantation was performed on day 2 after the third BCG injection. For end-point analysis, DLBCL cells were injected in 100 µl PBS. For analysis at day 7 post transplantation, DLBCL cells were injected in 300 µl of 50% Matrigel in PBS. Transplanted mice were monitored daily for tumor growth and signs of failing health (Young adult (2-4-month-old) LMC and M-g health . At the Dissected lymphomas or subcutaneous Matrigel plugs were cut into small (< 3 mm) pieces and incubated in 1-5 ml of enzyme dissociation solution  at 37°C with shaking for 40 min, vortexing every 5 minutes during the incubation. Dissociated tumor cells were filtered through a 70 μm cell strainer to remove residual tissues and then washed twice with PBS before immunofluorescence staining for FACS analysis.Traf3-/- (CD45.2) mice (8-12-wk-old) and mixed at a 1:1 ratio. Each C57.SJL recipient mouse was injected i.v. with 1×107 of mixed BM cells in 100 µl PBS. At 8 weeks after reconstitution, the chimerism in PBL was examined by FACS. A cohort of mixed BM chimeras were subjected to repeated injections of heat-killed BCG and analyzed on day 2 after the 3rd BCG injection. Another cohort of naïve BM chimeras were directly sacrificed and analyzed at the same time with the BCG-treated chimeras. The ratios of M-Traf3-/- (CD45.2) versus WT (CD45.1/CD45.2) CD11b+Gr1+ cells, B cells, and T cells in the spleen, PBL, and BM were analyzed by FACS and compared between naïve and BCG-treated BM chimeras.C57.SJL (CD45.1) recipient mice (8-12-wk-old) were subjected to whole-body X-ray radiation with a single dose of 1,100 rads at 24 h before BM transfer. The radiation was delivered at the rate of 0.28 Gy/min using an aluminum filter at 100 kVp  . Total BTraf3-/- mice at day 1 or day 2 after the 2nd and 3rd BCG injections. Serum levels of 40 cytokines and chemokines were measured using a Mouse Cytokine Array Panel A kit  following the manufacturer’s instructions  twice at a 1-week interval. Mice were i.p. injected with BrdU (3 mg in 0.3 ml of PBS) twice at 19 h and 16 h before sacrifice. Mice were euthanized on day 2 after the 2nd BCG injection. BrdU+ cells were detected by intracellular staining with an APC-conjugated anti-BrdU antibody using the BrdU Flow Kit (BD Pharmingen™) following the manufacturer’s protocol.Gender-matched, young adult (8-12-wk-old) mice were Traf3-/- mice (8-12-wk-old) were injected i.p. with anti-G-CSF neutralizing antibody , anti-CXCL-13 neutralizing Ab , or Ig isotype control, beginning at 1 day after the 1st injection of heat-killed BCG and continuing thrice weekly for 2 weeks. On day 2 after the 3rd BCG injection, spleens and BM cells were harvested from the treated mice for FACS analysis.LMC and M-Traf3-/- mice (2-4-month-old) and cultured in the BM cell medium , in the absence or presence of 10 ng/ml GM-CSF and 100 ng/ml LPS for 24 h. Production of G-CSF was detected by intracellular staining with Alexa Fluor 647-anti-G-CSF and expression of chemokine receptors  were determined by immunofluorescence surface staining, followed by FACS analysis.BM cells were harvested from the femurs of naïve LMC and M-Traf3-/- mice (8-12-wk-old). BMDMs were prepared as previously described . Each reaction also included the primers and probe (VIC-labeled) specific for mouse Actb, which served as an endogenous control. Reactions were performed on a QuantStudio™ 3 Real-Time PCR System  (BM cells were harvested from the femurs and tibias of LMC and M-escribed  and cultsystems) . Real-tisystems) . Relativsystems) .Traf3-/- mice (2-4-month-old) and labeled with 0.5 µM of a cell proliferation dye eFluor 670 (eBioscience) at 37°C for 10 min according to the manufacturer’s protocol  as previously described  for meas-/- mice (8-12-wk-old). B cells, T cells, and RBCs were depleted from BM cells using anti-B220, anti-CD19, anti-CD90.2, and anti-Ter119 magnetic beads (Miltenyi Biotec), and then Gr1+ BM cells were purified from the B220-CD19-CD90.2-Ter119- fraction using anti-Gr1-PE followed by anti-PE beads (Miltenyi Biotec). The purity of the resultant Gr1+ populations was verified to be greater than 92% of B220-CD19-CD3-Ter119-CD11b+Gr1+ as determined by FACS. Purified CD11b+Gr1+ cells were stimulated with 20 ng/ml GM-CSF at 37°C and total cellular protein lysates were prepared at different time points  as previously described (BM cells were harvested from the femurs and tibias of LMC and M-Traf3escribed  for meas7 cells/condition) were harvested and total cellular proteins were lysed in 1% CHAPS Lysis Buffer   , sonicatibitors) . Immunopibitors) . For SA ibitors) . For FLAibitors) . All theTraf3-/- mice, statistical significance was determined with the unpaired t test for two-tailed data. For comparison of three or more groups of data, a one-way analysis of variance (ANOVA) was used to determine the statistical significance. P values less than 0.05 are considered significant (*), P values less than 0.01 are considered very significant (**), and P values less than 0.001 are considered highly significant (***).Statistical analyses were performed using the Prism software . Survival curves were generated using the Kaplan-Meier method and were compared using a log-rank (Mantel-Cox) test to determine whether differences are significant. For direct comparison of cell subset frequencies or numbers, cytokine or chemokine levels, or expression levels of specific proteins between LMC and M-Traf3-/- mice (15-22-month-old) exhibit a spontaneous disease phenotype, characterized by chronic inflammation and tumor development that affect multiple organs  with spontaneous diseases exhibited potent immunosuppressive activity on syngeneic CD8 T cell proliferation in co-culture assays  mice  spontaneously developed in two aging M-Traf3-/- mice. Naïve young adult (2-4-month-old) LMC and M-Traf3-/- mice were able to reject the transplanted DLBCL cells , and tumor microenvironment (TME)  of MDSCs harvested from the same anatomical location , a known target of MDSCs  pivotal for MDSC expansion  labelling  and splenocytes for G-CSF-producing cells. We detected significantly increased percentages of G-CSF-producing CD11b+ myeloid cells in PBMCs and splenocytes of M-Traf3-/- mice after BCG injections  is a direct target gene of NF-κB  of gated CD11b+G-CSF+ monocytes and macrophages . We next tested an alternative possibility that TRAF3 inhibits the chemotaxis and trafficking of G-CSF-producing monocytes from the BM to blood circulation by down-regulating the expression of key chemokine receptors important for monocyte migration. For this purpose, we examined the expression levels of the following chemokine receptors: CCR2 - the primary receptor for monocyte emigration from the BM  and CXCLs (CXCL-1~2 and 9~13). Among these, CCL-2 and CCL-12 are two ligands for CCR2 and known as the principal chemotactic factors for the emigration of monocytes from the BM (Traf3-/- mice except for the aforementioned CXCL-13 and IP-10 (CXCL-10)  on a per cell basis. We detected drastically augmented transcript levels of Ccl22 in M-Traf3-/- BMDMs after treatment with LPS  as well as Ccl3 (encoding a ligand for CCR1 and CCR5) were down-regulated in M-Traf3-/- BMDMs   and endogenous TRAF3 was similarly co-immunoprecipitated with FLAG-tagged STAT3 (STAT3-FLAG) in transfected 293T cells –76. Intein vitro and induction of MDSCs in vivo are also being developed for conditions where immune suppression is desired, such as organ or stem cell transplantation and autoimmune disorders  (via the cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) to dampen the functional responses of T cells and NK cells  –41. KLRGd cADPR) , 43. UpoNK cells , 43. BotNK cells , 43, 93.NK cells , 93. ThuIn summary, our findings provide novel mechanistic insights into how TRAF3 functions as a critical immune checkpoint in myeloid cells to restrain MDSC expansion, thereby preventing overzealous inflammation and indirectly promoting anti-tumor immunity. This knowledge will inform future translational and clinical efforts aimed at developing novel immunotherapies to manipulate MDSCs. Pharmacological stabilization of TRAF3 or reconstitution of TRAF3 expression in myeloid cells by gene therapy could be novel therapeutic approaches to prevent or reduce MDSC expansion. Discovery of the GM-CSF-STAT3-TRAF3-PTP1B signaling axis in MDSCs and the TLR4-TRAF3-CCL22-CCR4-G-CSF axis in inflammatory macrophages and monocytes provides a framework for targeting these crucial pathways by developing new drugs or by repurposing available drugs specific for STAT3, PTP1B, CCR4, or CCL22. Furthermore, the novel link between MDSCs and up-regulated expression of CD38 and KLRG1 on T cells and NK cells identified in this study suggests a wider scope of applications for CD38- and KLRG1-blocking drugs in immunotherapies of cancers and inflammatory diseases. These new therapeutic strategies in combination with current therapies would help to improve patient outcome.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Rutgers University IACUC.in vivo BrdU labeling experiments, the cytokine array assay, and initial GM-CSF signaling experiments. JJ helped to perform the lymphoma transplantation, in vivo neutralization, and cultured BMDM experiments. SZ, AL, JJ, and PX analyzed the data and performed the statistical analyses. PX, SZ, and AL wrote the manuscript. All authors contributed to the article and approved the submitted version.PX, SZ, AL, and SO-R designed the experiments of this study. DS’A and PX designed the mixed BM chimera experiments. KL, DS’A, and LC provided guidance on lymphoma transplantation and T cell analyses. HY supervised the cytokine analyses and macrophage experiments. SZ performed most experiments of this study. AL established the BCG-induced chronic inflammation model, the DLBCL cell lines for transplantation, and the co-culture model of MDSCs and CD8 T cells, and also performed the


H domainantibody candidates, 87 (GPNMB) and 1B2 (VCAM-1), were isolated fromour in-house proprietary phage-displayed human VH antibodydomain libraries. The avidity was increased after conversion to VH-Fc.Domain-based bispecific T-cell engagers (DbTE) based on these twoantibodies combined with the anti-CD3ε OKT3 antibody exhibitedpotent killing against GPNMB and VCAM-1-positive cancer cells, respectively.Hence, these two domain antibodies are promising therapeutic candidatesfor cancers expressing GPNMB or VCAM-1.The elevated expression of GPNMB and VCAM-1 has beenobserved inmany cancers including breast cancer, melanoma, and prostate cancers.Such overexpression of GPNMB and VCAM-1 has been associated with poorprognosis and increased cancer metastasis. Thus, GPNMB and VCAM-1are potential targets for immunotherapies across multiple cancers.In this study, two high-affinity specific human V Inthe tumor microenvironment, GPNMB functionally promotes tumor progressionand invasion, cell adhesion and differentiation,9 endothelial cell recruitment, and metastasis through multiplemechanisms including interaction with syndecan-4 to block the proliferationand activation of T cells,10 interactionwith integrin by the extracellular domain of GPNMB to induce trans-endothelialmigration,11 interaction with the C-terminusof EGFR  to activate EGFR pathway,which further activate STAT3  signaling and promote cancer metastasis,13 and activation of MMP-3 , which is involvedwith cancer cell migration, invasion, and inflammation.14Glycoprotein nonmetastaticmelanoma protein B (GPNMB), also knownas osteoactivin (OA), dendritic cell-heparin integrin ligand (DC-HIL),or hematopoietic growth factor inducible neurokinin-1 type (HGFIN),is a type 1 transmembrane protein. It is highly expressed in manytumors including melanoma,15 As an immunoglobulin superfamilymember, it plays an important role in the immune surveillance of manydiseases. Recently, studies have shown that the elevated expressionof VCAM-1 was involved in tumor cell adhesion on endothelium cellsrelated to metastasis.17 Like GPNMB, this overexpressionwas associated with poor prognosis in many cancers including breastcancer,18 melanoma,19 colorectal cancer,20 ovariancancer,21 and prostate cancer.22 Overall, the high expression of GPNMB and VCAM-1in cancers and their functional association with cancer cell growthand metastasis make them important targets for development of antitumortherapeutics.Vascular cell adhesion molecular 1 (VCAM-1),also named CD106,shows similar behavior in cancerous tissues. VCAM-1 is expressed onthe luminal and lateral side of endothelial cells under inflammatorystimulation.23 Inrecent decades, the field has seen a growing interest in antibodydomains used as diagnostics and therapeutics due to their small size,low immunogenicity, and efficient infiltration into solid cancer tissues.These antibody domains/fragments enable treatments to target new epitopesthat are not accessible to full-size (IgG) antibodies or large antibodyconstructs. Recent studies show that variable domain antibodies caneven pass through the blood–brain barrier.24 Thus, the use of variable domain antibodies may be a powerfultool in the development of cancer immunotherapies.Antibody-based immunotherapies have become increasinglyattractiveoptions in cancer treatment due to their high affinity for targetproteins and relatively low toxicity compared to other therapies.More than 100 mAbs have been approved by the FDA (Food and Drug Administration)for immunotherapies of many different diseases like autoimmune andinflammatory diseases and cancers.H domain antibodiesthat target GPNMB and VCAM-1. These binders werecharacterized for their affinity and specificity. The domain-basedbispecific T cell engagers (DbTE) constructed by these two bindersshowed potent killing effects on GPNMB and VCAM-1-expressing cancercells, respectively. This is the first report of GPNMB or VCAM-1-specifichuman VH domain antibodies as candidates for cancer immunotherapy.In our currentstudy, we identified two potent human V22.1H librarywas used against human IgG1 Fc fused recombinant GPNMB and VCAM-1separately. The panning was performed as previously described.25 The VH phage library was first incubatedwith 50 μL Protein G magnetic beads (Thermo Fisher Scientific)to remove nonspecific binders. The phage was then blocked with 5%milk and incubated with 5 μg of GPNMB-Fc or VCAM-1-Fc protein.The libraries were then incubated with Protein G magnetic beads toseparate the antigen-bound phages. The beads were washed with PBSTfollowed by PBS before directly infection of log phase TG1 E. coli cells for phages expression and amplification. Threemore rounds of subsequent panning were performed, which reduced theGPNMB-Fc or VCAM-1-Fc concentration one fold each round. After fourrounds of panning, 192 individual clones were screened for bindingGPNMB-His or VCAM-1-His protein by ELISA.Human GPNMB-Fc, VCAM-1-Fc, and VCAM-1-His recombinant proteinswere purchased from R&D systems. Human GPNMB-His was purchasedfrom Acro Biosystems. To perform panning antibody candidates againstGPNMB and VCAM-1, a large phage-displayed human V2.2H antibodycandidates to VH-Fc format, the VH domain wasamplified and cloned into the pcDNA-IgG1 Fc vector. For the constructionof DbTE, humanized OKT326 was insertedat the C terminal of VH followed by the IgG1 Fc with LALAPGmutation. The expression and purification were performed as previouslydescribed.25 Briefly, the VH-Fc and DbTE were transiently transfected and expressed by the Expi293expression system and purified by protein A resin (Thermo Fisher Scientific).The VH binder was expressed in the E. coli TopF′ expression system and purified on Ni-NTA columns .To convert V2.325 Briefly, DPBS was used toestablish a baseline for 30 s, and streptavidin biosensors were coatedwith 16.7 μg/mL recombinant GPNMB-Biotin or VCAM-1-Biotin for2 min. Different doses of VH and VH-Fc wereused for association and monitored for 2 min to measure the affinityand avidity. Antibody dissociation was monitored in DPBS for 4 min.Antigen-coated biosensors in PBS served as a reference control.Theaffinity and avidity of the anti-hGPNMB and anti-hVCAM-1 antibodieswere detected by biolayer interferometry BLItz (ForteBio) as describedpreviously.2.4Expi293 cells (ThermoFisher Scientific) were maintained in an Expi293 expression mediumsupplemented with 0.4% penicillin–streptomycin (P/S). 293Tcells and SK-MEL-28 cells were purchased from ATCC and maintainedin DMEM or EMEM supplemented with 10% FBS and 1% P/S. HuT-78 cellspurchased from ATCC were maintained in IMDM supplemented with 20%FBS and 1% P/S. 293T-GPNMB/VCAM-1 cell lines were obtained by transfectionwith the plasmid containing GPNMB gene or VCAM-1 gene linked withthe Zeocin resistant. The stable cell line of 293T-GPNMB/VCAM-1 wasselected by using a high concentration of Zeocin antibiotic (500 μg/mL)for 7 days, and stable cells were maintained with 100 μg/mLof Zeocin.2.527 T cells were isolated from healthy donor’s PBMCs (Zen-Bio)by using the human Pan T cell isolation kit (Miltenyi Biotec) andactivated by CD3/CD28 T cell activator Dyna beads  at 1:1 cell/bead ratio for 48 h. The activated T cellswere used for the cytotoxicity assay of the DbTE antibody.T-cell isolationwas performed as previously described.2.65 cells/test were stained with anti-hGPNMB-PEor anti-hVCAM-1-PE antibody for 30 min at 4 °C. To verify the cell surface binding of theisolated antibody, cells were incubated with 50 nM VH-Fc87, VH-Fc 1B2, 1 μM VH 87, or VH 1B2 for 30 min at 4 °C, respectively. Cells were then stainedwith a secondary antibody, goat antihuman IgG-PE (Sigma-Aldrich) 1:250for VH-Fc, or 2 μL of anti-Flag-APC (Miltenyi Biotec)for VH. Irrelevant VH-Fc and VH wereused as isotype controls.The cell surface expressionlevel of GPNMB and VCAM-1 protein was detected by a commercial antibody.For this, 2 × 102.7Total proteins wereextracted from 293T, Sk-mel-28, and HuT-78 cells by RIPA buffer containing1× protease inhibitors (Thermo Fisher Scienctific). 20 μgof total proteins of each sample was loaded into a 4 to 12% Bis-Trismini protein gel (Thermo Fisher Scienctific). Proteins were then transferredto a 0.2 μm PVDF membrane (BioRad). The membrane was blockedin 5% no-fat milk for 1 h at room temperature, followed by incubationwith rabbit antihuman GPNMB antibody at 1:1000 dilution , mouse antihuman VCAM-1 antibody at 2 μg/mL (R&Dsystems), or β-Actin antibody at 1:1000 at 4 °C overnight. After three washes with TBST, the membraneswere incubated with goat antirabbit HRP secondary antibody at 1:3000dilution or goat antimouse HRP secondary antibody at 1:5000 dilution(Thermo Fisher Scienctific) for 1 h at room temperature. The membraneswere visualized by using ECL Western blot substrate (Thermo FisherScienctific) and imaged by a ChemiDoc XRS+ imaging system (Bio-Rad).2.84 cells/well) and activated Tcells were seeded in a 96-well plate at an E/T ratio 10:1, mixed withserially diluted DbTE antibodies in a growth medium, and incubatedfor 24 h at 37 °C in 5% CO2 humidified atmosphere.The final volume was 100 μL/well. The cell supernatant was diluted20-fold and incubated for 50 min for LDH assay setup. The calculationof relative % cytotoxicity is as follows: relative % cytotoxicity= 100 × /(target and effector maximum LDH release control –Target and effector cell only).The cell cytotoxicityof anti-GPNMB and anti-VCAM-1 DbTE was measured by LDH-Glo cytotoxicityassay kit (Promega) following the manufacturer’s instructions.Target cells values for binding to rhGPNMB or rhVCAM-1 were 13.4 nM and 2.8 nM,respectively, as determined by BLItz values of VH-Fc 87 and VH-Fc 1B2 as measuredby BLItz were 9.3 nM and 1.9 nM, respectively  or VCAM-1 (293T-VCAM-1), and cancer cell linessk-mel-28 (human melanoma cell line) and HuT-78 (human cutaneous T-lymphocytecancer cell line), which intrinsically express GPNMB or VCAM-1, respectively.The cells were tagged with a commercial antihuman GPNMB antibody andantihuman VCAM-1 antibody. Among these cell lines, 293T-GPNMB cells(MFI of isotype versus positive GPNMB Ab is 35.4 vs 1304) and sk-mel-28cells (MFI of isotype vs positive GPNMB Ab is 120 vs 479) moderatelyexpressed GPNMB. 293-VCAM-1 cells (MFI of isotype vs positive VCAM-1Ab is 31.8 vs 114) and HuT-78 cells (MFI of isotype vs positive Abis 21.8 vs 1852) showed a high expression level of VCAM-1. There wasno obvious expression of GPNMB and VCAM-1 on 293T cells (MFI of isotypevs GPNMB/VCAM-1 positive Ab is 60.3 vs 60.2/75.6) as observed by flowcytometry testing 1C,D. The3T cells1C. Similed above 1D. MoreoH-Fc1B2 1F.Taken together, the specific binding to membrane-bound GPNMB andVCAM-1 by each binder warrants further development as DbTEs for immunotherapyagainst GNNMB and VCAM-1 overexpressing cancers.3.2H 87 or VH 1B2 values of DbTE 87 and DbTE 1B2 as measured by BLItzwere 2.6 nM and 0.1 nM, respectively . To constructthese DbTEs and extend its half-life, humanized anti-CD3 antibodyOKT3 single chain variable fragment (scFv) fused with IgG1 Fc wasinserted in the C-terminal of Vr VH 1B22A. The Eectively 1A,B. Theectively 1. The spectively 2B. Next,ectively 2C. Nonspectively 2D. It shectively 2D and 1Aectively 2E,F. HowIn summary, these data support the utility of targetingGPNMB orVCAM-1 expressing tumor cells by these two variable domain antibodies.430 and shown tobe well-tolerated in melanoma, osteosarcoma, or triple-negative breastcancer patients, but antitumor activity is still limited. In solidcancer therapeutic development, the full length antibody still facingthe limitations for tumor penetration. The antibody fragments especiallyvariable domain antibodies showed advances since the two fragmentantibody, blinatumomab31 and caplacizumab,32 were approved.GPNMB and VCAM-1 are bothpotentially powerful targets in the treatmentof several types of cancer because of their high expression and functionin metastasis. High expression of both GPNMB and VCAM-1 has been associatedwith poor prognosis in patients, indicating a need for immunotherapiestargeting these proteins on cancer cells. Currently, only one ADCtargeting GPNMB named CDX-011 has been conducted in phase II or phase2b clinical trialH domain antibodiesthat target human GPNMB and VCAM-1 separately.Both antibodies showed high KD affinitiesin the nanomolar range. The avidities of these two antibodies werefurther enhanced after converting to VH-Fc (In this study, we selectedand characterized two fully human Vto VH-Fc 1. Detectto VH-Fc 2. The mo34 With further development, these antibodies may be promising therapycandidates for such aging-related diseases. Taken together, the describedanti-GPNMB antibody and anti-VCAM-1 antibody show significant potentialas variable domain antibody-based immunotherapy candidates for diseasesassociated with the elevated expression of GPNMB and VCAM-1.Moreover, recent studies have shown that GPNMBand VCAM-1 are associatedwith age-related diseases such as Alzheimer’s disease, Parkinson’sdisease, and age-related neurodegeneration.


Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways, including Wnt, Notch, and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling, resulting in the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-catenin activation induced by E. chaffeensis, rTRP120, and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM, and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors, Yap, and transcriptional enhanced associate domain (TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, the TRP120-Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1  was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis. Ehrlichia chaffeensis is a Gram-negative, obligatory intracellular bacterium and the etiologic agent of the life-threatening tick-borne disease, human monocytic ehrlichiosis (HME) (E. chaffeensis 120 kDa tandem repeat protein (TRP120) has emerged as a model moonlighting effector that functions as a nucleomodulin, ubiquitin ligase, and ligand mimic to reprogram the mononuclear phagocyte and escape host innate immune defenses (short linear motifs (SLiMs) within the TRP domain (is (HME) , 2. It pis (HME) . In the defenses -5. TRPP domain -10. LiP domain .E. chaffeensis activates canonical Wnt signaling by direct interaction between a TRP120-Wnt-SLiM ligand mimic and the host cell Wnt Frizzled 5 (Fzd5) receptor  effector to stabilize mitochondria by regulating mitochondrial matrix protein manganese superoxide dismutase (MnSOD) to induce antioxidative protection and inhibit apoptosis  levels and mitochondrial stability , which is regulated by the Hippo pathway ] upregulation, thereby promoting glycolysis and cell growth and apoptosis inhibition , Hepatitis C virus (HCV), Human papillomavirus (HPV), Epstein–Barr virus (EBV), and Kaposi Sarcoma-associated herpesvirus (KSHV) . Howevernd CYR61 , 38. HPVnd CYR61 .Ehrlichia Wnt SLiM ligand mimic that activates Wnt signaling. Therefore, since Hippo signaling is initiated through Wnt Fzd receptors, we considered that Hippo signaling may be regulated through the same ligand–receptor complex during infection . Moreover, significant nuclear accumulation of active Yap was observed over 48 hpi compared to uninfected controls based on intensity graphs  compared to the uninfected control (E. chaffeensis in Hippo deactivation (Yap activation), we examined Hippo pathway gene transcription using a human Hippo signaling PCR array  of Hippo genes being upregulated, including major Hippo and Wnt components YAP, TAZ, TEAD1, TEAD2, TEAD3, TEAD4, and DVL2 compared to controls , and 16% were downregulated , and Yap activation was examined using confocal microscopy . Active egulated . In compegulated . Though E. chaffeensis pathobiology, including posttranslational modification motifs, DNA-binding motifs, and ubiquitin ligase catalytic motifs .TRP120 contains a tandem repeat domain (TRD), with four tandem repeats, flanked by N- and C-terminal domains. Various TRP120 SLiMs have been reported within the TRD and C-terminus that are relevant to c motifs . We prevpathways , 9, 10. pathways . Howeverpathways , we idenE. chaffeensis and Wnt5a, confirming that the 6 aa Wnt SLiM was completely responsible for Wnt signaling activation and the amino acids flanking the 6 aa Wnt SLiM were not significant.To investigate Wnt SLiM activation of Yap, THP-1 cells were treated for 6 h with two peptides that contained the following sequences: TRP120-Wnt-SLiM (6 aa) and TRP120-TR-Wnt5a (19 aa); and two control peptides that did not contain Wnt SLiM: TRP120-Wnt-SLiM-mut  and TRP120-TR (−)   and Yap Further, we treated THP-1 cells with a single amino acid TRP120-Wnt-SLiM histidine deletion mutant (QDVAS) and observed no significant activation of Yap or β-catenin, indicating that the 6 amino acid TRP120-Wnt-SLiM containing histidine is essential for activation .YAP, WNT1, DVL2, TEAD1, and TEAD4, consistent with E. chaffeensis, TRP120-, and WNT5a (10 ng/mL). Moreover, all Hippo genes were upregulated in response to a 100-fold higher TRP120-Wnt-SLiM concentration .To investigate if TRP120-Wnt-SLiM regulates Hippo target genes, THP-1 cells were treated with TRP120-Wnt-SLiM , and significant Hippo gene activation was detected in a concentration-dependent manner . TRP120-E. chaffeensis survival depends on Hippo transcriptional components, we used RNAi to individually silence genes for YAP, TEAD1, TEAD3, and TEAD4  (To further elucidate the role of TRP120-Wnt-SLiM during control) .E. chaffeensis TRP120 TRD directly binds the Fzd5 receptor  in SLC2A1-siRNA-transfected cells compared to the scramble control transfected cells  to determine whether E. chaffeensis infection or TRP120-Wnt-SLiM regulated GLUT1, BCL-xL, and Bax levels during Yap inhibition. During infection, there was a significant reduction in GLUT1 in the presence of Verteporfin, demonstrating that E. chaffeensis depends on Yap activation to increase GLUT1  at 24 hpi compared to uninfected Verteporfin-treated cells and E. chaffeensis-infected and uninfected DMSO-treated cells, supporting the conclusion that E. chaffeensis activates Yap to prevent apoptosis. Additionally, ehrlichial survival was significantly reduced in the presence of Verteporfin compared to the control (DMSO) .Our results support the importance of the anti-apoptotic Yap-GLUT1-BCL-xL axis during infection and demonstrate that  or DMSO . E. chafl (DMSO) . Furtherteporfin . To defiteporfin . E. chafE. chaffeensis contains a Wnt-SLiM and depends on the Wnt-Fzd5 receptor to activate the transcription co-activator Yap, which promotes a significant upregulation in genes critical for Hippo and Wnt signaling. This is the first report of a single eukaryotic SLiM mimic in bacteria that can regulate multiple conserved signaling pathways. This study also reveals a novel strategy utilized by obligate intracellular bacteria to extend host cell lifespan and highlights the importance of pathogen utilization of eukaryotic cellular signaling motifs for reprogramming the host cell to promote infection.Based on the premise of Hippo-Wnt crosstalk and the regulation of Wnt signaling by the TRP120-Wnt-SLiM, we sought to identify whether the TRP120-Wnt-SLiM deactivates Hippo, leading to Yap activation. Indeed, we reveal that TRP120-Wnt-SLiM regulates Hippo signaling and identifies the downstream effects directed at inhibiting intrinsic host-cell apoptosis. E. chaffeensis regulates Hippo signaling during infection. Indeed, we confirmed that infection induces Yap activation and transcriptional induction of Hippo pathway genes. Although there was differential expression of Hippo pathway genes in TRP120 and Wnt5a-treated cells, we discovered that many Hippo target genes were upregulated by both TRP120 and Wnt5a, which supports TRP120 mimicry of Wnt5a. Some differences between TRP120 and Wnt5a were expected since the biological functions of various Wnt ligands differ despite a highly similar amino acid sequence  to target Wnt signaling and determined that it does activate both Hippo and Wnt signaling, consistent with known Hippo-Wnt receptor overlap and crosstalk . In contrast to our finding that Hippo relies completely on the Fzd5 receptor for signaling, β-catenin activation was only significantly reduced (~50%) in the Fzd5 receptor KO cells. This is consistent with reports demonstrating that multiple Wnt ligands and Fzd receptors are involved in β-catenin activation. Similarly, we have observed interactions between TRP120 and other Fzd receptors known to activate β-catenin , supplemented with 10% fetal bovine serum, and incubated at 37°C in a 5% CO2 atmosphere. Peripheral blood mononuclear cells were obtained from deidentified healthy human donors , and PHMs were isolated using MACS negative selection . E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells and PHMs as previously described  or PHMs were propagated in RPMI 1640 with The NCBI Protein Basic Local Alignment Search Tool (Protein BLAST) was utilized for sequence alignment of TRP120 (NCBI accession number: AAO12927.1) and Wnt5a and Wnt3a amino acid sequences (NCBI accession numbers: AAH74783 and EAW69829).E. chaffeensis recombinant full-length TRP120 (rTRP120-FL), TRP120 TRD (rTRP120-TR), or thioredoxin  were expressed in Escherichia coli and purified as described previously (eviously . rWnt5a QDVASHESGVSDQPAQV) (Antibodies used in this study include α-disulfide bond formation protein (Dsb) , α-TRP12SDQPAQV) , α-TRP12SDQPAQV) , α-activE. chaffeensis or TRP120-Wnt-SLiM were incubated for 1 h or overnight, respectively, with 1.5 µg/mL of either α-TRP120-Wnt-SLiM antibody (targets TRP120 sequence SKVEQEETNPEVLIKDLQDVAS) or α-TRP120-PIS antibody (control), and then THP-1 cells were subsequently treated with each mixture for 10 h.E. chaffeensis (MOI 100) 24 h post-transfection. Cells were harvested at 24 hpi, and the ehrlichial load was determined using qPCR. All knockdowns were performed with three biological and technical replicates, and significance was determined using t-test analysis.All siRNAs were ON-TARGETplus SMARTpool . siRNA KD was performed as previously described , 10. ScrE. chaffeensis-infected (MOI 100) and uninfected THP-1 cells were seeded in T-150 flasks  and collected at 0, 4, 10, 24, and 48 hpi. THP-1 cells were treated with rTRP120-FL, rTrx (-), rWnt5a, TRP120-TR-Wnt5a, TRP120-TR (-), TRP120-Wnt-SLiM, or TRP120-Wnt-SLiM-mut (1 µg/mL) and collected 6 hpt for confocal microscopy as previously described , rWnt5a, and TRP120-Wnt-SLiM and TRP120-Wnt-SLiM-mut peptide-treated PHMs were seeded in 12-well plates (Corning) containing a coverslip and incubated for 10 h. Cells were prepared for confocal microscopy as previously described (n = 10) were used to detect active Yap. Experiments were performed with three biological and technical replicates.cted MOI 0 and unicted MOI 0 and uniE. chaffeensis-infected (MOI 100), uninfected, rTRP120-FL, rTrx (-), rWnt5a, and TRP120-Wnt-SLiM (10 ng/mL or 1 µg/mL) cells were harvested at 24 h. Uninfected/untreated or rTrx (-)-treated cells were used as controls for infection and protein/peptide treatments. RNA isolation and cDNA synthesis were performed as previously described  using the QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific). PCR data analysis was performed as previously described  was used to determine the expression of 84 key Hippo target genes. PCR arrays were performed according to the manufacturer’s protocol (Qiagen). Real-time PCR was performed using the RTescribed .t-test analysis. All blots are presented in their original form but may have a loss of resolution due to image compression from the dock system, Chemidoc-it2 Imager.Briefly, THP-1 cells were harvested, and lysates were prepared using CytoBuster protein extraction reagent  supplemented with a complete mini EDTA-free protease inhibitor  and phenylmethene-sulfonylfluoride PMSF 10 mM) (Sigma-Aldrich). Cell lysate protein concentrations were determined, and Western blots were performed as previously described ( mM (SigmE. chaffeensis (MOI 100) or treated with rWnt5a, TRP120-Wnt-SLiM, or TRP120-Wnt-SLiM-mut (1 µg/mL) and collected at 6 h for confocal and Western blot analysis.CRISPR/Cas9 Fzd5 receptor KO THP-1 cells were obtained from a commercial source  and serially diluted to isolate a clonal population. Normal and Fzd5 receptor KO THP-1 cells were infected with SLC2A1 gene expression during infection was determined using real-time qPCR. THP-1 cells were infected with E. chaffeensis (MOI 100). Cells were harvested at 0, 3, and 24 hpi to examine gene expression during the entry and early replication phases. The fold change in SLC2A1 from 0 to 3 or 24 hpi was calculated using the 2CT−ΔΔ method and TC values for host SLC2A1 and GAPDH genes as previously described , uninfected, TRP120-Wnt-SLiM- and TRP120-Wnt-SLiM-mut-treated THP-1 cells were incubated with DMSO or Verteporfin (7 µg/mL) for 24 h, then cells were harvested for Western blot and Diff-Quik staining (Thermo Fisher Scientific). Diff-Quik images are in their original form, taken with a DP25 camera using cellSens. However, this software can create artifacts. Ehrlichial load was determined using qPCR as described above. Cell counts and viability were determined by the Cellometer Mini Brightfield Automated Cell Counter .


Lastly, we found CD8+ TEFF from HBV+ ESCC patients showing higher fraction of Exhaustionhi T than their HBV– counterpart. In summary, anti-PD-1 therapy on HBV+ non-liver cancer patients is safe and achieves better outcomes than that on HBV– non-liver cancer patients, potentially because HBV+ patients had higher fraction of Exhaustionhi T, which made them more efficiently respond to anti-PD-1 therapy.Anti-PD-1 therapy has shown promising outcomes in the treatment of different types of cancer. It is of fundamental interest to analyze the efficacy of anti-PD-1 therapy in cancer patients infected with hepatitis B virus (HBV) since the comorbidity of HBV and cancer is widely documented. We designed a multicenter retrospective study to evaluate the efficacy of anti-PD-1 therapy on non-liver cancer patients infected with HBV. We found anti-PD-1 therapy achieved much better outcomes in HBV+ non-liver cancer patients than their HBV– counterparts. We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) from esophageal squamous cell carcinoma (ESCC) patients. We found both cytotoxicity score of T cells and MHC score of B cells significantly increased after anti-PD-1 therapy in HBV+ ESCC patients. We also identified CX3CR1 Immune checkpoint blockade (ICB) therapy is a promising immunotherapy approach, among which anti-programmed cell death-1 (anti-PD-1) therapy yielded promising outcomes in treatment of dozen of types of cancer, including melanoma, hepatocellular carcinoma (HCC), gastric cancer, and esophageal cancer . HoweverSimultaneously, the success of anti-PD-1 in cancer therapy potentially indicates that anti-PD-1 might be effective for treating infectious diseases since chronic infection also showed high expression of PD1 and T-cell exhaustion . Over 35Here, we performed a multicenter retrospective study on 84 non-liver cancer patients, including 35 HBV+ patients and 49 HBV– patients. We further performed single-cell RNA sequencing (scRNA-seq) on six peripheral blood mononuclear cells (PBMCs) samples from three esophageal squamous cell carcinoma (ESCC) patients (pre- and post-anti-PD-1 therapy), to analyze their response to anti-PD-1 therapy. We further analyzed the differences in the immune microenvironment between HBV+ ESCC patients and HBV– ESCC patients to explore the mechanism underlying their different response to anti-PD-1 therapy.p-value = 0.499) , with 15 (42.86%) HBV+ non-liver cancer patients showing partial response (PR) while only 7 (14.29%) HBV– patients showing PR  , the odds ratio (OR) of HBV+ non-liver cancer patients compared with their HBV– counterparts was 4.41 . HBV+ patients showed much longer overall survival (OS), further indicating that HBV+ patients have much better outcome than their HBV– counterparts Fig. . Althoug15) Fig.  potentia+ TN, CD4+ TM, CD8+ TN, BN, and BM consistently decreased across patients after anti-PD-1 therapy, while the fraction of cMo increased in both HBV–#1 and HBV+#2  and two HBV-positive patients (HBV+#1 and HBV+#2), were enrolled from the Southwest Hospital for exploring their response to anti-PD-1 therapy. HBV+#1 and HBV+#2, both with hepatitis B surface antigen (HBsAg)-positive and hepatitis B core antibody (HBcAb)-positive, had 1910 IU/mL HBV-DNA and 48,600 IU/mL HBV-DNA in blood, respectively  between samples pre- and post-anti-PD-1 therapy in each of the 6T cell subsets, namely CD4–#1 Fig. .Fig. 3Ch+ TEFF since it has the highest increase of population size and the highest increase of cytotoxicity score in HBV+#2. We clustered CD8+ TEFF and identified two subsets, namely CX3 CR1hi TEFF and CX3 CR1low TEFF  family genes , which were cancer-related transcription factors [low TEFF highly expressed regulators of G protein signaling (RGS1 and RGS2) that was related to lower infiltration to cancer [CXCR4  patients in The Cancer Genome Atlas (TCGA) [low TEFF-specific genes, namely CX3CR1low TEFF signature high patients, had lower survival than CX3CR1low TEFF signature low patients  . We founnts Fig. . FurtherBN and BM in HBV+ patients after anti-PD-1 therapy, while it significantly decreased in HBV–#1  and HBcAb-positive . Furthermore, HBV-DNA was detected in all the 35 HBV+ patients, indicating HBV was active in those patients , while the other 9 patients received anti-PD-1 monotherapy. The anti-PD-1 antibodies used in this study include Toripalimab, Sintilimab, Pembrolizumab, Camrelizumab, Nivolumab, and Tislelizumab. Similarly, 39 out of the 49 HBV– non-liver cancer patients received anti-PD-1 combination therapy, while the other 10 patients received anti-PD-1 monotherapy. In addition, some patients were followed up for obtaining survival information after treatment (Supplementary Tables Patients enrolled in this study did not show any immune-related adverse event (irAE) or low grade (1 grade or 2 grade) irAEs during and after anti-PD-1 therapy, except three 3-grade irAEs, namely maculopapular rash in a HBV+ patient, which may be caused by drug treatment , cardiotWe performed scRNA-seq of 6 PBMCs samples derived from the three ESCC patients. This study was approved by IRB at the Southwest Hospital, AMU (ID: KY2021112). All individuals signed an informed consent form approved by the IRBs at the time of enrollment. Patients received the treatment according to clinical routine. The scRNA-seq library preparation and data analyses following our previous studies –47.Approximately 5 mL blood sample was phlebotomized from each of the three ESCC patients pre- and post-treatment and was transported to the laboratory in an EDTA- containing anticoagulant tube . Blood samples were centrifuged at 1750 rpm for 8 min to collect the white blood cells. Lymphocyte Separation Medium (Human)  was added to substratum cell precipitation to obtain PBMCs after centrifugating at 1950rpm for 20 min and washed in 0.04% BSA of PBS  for twice with centrifuging at 1000 rpm for 5 min. Trypan blue  staining was used to check for cell viability. The single-cell RNA sequencing libraries of PBMCs were generated using Chromium Next GEM Single-Cell 3’ Reagent Kit v3.1 from 10x genomics. Briefly, 20,000 cells were loaded into each channel of the 10x Chromium controller to generate single-cell GEMs. Single-cell RNA-seq libraries were prepared using the Chromium Single-Cell 3’ Gel Bead and Library Kit (10x Genomics), according to manufacturers’ instructions. Sequencing libraries were loaded at 2.4 pM on an Illumina NovaSeq 6000 with 2× 75 paired-end kits.NormalizeData function. We identified the top 3,000 highly variable genes using the FindVariableFeatures function and further scaled the gene expression matrix using the ScaleData function. We conducted principal component analysis (PCA) for linear dimension reduction using the RunPCA function and the top 30 PCs were used for further analyses. The RunHarmony function in Harmony package was used to remove the batch effect in our data. Finally, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) were conducted using the RunUMAP function.Briefly, we mapped the reads to the human reference genome GRCh38 and aggregated the gene expression matrixes using Cell Ranger (V.5.0.0). We constituted a Seurat object using the filter expression matrixes from Cell Ranger. Seurat package (V.3.2.3)  and HarmFindNeighbors and FindClusters functions based on the normalized gene expression matrix. We identified 17 clusters that were annotated according to their specific expression of classic markers, including 9T cell subsets (CD3D), 2 natural killer cell (NK) subsets (KLRF1), 2 monocyte subsets (FCN1), 2 B-cell subsets (CD19), mDC (CLEC10A) and plasma cell (MZB1)  (TCL1A+) and memory B cells (BM) (CD27+). The 2 monocyte subsets are classic monocyte (cMo) (CD14+) and non-classical Monocyte (ncMo) (FCGR3A+). The 9T cell subsets are CD4+ Naive T cells (CD4+ TN) (CD4+CCR7+), CD8+ Naive T cells (CD8+ TN) (CD8+CCR7+), CD4+ memory T cells (CD4+ TM) (CD4+SELLloCCR7lo), CD8+ memory T cells (CD8+ TM) (CD8+SELLloCCR7lo), CD4+ effector T cells (CD4+ TEFF) (CD4+GZMA+GZMB+), CD8+ effector T cells (CD8+ TEFF) (CD8+GZMA+GZMB+), CD4+ regulatory T cells (CD4+ Treg) (CD4+FOXP3+), γδT (TRDC+TRGC1+) and mucosal-associated invariant T-cell (MAIT) (ZBTB16+SLC4A10+)  Fig. .(Fpost – Fpre)/Fpre, where Fpre is the fraction of a cell subset in PBMCs before anti-PD-1 therapy, and Fpost is the fraction of the same cell subset in PBMCs after anti-PD-1 therapy. A positive value of PopIndex indicates the increase of population size after anti-PD-1 therapy, and a negative value indicates population size decrease after anti-PD-1 therapy.We calculated the PopIndex of each cell subset to measure the relative change of population size after anti-PD-1 therapy. The PopIndex was defined as PopIndex = FindMarkers function was utilized to find DEGs between the cell subsets. DEGs with bonferroni-adjusted p-values < 0.05 and average Log2 (fold-change) (avg_log2FC) > 0.2 were kept for further analyses.The AddModuleScore function. All the scores were measured according to expression of specific functional genes. For instance, the cytotoxicity score was calculated based on 12 cytotoxicity-associated genes: PRF1, IFNG, GNLY, NKG7, GZMB, GZMA, GZMH, KLRK1, KLRB1, KLRD1, CTSW, and CST7. The exhaustion score was calculated based on 6 exhaustion-associated genes: LAG3, TIGIT, PDCD1, CTLA4, HAVCR2 and TOX. The MHC-I score was calculated based on 9 MHC-I-associated genes: HLA-A, HLA-B, HLA-C PSMB8, PSMB9, TAP1, TAP2, TAPBP and B2M. The MHC-II score was calculated based on 11 MHC-II-associated genes: HLA-DRA, HLA-DRB1, HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DMA, and HLA-DMB.In order to evaluate the changes of functional features of each cell subset pre- and post-anti-PD-1 therapy, we calculated the cytotoxicity score, exhaustion score, major histocompatibility complex (MHC)-I score and MHC-II score of each cell using the p-value of clinical statistical analyses were calculated using Fisher’s exact test. The statistical significance of the score of gene sets was determined by Student’s t-test. Log-rank-test was utilized to calculate the p-value of the survival analyses. Kruskal–Wallis test was used to determine whether the proportion of Exhaustionhi T were significantly different between two samples.The OR value and Supplementary figureTable S1Table S2Table S3Table S4Table S5Table S6Table S7Table S8


Investigation of downstream mechanisms using RNA immunoprecipitation and luciferase reporter assay revealed that lncRNA PRRT3-AS1 acted as a competing endogenous RNA by adsorbing microRNA-507 (miR-507) and enhanced the expression of its target gene, homeobox B5 (HOXB5), in NSCLC. Furthermore, miR-507 downregulation or HOXB5 upregulation eliminated the cancer-inhibiting effects of lncRNA PRRT3-AS1 depletion in NSCLC cells. To conclude, the lncRNA PRRT3-AS1/miR-507/HOXB5 pathway acts as a promoter of malignant characteristics in NSCLC, and this newly identified competing endogenous RNA pathway may be an effective diagnostic, prognostic, and therapeutic target in NSCLC.Long noncoding RNAs (lncRNAs) act as key regulators controlling complex cellular behaviors in nonsmall cell lung cancer (NSCLC). We investigated the expression of lncRNA PRRT3 antisense RNA 1 (PRRT3-AS1) in paired samples of NSCLC and adjacent normal tissues from a patient cohort in our hospital using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and found that it was significantly higher in NSCLC tissue than in normal tissue, consistent with The Cancer Genome Atlas database. Furthermore, functional investigation revealed that lncRNA PRRT3-AS1 depletion inhibited NSCLC-cell proliferation, colony formation, invasion, and migration, whereas its overexpression exerted the opposite effects. Moreover, PRRT3-AS1 knockdown suppressed  Among all cancers in humans, lung cancer has the highest incidence and mortality rate globally , with apLong noncoding RNAs (lncRNAs) are a family of RNA molecules comprising 200 or more nucleotides with no protein-coding ability ,6. In thMicroRNAs (miRNAs) are a family of short (approximately 17–23 nucleotides) endogenous noncoding RNA molecules –26. DuriSeveral lncRNAs are known to be key regulators of NSCLC malignancy. We investigated the expression and detailed functions of PRRT3 antisense RNA 1 (PRRT3-AS1) in NSCLC, in addition to the downstream mechanisms.We obtained NSCLC tissues from 53 patients in our hospital who had not undergone chemotherapy or radiotherapy before surgery. Immediately after tissue excision, all tissue samples were immersed and stored in liquid nitrogen until further analysis. This research was approved by the ethics committee of the Affiliated Zhongshan Hospital of Dalian University. In addition, all participating patients provided written informed consent.2 at 37°C.We purchased human NSCLC cell lines A549, H460, SK-MES-1, and H1299 from the American Type Culture Collection . Bronchial epithelial cell growth medium  was adopted for culturing a human nontumorigenic bronchial epithelial cell line BEAS-2B (ATCC). Cell lines A549 and H1299 were grown in RPMI-1640 medium (Gibco). SK-MES-1 and H460 cell lines were grown in F-12K and MEM (Gibco), respectively. Each of the above culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco). All cells were grown in a humidified atmosphere of 5% COWe purchased small interfering RNAs (siRNAs) against PRRT3-AS1 (si-PRRT3-AS1) and homeobox B5  and negative control (NC) siRNA (si-NC) from GenePharma . In addition, we used the miR-507 mimic and miR-507 inhibitor  to knock down and upregulate endogenous miR-507 levels, respectively. We used miRNA mimic (miR-NC) and inhibitor NCs as controls. The PRRT3-AS1 overexpression plasmid pcDNA3.1-PRRT3-AS1 (pc-PRRT3-AS1) and HOXB5 overexpression plasmid pcDNA3.1-HOXB5 (pc-HOXB5) were designed and synthesized by GenePharma. NSCLC cells were seeded into 6-well plates, and cell transfection was executed when cell growth reached 80% confluence. Cellular transfection was done by applying Lipofectamine® 2000  and the vectors to the wells.−ΔΔCq formula.We used the TRIzol® reagent (Invitrogen) to extract total RNA. Total RNA was reverse-transcribed by using the Mir-X miRNA First-Strand Synthesis Kit . With the obtained cDNA as a template, we performed quantitative polymerase chain reaction  to determine miR-507 expression by using the Mir-X miRNA quantitative reverse transcription PCR (qRT-PCR) TB Green® Kit (TaKaRa). U6 served as the endogenous control for miR-507. To measure PRRT3-AS1 and HOXB5 expression, we used the PrimeScript® RT Reagent Kit for cDNA synthesis and SYBR® Premix Ex Taq™ II (TaKaRa) for qPCR. PRRT3-AS1 and HOXB5 levels were normalized to GAPDH the internal control, and reported as log-fold changes using the 22 atmosphere. After incubating with 10 µL of the cell counting kit-8 (CCK-8) solution (Beyotime), we estimated the proliferation rate by the measurement of optical density at 450 nm. We performed the assay every day until day 4 and plotted the results as a growth curve.We inoculated transfected NSCLC cells into 96-well plates with a density of 2000 cells per well and incubated them at 37°C in a 5% COFor colonies, a cell suspension was prepared, and 2 ml of the cell suspension containing 600 cells was added into each well of 6-well plates. After 14 days of cultivation, the newly formed colonies were fixed with 100% methanol and stained with 0.1% crystal violet. The cells in the colonies were manually counted using an inverted microscope (x200 magnification).4 of transfected cells, and plated 600 μL of culture medium supplemented with 10% FBS into the lower chambers. We allowed the NSCLC cells to penetrate the pores in the submembrane surface for 24 h, then fixed them with 100% methanol, and stained them with 0.1% crystal violet. The cells that invaded the lower chambers were counted in five randomly chosen regions (in each well) under an inverted microscope. For the migration assay, we did not apply Matrigel, but the remaining steps were the same as those in the Matrigel invasion assay.We explored the invasive ability of NSCLC cells using Transwell inserts (BD Biosciences). We added 100 µL of Matrigel (BD Biosciences) into the inner sides of the Transwell inserts and performed polymerization by incubating them at 37°C for 2 h. Next, we loaded the upper chambers with 100 μL of FBS-free culture medium containing 4 × 106 PRRT3-AS1-knockdown H460 cells were subcutaneously inoculated into each mouse in the sh-PRRT3-AS1 group. We monitored the width and length of tumor xenografts from 1 week after cell inoculation and then every 5 days. Thirty-two days later, all mice were anesthetized by means of cervical dislocation, and the tumor xenografts were completely detached and weighed.The short hairpin RNA (shRNA) targeting PRRT3-AS1 (sh-PRRT3-AS1), and NC shRNA (sh-NC) were designed and chemically generated by GenePharma. We inserted the shRNAs into a lentiviral vector to transfect the shRNAs into H460 cells. The PRRT3-AS1-knockdown H460 cells were selected by cultivating them with puromycin. Next, a total of 8 BALB/c nude mice  were obtained from HFK Bioscience  and randomly divided into sh-PRRT3-AS1 and sh-NC groups. A total of 2 × 10All animal experiments were approved by the Animal Care and Use Committee of Affiliated Zhongshan Hospital of Dalian University. The humane endpoints were tumor diameter >1.5 cm, tumor ulceration, abnormal feeding, weight loss, ascites and cachexia. Meanwhile, no anesthesia was applied in the animal experiments.In Situ Hybridization (FISH) Kit . In detail, after washing with phosphate-buffered saline, H460 and A549 cells were fixed with 4% paraformaldehyde, followed by the hybridization treatment specifically targeting PRRT3-AS1  at 37°C without light. On the next day, DNA staining was performed using Hoechst solution. The cells were, imaged under a confocal laser scanning microscope .The subcellular location of PRRT3-AS1 in NSCLC cells was examined using the Fluorescent http://mirdb.org/) database for PRRT3-AS1’s target miRNAs. To predict the downstream target of miR-507, we used Targetscan (https://www.targetscan.org/vert_80/) applied to miRDB.We searched the miRNA Target Prediction DataBase (miRDB) (We performed a RNA immunoprecipitation (RIP) assay using the Magna RIP RNA-Binding Protein Immunoprecipitation kit . Briefly, we harvested 80% confluent NSCLC cells and lysed them using the RIP lysis buffer. Next, we performed Argonaute 2 (Ago2) immunoprecipitation by incubating the cell lysate with magnetic beads conjugated with anti-Ago2 antibody or normal immunoglobulin G (IgG) . Ago2 is an important element of RNA-induced silencing complex, and it can promote the degradation of target mRNAs via its catalytic activity in gene silencing processes induced by miRNAs. After overnight incubation at 4°C, we extracted the immunoprecipitated RNA and evaluated the abundance of lncRNA PRRT3-AS1, miR-507, and HOXB5 mRNA using qRT-PCR.The PRRT3-AS1 and HOXB5 wild-type (WT) sequences, both enclosing the predicted miR-507-binding sequences, were amplified by GenePharma, and fused to the psiCHECK™-2 luciferase reporter vector (Promega), generating the WT-PRRT3-AS1 and WT-HOXB5 reporter vectors. Similarly, the mutant (MUT) luciferase reporter vectors MUT-PRRT3-AS1 and MUT-HOXB5 were built by inserting PRRT3-AS1 and HOXB5 mutant sequences into the psiCHECK™-2 vector. Next, NSCLC cells were transfected with WT or matched MUT reporter vectors alongside the miR-507 mimic or miR-NC. On post-transfection day 2, we detected luciferase activity by applying the dual-luciferase reporter assay (Promega).We performed total protein extraction and quantification using the Pierce RIPA lysis buffer and the Pierce™ bicinchoninic acid (BCA) Kit , respectively. Protein samples were separated by 10% SDS-PAGE and blotted onto PVDF membranes. After blocking them with 5% defatted milk powder, the membranes were incubated at 4°C overnight with the primary antibodies against Hoxb5 (ab109375) or Gapdh (ab128915), followed by incubation with the HRP-conjugated anti-rabbit secondary antibody . Finally, the bands of target proteins were developed by treatment with an enhanced chemiluminescence reagent (Beyotime).t-test or ANOVA. p < 0.05 was considered statistically significant.All experiments were independently repeated three times. Results are reported as the mean ± 1 SD. The between-group differences were compared using Student’s th overexpressed lncRNA in lung adenocarcinoma , which indicated that it was mostly located in the cytoplasm (To determine downstream mechanisms of PRRT3-AS1 activation, we detected the subcellular localization of PRRT3-AS1 in NSCLC cells. First, to predict lncRNA PRRT3-AS1’s subcellular location, we used lncLocator (ytoplasm . We veriytoplasm . This obytoplasm . A searcWe investigated the regulatory effects of lncRNA PRRT3-AS1 on the six miRNAs and found that miR-507 was the one that changed the most . LuciferNext, we determined the roles of miR-507 overexpression  in NSCLCGiven that lncRNA PRRT3-AS1 sequestered miR-507 in NSCLC, we hypothesized that lncRNA PRRT3-AS1 indirectly modulated HOXB5 expression through sequestering miR-507. In fact, HOXB5 levels were decreased in H460 cells after lncRNA PRRT3-AS1 depletion, whereas HOXB5 levels were increased in pc-PRRT3-AS1-transfected A549 cells –5H. In aWe designed rescue experiments to determine whether lncRNA PRRT3-AS1 performed tumor-promoting actions in NSCLC cells by controlling the miR-507/HOXB5 axis. First, we tested the transfection efficiency of the miR-507 inhibitor in NSCLC cells, verifying that miR-507 significantly decreased in miR-507 inhibitor-transfected cells . MeanwhiNext, si-PRRT3-AS1 was combined with the miR-507 inhibitor or the pc-HOXB5 to transfect H460 cells. PRRT3-AS1-overexpresed A549 cells underwent cotransfection with the miR-507 mimic or si-HOXB5. The cell proliferation inhibited by si-PRRT3-AS1 was reversed by miR-507 inhibition or HOXB5 upregulation . ConversIn addition, the introduction of the miR-507 inhibitor or pc-HOXB5 offset the impaired H460 cell migration and invasion  induced in vivo. In contrast to tumors in the sh-NC group, the sh-PRRT3-AS1-transfected subcutaneous tumors grew significantly slower (in vivo.Xenograft tumor assay was performed to illustrate whether lncRNA PRRT3-AS1 affected NSCLC tumor growth y slower  and 8B. y slower . Furthery slower  and HOXBy slower  levels wy slower . Taken tMany lncRNAs have been considered as crucial promoters or inhibitors of NSCLC progression , but morin vitro, whereas PRRT3-AS1 overexpression exerted the opposite effects. These results suggest that PRRT3-AS1 could be considered as a possible target for NSCLC diagnosis, prognosis, and management.PRRT3-AS1 is overexpressed in prostate cancer, and the knockdown of PRRT3-AS1 suppresses cell viability, migration, and invasion, while promoting cell apoptosis . Howeverin situ hybridization, we determined that the subcellular localization of lncRNA PRRT3-AS1 was primarily in the cytoplasm of NSCLC cells, offering a theoretical basis for lncRNA PRRT3-AS1 functions as a ceRNA.Next, we determined the mechanisms underlying the effect of lncRNA PRRT3-AS1 on malignant properties. The extensive research on the potential mechanisms of lncRNA function has mostly been dependent on the cellular localization of lncRNAs. Nuclear lncRNAs directly bind to proteins and regulate gene expression at the transcriptional level . In contBioinformatic analysis of downstream targets of lncRNA PRRT3-AS1 predicted miR-507 as a possible lncRNA PRRT3-AS1-sequestered miRNA. Subsequent luciferase reporter and RIP assays confirmed that lncRNA PRRT3-AS1 acted as a miR-507 sponge in NSCLC cells. Mechanistic investigation also identified HOXB5 as a direct target of miR-507 in NSCLC cells, showing that lncRNA PRRT3-AS1 indirectly modulated HOXB5 in NSCLC cells by sponging miR-507. Therefore, we hypothesize that PRRT3-AS1, miR-507, and HOXB5 coexist in the same RNA-induced silencing complex. Overall, lncRNA PRRT3-AS1 acts as a ceRNA to directly interact with miR-507 and indirectly regulate HOXB5 expression in NSCLC cells, thereby forming the novel lncRNA PRRT3-AS1/miR-507/HOXB5 ceRNA pathway.We have known that miR-507 is differentially expressed in several human cancers, including NSCLC . Here, win vivo. Secondly, other ways may be involved in the mechanisms underlying the oncogenic roles of PRRT3-AS1 in NSCLC. We will resolve the two limitations in the near future.Our study had two limitations. Firstly, we did not explore the effect of PRRT3-AS1 on metastasis To conclude, PRRT3-AS1, which is overexpressed in NSCLC, upregulates HOXB5 expression by sequestering miR-507 and aggravates the oncogenicity of NSCLC. Our findings advance the understanding of NSCLC pathogenesis and strongly suggest the PRRT3-AS1/miR-507/HOXB5 pathway as a promising novel therapeutic target.


KRAS G12C mutation (mKRAS G12C) is the most frequent KRAS point mutation in non-small cell lung cancer (NSCLC) and has been proven to be a predictive biomarker for direct KRAS G12C inhibitors in advanced solid cancers. We sought to determine the prognostic significance of mKRAS G12C in patients with NSCLC using the meta-analytic approach. A protocol is registered at the International Prospective Register for systematic reviews (CRD42022345868). PubMed, EMBASE, The Cochrane Library, and Clinicaltrials.gov.in were searched for prospective or retrospective studies reporting survival data for tumors with mKRAS G12C compared with either other KRAS mutations or wild-type KRAS (KRAS-WT). The hazard ratios (HRs) for overall survival (OS) or Disease-free survival (DFS) of tumors were pooled according to fixed or random-effects models. Sixteen studies enrolling 10,153 participants were included in the final analysis. mKRAS G12C tumors had poor OS  but similar DFS  compared to KRAS-WT tumors. Compared to other KRAS mutations, mKRAS G12C tumors had poor DFS  but similar OS . Compared to other KRAS mutations, high PD-L1 expression (>50%)  was associated with mKRAS G12C tumors. mKRAS G12C is a promising prognostic factor for patients with NSCLC, negatively impacting survival. Prevailing significant heterogeneity and selection bias might reduce the validity of these findings. Concomitant high PD-L1 expression in these tumors opens doors for exciting therapeutic potential. KRAS) is one of the most common oncogenic drivers in NSCLC, seen in over 30% of lung adenocarcinomas (LUAD), depending on ethnicity and tumor stage and associated with smoking and female patients   . Despitetentious ,9,10,11.KRAS status harbors a spectrum of distinctly mutated KRAS substitutions , though between-study heterogeneity was high   AKRAS G12C to KRAS G12D mutated tumors also showed similar outcomes  (p = 0.013) suggested the presence of publication bias. The trim-and-fill method led to the addition of three studies; however, the combined HR after adjustment remained statistically non-significant [adjusted HR (aHR) = 0.92 95% CI 0.73–1.17] (Studies that compared  = 0.66] . Sensiti = 0.66]  and sign73–1.17] .p = 0.69), and for non-Asians was 1.07  (p = 0.11), whereas, for PCR-based methods, it was 1.20   . Among t = 0.50) . Finally results .mKRAS G12C and other KRAS mutations     compared with other KRAS mutations. Finally, TP53 and STK11 were not associated with either mKRAS G12C or other KRAS mutant tumors. Overall, KRAS G12C mutated tumors harbor poor prognosis; however, considerable between-study heterogeneity existed in most of these analyses.Small molecule inhibitors of 1 trials ,52,53. TC tumors . Our sysmKRAS and survival in NSCLC patients is relatively weak and primarily restricted to advanced disease [KRAS mutation subtypes uniquely alters tumor biology and thus may influence distinct clinical behavior that may not be apparent in examining KRAS mutations in toto [KRAS mutation subtype in NSCLC and showed consistent results across ethnicities and testing methodologies.In prior meta-analyses, the overall link between  disease ,11. Diff disease . Further in toto ,56. Our mKRAS G12C than other KRAS mutations [mKRAS G12C and other KRAS mutations showed comparable OS rates in our study. In a study on early-stage NSCLC (n = 179), mKRAS G12C was associated with poor DFS (p = 0.006) compared with KRAS-WT; however, the statistical strength of this analysis dropped significantly for stage I patients, suggesting the influence of pathological stage on DFS [KRAS G12C inhibitors showed clinical efficacy in eradicating micrometastases and enhanced anti-tumor activity when combined with targeted agents [KRAS G12C inhibitor monotherapy or in combination with targeted agents in patients with early-stage mKRAS G12C tumors may open the door for exciting therapeutic paradigms.Surgically resected NSCLC are at a higher risk of disease relapse if associated with the utations ,49. In cd agents . Based oKRAS-comutations and PD-L1 expression may also modify the behavior of mKRAS tumors [mKRAS tumors exhibit co-mutations, of which TP53 (35–50%), STK11 (12–20%), and KEAP1 (7–10%) constitute the majority [mKRAS G12C tumors show similar concurrent genomic alterations, although they have also been associated with ERBB2 amplification and ERBB4 mutations [PTEN and PDGFRA mutations are less commonly observed in mKRAS G12C tumors [KRAS-comutations have been considered an adverse prognostic factor capable of predicting tumor progression and chemo-resistance [KRAS-comutations. However, data from clinical trials indicated that patients harboring STK11 and KRAS co-mutations had a higher objective response rate (ORR) (63% vs. 33%), whereas KEAP1 and KRAS co-mutation were associated with a lower ORR (20% vs. 44%) following KRAS G12C inhibitor therapy [mKRAS. In this context, KRAS G12C inhibitors are being evaluated as a first-line therapeutic option in patients with NSCLC harboring STK11 and KRAS G12C co-mutations (KRYSTAL-1 and CodeBreaK 201).S tumors ,58. Apprmajority ,60. mKRAutations . PTEN anC tumors ,61,62. Ksistance ,64,65,66 therapy ,53. ThesmKRAS tumors, and tumors with high PD-L1 expression were associated with a dismal prognosis [mKRAS G12C tumors. In preclinical data, KRAS G12C direct inhibitors have been shown to upregulate a pro-inflammatory tumor microenvironment and increase anti-tumor T-cell activity [KRAS G12C inhibitors have been shown to increase intratumoral chemokine concentrations, potentially increasing T-cell infiltration and enhancing immunosurveillance [KRAS G12C and anti-PD-1 combination therapy led to a synergistic response compared with either monotherapy [mKRAS G12C received anti-PD-1/PD-L1 therapy along with sotorasib and demonstrated an ORR of 32.2%, and a median PFS of 6.3 months [mKRAS G12C [KRAS G12C inhibitor combinations for advanced solid tumors.The degree of PD-L1 expression in tumor cells corresponds to the prognosis in patients with rognosis ,67,68. Oactivity . Furthereillance . In CT-2otherapy . In the 3 months . LikewisRAS G12C . KRYSTALmKRAS-G12C in the NSCLC and mKRAS cohorts reflected the real-world data that were previously reported, suggesting a representative population and thus more likely to reflect prognosis in clinical practice [We found that the frequency of practice ,70. Acknpractice . ApproxiKRAS G12C mutation in early-stage NSCLC; thus, further investigations are needed.Trials like ADAURA and PACIFIC have provided sufficient evidence for the role of targeted agents in early-stage NSCLC. In addition, ANVIL (NCT02595944), PEARLS/Keynote-091 NCT02504372), and IMpower010 (NCT02486718) trials are currently exploring the role of immune checkpoint inhibitors in the adjuvant setting , and IMp. HoweverKRAS mutations in patients with NSCLC. Extrapolating these findings, the following recommendations may be made for clinical practice: 1) Inclusion of KRAS status, especially KRAS G12C mutation, as a routine test using a comprehensive molecular gene panel. Similarly, future studies should focus on 1) Evaluation of KRAS G12C inhibitors-anti-PD-1/PD-L1 inhibitor combination therapy in advanced NSCLC; 2) evaluation of adjuvant KRAS G12C inhibitor in surgically resected NSCLC with mKRAS G12C; and 3) assessment of the impact of KRAS G12C-comutations including TP53, STK11, and KEAP1, on prognosis.Our findings contribute to the evolving landscape of mKRAS G12C were associated with worse DFS than tumors with other KRAS mutations and worse OS than tumors with KRAS-WT. The presence of significant heterogeneity and publication bias collectively undermines the validity of these findings. However, these outcomes reflect real-world prognoses and may be utilized in clinical practice to stratify high-risk patients and provide more effective therapeutic strategies for patients with NSCLC.Our meta-analysis on NSCLC found that tumors with


Pre-exposure prophylaxis (PrEP) with a combination of long-acting monoclonal antibodies with tixagevimab/cilgavimab (T/C) have provided viral neutralizing activity against SARS-CoV-2 and protected solid organ transplant recipients (SOTRs) that produced poor immunogenicity after COVID-19 immunization. However, real-world data on the effectiveness and safety of T/C in SOTRs during the Omicron wave is limited.A prospective study at a single transplant center was conducted to evaluate the effectiveness and adverse events (AE) of T/C among SOTRs during the BA.2.75 Omicron lineages period (August 2022 to February 2023). T/C of 150/150 and 300/300 mg doses were provided from August 2022 to December 2022 and after, respectively.A total of 39 SOTRs comprising kidney (n= 29), liver (n=2), and heart (n=8) transplant recipients were included in the study. Thirty-three (85%) and 6 (15%) SOTRs received 150/150 and 300/300 mg of T/C, respectively. The median (IQR) age was 52 (35-62) years. Twenty-two (56%) had a prior history of COVID-19. The median (IQR) number of COVID-19 vaccine was 4 (3-4) doses. During the median follow-up of 6.35 (5-8.33) months, COVID-19 developed in 5 (12.8%) out of 39 SOTRs with a median (IQR) time of 73 (36-115) days from receiving T/C. None of those who received a 300/300 mg dose developed COVID-19. There was no severe COVID-19 or death observed. Five (12.8%) SOTRs reported mild and reversible AEs with pain at injection (n=2), fever (n=3), and muscle aches (n=1). There was no allograft rejection reported.Breakthrough SARS-CoV-2 infection remains among fully vaccinated SOTRs receiving T/C as PrEP and subjects to concurrent circulating strain in the community. However, the disease severity appears mild, and the short-term safety profile seems tolerable.All Authors: No reported disclosures


The predicted PCNAΔSL47 structure shows the potential distortion of the central loop and reduced hydrophobicity. PCNAΔSL47 shows a defective interaction with PCNAWT leading to defects in homo-trimerization in vitro. PCNAΔSL47 is defective in the FEN1 and LIG1 interaction. PCNA ubiquitination and DNA-RNA hybrid processing are defective in PCNAΔSL47-expressing cells. Accordingly, PCNAΔSL47-expressing cells exhibit an increased number of single-stranded DNA gaps and higher levels of γH2AX, and sensitivity to DNA-damaging agents, highlighting the importance of PCNA Ser46-Leu47 residues in maintaining genomic integrity.Proliferating cell nuclear antigen (PCNA) is a maestro of DNA replication. PCNA forms a homotrimer and interacts with various proteins, such as DNA polymerases, DNA ligase I (LIG1), and flap endonuclease 1 (FEN1) for faithful DNA replication. Here, we identify the crucial role of Ser46-Leu47 residues of PCNA in maintaining genomic integrity using  The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), is a master coordinator of DNA replication fork-associated processes. PCNA plays a crucial role in DNA replication, DNA damage bypass, DNA repair, chromatin remodeling, cohesion establishment, and sister chromatid exchange –4. PCNA pol30 mutations at E113G, L151S, or G178S, which cannot form trimeric complexes properly, inhibit translesion synthesis (TLS) and prevent mutagenesis, but not RAD6/18-dependent PCNA mono-ubiquitination [The three PCNA monomers form a homo-trimeric complex with a ring-shaped structure for faithful DNA replication and DNA synthesis . Defectstination . The PCNtination . Therefo41DSSH44) and inter-domain connecting loop (IDCL) (121LDVEQLGIPEQE132) of PCNA are evolutionarily conserved and located on the front part. These residues play crucial roles in the binding of multiple PCNA-associated proteins. Both the center loop and IDCL do not contain a positively charged arginine (R) or lysine (K) residue and therefore serve as the hydrophobic core. [1–βD1 loop containing the central loop, and this loop is known to be important for PCNA loading onto DNA by RFC in vitro [pol30 (human PCNA) at DD41, 42AA cause defects in Pol δ stimulation and Pol ε-mediated DNA synthesis, resulting in growth defects [The central loop , structurally distorts the central loop region and reduces its hydrophobicity. The PCNAΔSL47 mutant-expressing cells are defective in interaction with FEN1 and LIG1, and PCNA ubiquitination. PCNAΔSL47-expressing cells show an increased level of cellular DNA-RNA hybrids and γH2AX. Accordingly, PCNAΔSL47-expressing cells are sensitive to genotoxic stress, which highlights the importance of Ser46-Leu47 residues of PCNA in preserving genomic integrity.Herein, we identified the role of conserved and crucial PCNA residues Ser46-Leu47 in PCNA trimerization and ubiquitination using structure prediction, pcna gene using the CRISPR/Cas9 system and acquired several zebrafish homozygous mutants [pcnaΔSL47/ΔSL47 mutants. PCNA Ser46-Leu47 residues were adjacent to the central loop and conserved across species  and Danio rerio  using wild-type PCNA (PCNAWT) and PCNAΔSL47 using Alphafold2  WT-WT-WT; (2) WT-WT-ΔSL47; (3) WT-ΔSL47-ΔSL47; (4) ΔSL47-ΔSL47-ΔSL47. Consistent with the monomer prediction results, when PCNAΔSL47 mutants were included during PCNA trimerization, the exposed surface and length of the PCNA central loop region were reduced compared to the PCNAWT homotrimer  than its molecular weight (90 kDa). In the case of PCNAΔSL47, we observed a major band (75 kDa) appearing at a larger size than PCNAWT. PCNAΔSL47 protein had a lower migration speed than PCNAWT, despite the fact that amino acids are deleted compared to PCNAWT homo-trimer. Furthermore, unlike with PCNAWT, abundant protein bands were visible at sizes above the major band (75 kDa) in PCNAΔSL47 , we performed gel filtration analysis. PCNAWT proteins eluted mostly at ~120 kDa, similar to the PCNA homotrimer molecular mass (Lane 7–10) (ΔSL47 mutant proteins eluted in a broad range of molecular masses (Lane 1–6), and the expected sizes were higher than that of the PCNAWT protein. PCNAΔSL47 protein was eluted more quickly compared to PCNAWT, indicating that PCNAΔSL47 forms the higher order of oligomers or aggregation (To test PCNAne 7–10) , consistregation .ΔSL47 mutant monomers form a trimer with PCNAWT in cells. To test this hypothesis, we performed an immunoprecipitation assay using anti-c-Myc-agarose while co-overexpressing Myc-tagged PCNAWT with hemagglutinin (HA)-tagged PCNAWT or PCNAΔSL47. Interestingly, Myc-tagged PCNAWT interacted with HA-tagged PCNAWT but not with the HA-tagged PCNAΔSL47 mutant  and expressed either HA-tagged PCNAWT or PCNAΔSL47 in the cell. The siRNA-mediated knockdown efficiency and expression of HA-PCNAWT and HA-PCNAΔSL47 were confirmed by immunoblotting  to measure the single-stranded DNA gaps , and theondition . We alsong cells . Taken t41DSSHV45 amino acids [ΔSL47 on a PCNASHV43AAA mutant. Therefore, we substituted human PCNA Ser43-His44-Val45 residues into alanine (A) and generated PCNASHV43AAA as previously reported [SHV43AAA and PCNAΔSL47 double mutant   deletion, rather than alanine substitution. Ub-PCNA levels were further characterized with each mutant by immunoblotting with whole-cell lysate. As a result, cells expressing PCNAΔSL47, PCNASHV43AAA, and PCNASHV43AAA;ΔSL47 mutants impaired PCNA ubiquitination, without synergistic effect in PCNASHV43AAA;ΔSL47 mutant-expressing cells  . With thtination . Consistion Figs , we obse mutants . We suspng cells .SHV43AAA mutant-expressing cells showed a higher level of DNA-RNA hybrids as well as γH2AX levels compared to PCNAWT-expressing cells , we observed central loop distortion , these mutants do not show synergistic effects in PCNA ubiquitination, regulation of DNA-RNA hybrids and γH2AX levels, and putative central loop distortion . Previous intact . In our ΔSL47 mutant is defective in FEN1 and/or LIG1 interaction  gaps. It is noteworthy that cells expressing PCNA K164R mutant and RAD18 knockout cells, which are defective in PCNA ubiquitination, also exhibit OFM failure [FEN1 and LIG1 interact with PCNA through its PCNA-interacting protein-box (PIP-box) motif, and their processing and ligase activities are crucial for Okazaki fragment maturation (OFM) ,49. Our eraction , which mng cells . This su failure . Notably failure .ΔSL47 protein adducts cannot be unloaded after replication fork passage in ATAD5-deficient cells. However, at present, it is unknown whether ATAD5-RLC can unload PCNAΔSL47 protein adducts even though PCNAΔSL47 can interact with RFC subunits. We suspect that PCNAΔSL47 may show a severe phenotype in ATAD5-deficient cells, and further studies are required to confirm this.After DNA synthesis or damage bypass, PCNA and/or Ub-PCNA are unloaded by the ATAD5-RFC-like complex (RLC) –9. Therein vitro experimental results, PCNA should be loaded onto chromatin for its ubiquitination, since soluble PCNA which is not loaded onto DNA cannot be ubiquitinated [ΔSL47 mutants are loaded onto DNA to a similar extent to PCNAWT [ΔSL47-expressing cells. Not only SUMOylation, but also other PCNA post-translational modifications (PTMs) such as acetylation , methylation , and phosphorylation (Y211) and their role have been characterized [ΔSL47 on PCNA PTMs in addition to its ubiquitination, further studies are required to characterize the remained mechanisms.According to itinated ,38. Conso PCNAWT , but chro PCNAWT ,53, may ylation K0, K248, cterized –57. AlthΔSL47 loading onto DNA by RFC in vitro PCNA loading assay, while interaction between PCNAΔSL47 and RFC1 or RFC4 is intact in cells  First difference is about the presence or absence of PCNAWT monomers. In the in vitro assay, only PCNAWT or PCNAΔSL47 proteins are present respectively. Therefore, PCNAΔSL47 monomers form a trimer by themselves, without the presence of PCNAWT monomers. However, in cells, there are basal levels of endogenous PCNAWT monomers. 2) The other difference is about the usage of N-terminal deleted RFC1ΔN554 purified protein for in vitro PCNA loading assay. We purified RFC1ΔN554 protein, contains RFC1 C-terminus from 555–1148 and is reported to form the RFC complex with enhanced activity [in vitro PCNA loading reaction  that we have not tested, or the structure during docking with PCNAΔSL47, in the absence of PCNAWT monomer in vitro. Importantly, the deletion of additional N-terminal amino acids of RFC1 (RFC1ΔN604) abolishes PCNA loading while PCNA interaction is intact [ΔSL47in vitro. Further studies are required to determine how RFC opens and interacts with PCNAΔSL47in vitro.We observed that abolished PCNAlls Figs . There aactivity , for an reaction . In partin cells , we are s intact , suggest2 at 37°C. Plasmids expressing HA-tagged PCNA constructs were cloned into the pcDNA5 FRT-TO, and S-tagged PCNA constructs were cloned into the pcDNA3 mammalian expression vector. The V5(GKPIPNPLLGLDST) epitope-tagged FEN1 and LIG1 constructs were cloned into a pcDNA5 FRT-TO mammalian expression vector. Transfection of plasmid DNA was performed using X-tremeGENE™ HP  or Transporter 5  and siRNAs were transfected using RNAiMAX . Cells were analyzed 48 h after transfection.HEK293T and HeLa cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (HyClone), 100 U/mL penicillin G (Life Technologies), and 100 mg/mL streptomycin (Life Technologies) in a humidified atmosphere of 5% COTM (#A-003289-25).Synthetic duplex small interfering RNA (siRNA) was purchased from Bioneer and used as control siRNA (Bioneer #SN-1002). PCNA 3’-untranslated region (UTR)-targeting siRNA was purchased from Dharmacon2O2) (H1009) (Sigma-Aldrich), formaldehyde (P2031), and formamide (FC1014) (Biosesang). The following antibodies were used: anti-PCNA (ab18197), anti-LIG1 (ab615) (Abcam), anti-ubiquityl-PCNA (Ub-PCNA) (Lys164) (13439S), anti-histone H3 (9175), anti-CHK1 (2360), anti-pCHK1 (S345) (2348), anti-γH2AX (9718) for immunoblotting , anti-FLAG (F3165), anti-HA (H9658), anti-V5 (V8137), anti-Myc (05–724), anti-S tag (SAB2702227), α-tubulin (T9026) (Sigma-Aldrich), anti-RFC1 (NBP2-54960) , anti-FEN1 (sc-13051) (Santa Cruz), anti-RPA32 (A300-244A), anti-pRPA32 (S4/S8) (A300-245A), anti-pRPA32 (S33) (A300-246A) (Bethyl), anti-S9.6 (ENH001) (Kerafast), and anti-γH2AX (05–036) for immunostaining (Millipore).The following chemicals were used in this study: ML323  (Selleckchem), and Dynabeads M-280 streptavidin (11206D) (Invitrogen). The following reagents were used in this study: Ultrapure 20X SSC (#15557044) (Invitrogen), hydrogen peroxide solution (HWT), ΔSL47 (PCNAΔSL47), and FLAG-tagged TALE proteins were expressed in BL21 cells . RFC  were purified using the Bac-to-Bac Baculovirus expression system  as previously described [ΔN554 protein because it is sufficient for in vitro PCNA loading assays [His-tagged Human PCNA wild-type (PCNAg assays . VirusesCTCTA TAAGA TATAG TCAAG TTCAG ACGTC CATGC CCTTA TCGGA GTCTC CGGCA AATGC AATGC TCAGC ATCTC CAGCC GCTTA GCATA CTTGC CGATG TACAT GAAAG CTTTG TCTGT CAGCA GGCCG). One oligonucleotide was a 5′ biotinylated 50-mer (CGGCC TGCTG ACAGA CAAAG CTTTC ATGTA CATCG GCAAG TATGC TAAGC) annealed to the 3′ portion of 130-mer oligonucleotides, and the other was a 70-mer (GCTGA GCATT GCATT TGCCG GAGAC TCCGA TAAGG GCATG GACGT CTGAA CTTGA CTATA TCTTA TAGAG) annealed to the 5′ portion of the 130-mer. The resulting DNA substrates contained a 10 nucleotides gap. The annealed primer-template DNA was attached to streptavidin-coated magnetic beads  and unbound DNA/oligonucleotides were washed out. Bead-attached DNA was resuspended in 300 mM potassium acetate KoAc Buffer H without magnesium acetate. Each substrate had a TALE-binding sequence on the opposite side of the biotinylation. 0.5 pmol DNA substrate was pre-incubated with 50 nM TALE in 40 μL reaction mixture  at 37°C for 30 min before the PCNA loading reaction.A 130-mer DNA substrate was prepared by annealing two oligonucleotides to 130-mer oligonucleotides  and incubated for 10 min at 4°C. After centrifugation, pellets were further lysed in RIPA buffer with Benzonase nuclease for 45 min at 4°C. For immunoblot analysis, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blot was blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) supplemented with 5% skim milk for 30 min and incubated with primary antibodies overnight. After washing with TBST buffer, the blot was incubated with HRP-conjugated secondary antibodies (Enzo Life Sciences) for 30 min. After washing with TBST buffer, the signal was detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific) and an automated imaging system .Protein extraction and immunoblot analyses were performed as previously described . For who2, 10 mM KCl, 10%glycerol, 1 mM DTT, 0.1 M PMSF, phosphatase inhibitors and protease inhibitors) and incubated for 8 min at 4°C. After centrifugation, nuclei were further lysed in buffer X  with Benzonase nuclease followed by sonication and centrifugation. Anti-FLAG M2 agarose , anti-c-Myc agarose , anti-HA agarose , anti-V5 agarose , and anti-S-protein agarose  affinity beads were used for immunoprecipitation, as described previously. Agarose beads were incubated with cell lysates for 2 h at 4°C, washed three times with Buffer X at 4°C, and bound proteins were eluted with FLAG-peptide  or collected by denaturation with 2X SDS loading buffer and resolved by SDS-PAGE and immunoblotting.Isolation of a nuclear protein fraction and a Triton X-100–insoluble fraction (chromatin-bound fraction), immunoprecipitation, and immunoblot analyses were performed as previously described, with slight modifications . For nuc2O2 for 4 h, the purple formazan crystal was dissolved with DMSO, and the colored solution was quantified using a plate reader. For the CellTiter-Glo® 2.0 Assay, cells were lysed with CellTiter-Glo® reagent and the luminescence signal was read using a plate reader.The cell survival assay was performed as previously described . BrieflyThe COMET assay was performed as previously described . In brieCells plated on LabTek™ chamber slides (Thermo Fisher Scientific) were fixed and stained as previously described  with sliΔSL47, PCNASHV43AAA, and PCNASHV43AAA;ΔSL47 mutants. The constructs were confirmed by sequencing.Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit  according to the manufacturer’s instructions to generate plasmid DNA for the PCNAhttps://www.uniprot.org/, accessed on March 27, 2022). AlphaFold2 was downloaded from GitHub and installed as previously described . Protein structure prediction was performed using recommended codes [https://www.pymol.org/, accessed June 25, 2022) was used to analyze the predicted protein structure.The PCNA amino acid sequences were downloaded from the UniProt database ; **** p < 0.0001, *** p < 0.001, ** p <0.01, * p < 0.05, and n. s. = not significant.Prism 9 (GraphPad Software) was used to generate graphs and analyze the data. Statistical analyses were performed using a two-tailed unpaired Student’s S1 Fig(A-C) Predicted trimerized PCNA structure analyzed with WT-WT-WT, WT-WT-ΔSL47, WT-ΔSL47-ΔSL47, ΔSL47-ΔSL47 combinations in zebrafish. PCNA WT-WT-WT homotrimer (Gray) are structurally aligned with either WT-WT-ΔSL47 (Green) (A), WT-ΔSL47-ΔSL47 (Cyan) (B), ΔSL47-ΔSL47-ΔSL47 (Yellow) (C). Either the front or side view is displayed (Top). The central loop regions are colored as indicated (Bottom).(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.S2 Raw images(TIF)Click here for additional data file.S3 Raw images(TIF)Click here for additional data file.S4 Raw images(TIF)Click here for additional data file.S5 Raw images(TIF)Click here for additional data file.