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Although most studies describing coronavirus disease 2019 vaccine responses have focused on antibodies, there is increasing evidence that T cells play a critical role. Here the authors evaluated T-cell responses in seronegative donors before and after vaccination to define responses to the severe acute respiratory syndrome coronavirus 2 reference strain as well as to mutations in the variant strains Alpha/B.1.1.7 and Beta/B.1.351. The authors observed enhanced T-cell responses to reference and variant spike strains post-vaccination.

Hematopoietic stem cell transplantation (HSCT) is being increasingly used as a curative approach for sickle cell disease (SCD). With the risk of graft-versus-host disease (GVHD), especially in the human leukocyte antigen−mismatched donors, intense immunosuppression is required leading to an increased risk of viral infection. Post-HSCT, adoptive transfer of virus-specific T-cell (VST) therapies have not been well-studied in patients with SCD. Here, we report the outcomes of patients with SCD at a single-center who received VSTs after transplant to prevent or treat viral infections. Thirteen patients who received HSCT from human leukocyte antigen-matched (n = 9) or -mismatched (n = 4) donors for SCD were treated with a total of 15 VST products for the treatment or prophylaxis of multiple viruses (cytomegalovirus, Epstein-Barr virus, adenovirus, BK virus, human herpes virus 6 +/− human parainfluenza virus 3). Of the patients evaluated, 46.2% (n = 6)) received VSTs as treatment for viral infection. Eighty percent of patients with active viremia (n = 4/5) achieved remission of at least 1 target virus. Seven additional patients (53.8%) received VSTs prophylactically and 6 of 7 (85.7%) remained virus-free after infusion. No immediate infusion-related toxicities occurred, and severe de novo acute GVHD occurred in only 2 (15.4%) patients. Given the good safety profile, high-rate of clinical responses and sustained remissions when administered with standard antiviral treatments, the routine use of VSTs after HSCT as prophylaxis or treatment may improve the overall safety of transplant for patients with SCD.

Background aims Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized. Methods Peptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes. Results PRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes. Conclusions TAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2–specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation., Key Points • Coronavirus-specific polyfunctional T cells can be expanded from convalescent individuals for use for patients after bone marrow transplant. • SARS-CoV-2 T-cell products target structural viral proteins, including commonly recognized regions in the C terminus of membrane protein.

Patients with hematologic malignancies relapsing after allogeneic blood or marrow transplantation (BMT) have limited response to conventional salvage therapies, with an expected 1-year overall survival (OS) of

T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an "off-the-shelf" setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-β) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors' group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells' functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-β. However, a direct comparison of the effects of TGF-β-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-β results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-β-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-β-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

Adoptive T cell immunotherapy has been used to restore immunity against multiple viral targets in immunocompromised patients after bone-marrow transplantation and has been proposed as a strategy for preventing coronavirus 2019 (COVID-19) in this population. Ideally, expanded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-virus-specific T cells (CSTs) should demonstrate marked cell expansion, T cell specificity, and CD8+ T cell skewing prior to adoptive transfer. However, current methodologies using IL-4 + IL-7 result in suboptimal specificity, especially in CD8

BACKGROUND: The ReMIND trial hypothesizes that autologous T-cells specific for three tumor-associated antigens (TAA) -WT1, PRAME, and survivin- will be safe and elicit anti-tumor immunity in pediatric patients with CNS cancer. METHODS: Patients (n=25) received autologous TAA-T for newly-diagnosed DIPG (Stratum A, up to 4x107/m2) or recurrent CNS malignancies (Stratum B, up to 8x107/m2) in this dose-escalation study (NCT03652545) and were monitored for toxicity and response. RESULTS: Autologous TAAT products were successfully manufactured from 28 patients. Using IFN-γ ELISPOT assay, 10/11 evaluable products had specificity for 1-3 TAAs. 25 patients received TAA-T (6 in Stratum A and 19 in Stratum B) and completed the 45-day safety monitoring period. Twenty-four (96%) had no dose limiting toxicities (DLT), but 1 (4%) patient with DIPG experienced a DLT related to potential immune-mediated pseudoprogression. Median overall survival for patients with DIPG (Stratum A) was 14 months (range, 6-32 months). Median progression-free survival (PFS) for Stratum B patients was 8 months (range, 2-26+ months), which exceeded their preceding median duration of disease stability of 2 months (range, 1-5 months). Plasma cytokine profiles demonstrated infusion-related immune cytokine responses. CONCLUSIONS: In summary, TAAT had a favorable toxicity profile (4%) especially compared to CAR-T therapy and may elicit anti-tumor immune responses that contribute to prolonged survival. Immunobiology studies and response assessments are ongoing for both strata. Based on these encouraging preliminary results, we have added a stratum that includes prescribed lymphodepletion pre TAA-T administration at a cell dose of 8x107/m2. Further, we plan to add an additional stratum to allow direct administration of TAA-T into the CNS via an Ommaya reservoir.

Ligustilide exerts potential neuroprotective effects against various cerebral ischaemic insults and neurodegenerative disorders. However, the function and mechanisms of LIG-mediated hippocampal neural stem cells (H-NSCs) activation as well as cognitive recovery in the context of post-operative cognitive dysfunction (POCD) remain elusive and need to be explored. Mice were subjected to transient global cerebral ischaemia and reperfusion (tGCI/R) injury and treated with LIG (80 mg/kg) or vehicle for 1 month. Morris water maze test and western blot were employed to assess cognitive function. Nissl staining and immunofluorescence (IF) staining were used to detect H-NSCs proliferation and neurogenesis in hippocampus. Subsequently, primary H-NSCs were treated with LIG, and the level of H-NSCs proliferation and neuronal-differentiation was examined by IF staining for Edu and β-Tubulin III. The protein levels of ERK1/2, β-catenin, NICD, TLR4, Akt and FoxO1 were examined using western blotting. Finally, pretreatment with the ERK agonist SCH772984 was performed to observe the change in ERK expression. LIG treatment promoted H-NSCs proliferation and neurogenesis, increased the number of neurons in the hippocampal subfields, and ultimately reversed cognitive impairment in tGCI/R injury. Furthermore, LIG also promoted primary H-NSCs proliferation and neuronal-differentiation, as well as ERK1/2 phosphorylation. Pretreatment with SCH772984 effectively reversed the ability of LIG to induce ERK1/2 phosphorylation and promote H-NSCs proliferation and neuronal-differentiation. LIG can promote cognitive recovery after tGCI/R injury by activating ERK1/2 in H-NSCs to promote their proliferation and neurogenesis in the hippocampus. Therefore, LIG has potential for use in the prevention and/or treatment of POCD.

Background: Despite similar rates of infection, adults and children have markedly different morbidity and mortality related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Compared to adults, children have infrequent severe manifestations of acute infection but are uniquely at risk for the rare and often severe Multisystem Inflammatory Syndrome in Children (MIS-C) following infection. The cause of these differences in presentation may relate to differences in the host response to infection between adults and children. We thus set out to measure the SARS-CoV-2-specific T cell (CST) response in convalescent adults versus children with and without MIS-C following SARS-CoV-2 infection. Methods: CSTs were expanded from blood collected from convalescent children and adults post SARS-CoV-2 infection and evaluated by intracellular flow cytometry, surface markers, and cytokine production following stimulation with SARS-CoV-2-specific peptides. Presence of serum/plasma antibody to spike and nucleocapsid was measured using the luciferase immunoprecipitation systems (LIPS) assay. Findings: Twenty-six of 27 MIS-C patients, 7 of 8 non-MIS-C convalescent children, and 13 of 14 adults were seropositive for spike and nucleocapsid antibody. CST responses were significantly increased in adults versus children with uncomplicated SARS-CoV-2 infection (P=0.008), while children with MIS-C displayed a CST response that was similar to convalescent adults. Interpretation: Age-related differences in the magnitude of CST responses suggest differing post-infectious immunity to SARS-CoV-2 in children compared to adults. Children with MIS-C have T cell immunity to SARS-CoV-2 that is similar to convalescent adults and greater than children with uncomplicated SARS-CoV-2, despite near uniform seropositivity. Funding: This study was funded by the Board of Visitors of Children’s National Hospital, the Connor Family Foundation, the Katzen Foundation, the National Institute of Allergy and Infectious Diseases, and the National Institute of Dental and Craniofacial Research. Declaration of Interest: C.M.B. is a co-founder and on the scientific advisory boards for Catamaran Bio and Mana Therapeutics with stock and/or ownership, is on the Board of Directors for Caballeta Bio with stock options and has stock in Neximmune and Repertoire Immune Medicines. M.D.K. is on a scientific advisory panel for Enzyvant. The other authors declare that there are no conflicts of interest. Ethical Approval: Peripheral blood mononuclear cells (PBMCs) were obtained from participants recruited from Children’s National Hospital (Washington, DC) and the National Institutes of Health under informed consent approved by the Institutional Review Board of both institutions in accordance with the Declaration of Helsinki.

BACKGROUND Patients with relapsed CNS malignancies or DIPG face terrible prognoses. We hypothesized that T cells specific for 3 tumor-associated antigens (TAA), WT1, PRAME and survivin, would be safe and elicit anti-tumor immunity. METHODS Patients (n=15) received autologous tumor antigen-associated T cells (TAAT) (up to 4x107/m2) for newly diagnosed DIPG (Group A) or recurrent CNS malignancies (Group B) on a Phase I dose-escalation study (NCT03652545) and were monitored for safety and response. RESULTS/DISCUSSION 15/15 patients who received TAAT completed the 45-day safety monitoring phase with no dose-limiting toxicities. Adverse events were minimal despite multiple pretreatments in Group B. Infused cells were predominantly CD3+ T cells (median 96%; range: 87–99%), with CD4+ and CD8+ comprising 16% (range: 5–87%) and 40% (range: 4–67%) respectively. Specificity for 1–3 TAAs was demonstrated in 13/15 TAAT by a-IFN-γ ELISPOT. Plasma cytokine and proteomic analyses are ongoing but have demonstrated dynamic post-infusion immune cytokine and protein responses. Increases in the inflammatory and immune-stimulatory cytokines IL-1b, IL-6, IL-2 and IL-7 were observed post-infusion in most patients evaluated. Infusion-related increases in regulatory cytokines IL-10 and IL-13 were also observed in 4/7 patients. These results are consistent with an infusion-mediated immune response in vivo. Of 9 patients who have been tested thus far, 29/92 plasma proteins showed significant differences between dose levels 1 and 2, including increased IL-7 (p < 0.0004) and CD40L (p < 0.046) and reduced IL-4 (p < 0.0004). T cell receptor sequencing data on in vivo TAAT persistence is pending. In summary, TAAT have thus far been safe and elicit immune responses in vivo. Clinical and immunologic response assessments are ongoing.

Background Patients with relapsed or refractory Wilms tumor (WT) have poor prognoses with limited treatment options.1–3 Immunotherapy offers a promising alternative for targeted therapy but has been limited by immune evasion tactics.4–6 Adoptive cell therapy with patient-derived tumor-associated antigen-specific T cells (TAA-T) targeting 3 antigens (WT1, PRAME, and survivin) has the potential to overcome antigen loss. The objective of this phase I clinical trial is to determine the safety of administering TAA-T to patients with high-risk, relapsed/refractory solid tumors. Secondary objectives include determination of clinical efficacy and immunobiology following infusion. Methods T cells expanded from patient peripheral blood are stimulated weekly with antigen presenting cells expressing an overlapping peptide library spanning the tumor antigens WT1, PRAME, and survivin. Following release testing, patients are infused with TAA-T on a dose escalation study, ranging from a dose of 1 x 107/m2 (dose level 1) to 4 x 107/m2 (dose level 3). Clinical and immunobiological studies performed post-infusion monitor for adverse effects and assess immune and disease responses. Results Therapy with TAA-T was shown to be safe and well-tolerated in patients with high-risk solid tumors on this dose-escalation study.7 A total of 18 patients have been infused, with WT as the predominant diagnosis, accounting for 10 patients. We elucidated a dose-response relationship, with a prolonged median time to progression for patients treated on dose level 3 (recommended dose level [RDL]) compared to those on dose level 1 and 2 combined (5.2 vs 2.8 months, respectively) (figure 1). Patients demonstrated prolonged progression-free survival (PFS) compared to therapy received just prior to TAA-T (figure 2). Best response observed was stable disease. A majority of patients demonstrated improved anti-tumor immunity as evidenced by antigen spreading (figure 3). Conclusions In long-term follow up, the 1-year progression-free survival (PFS) remains improved for patients treated at the recommended dose level compared to the PFS observed with therapy received immediately prior to TAA-T (29% vs 15%, respectively). Three patients are long-term (28–38 months) survivors without disease progression or further therapy. This unique immunotherapeutic has been well-tolerated without life-threatening cytokine release syndrome. To enhance TAA-T activity further in vivo, we will evaluate the safety of prescribed lymphodepletion prior to TAA-T infusion and assess anti-tumor immunity. We expect that lymphodepletion will enhance T cell persistence and expansion and recruit endogenous immune response with altered kinetics. Ethics Approval The study was approved by the Children’s National Hospital Institutional Review Board, approval number NCT02789228. References Dome JS, Graf N, Geller JI, Fernandez CV, Mullen EA, Spreafico F, Van den Heuvel-Eibrink M, Pritchard-Jones K. Advances in Wilms Tumor Treatment and Biology: Progress Through International Collaboration. J Clin Oncol 2015;33(27):2999–3007. Mullen EA, Chi YY, Hibbitts E, Anderson JR, Steacy KJ, Geller JI, Green DM, Khanna G, Malogolowkin MH, Grundy PE and others. Impact of Surveillance Imaging Modality on Survival After Recurrence in Patients With Favorable-Histology Wilms Tumor: A Report From the Children’s Oncology Group. J Clin Oncol 2018:JCO1800076. Malogolowkin MH, Hemmer MT, Le-Rademacher J, Hale GA, Mehta PA, Smith AR, Kitko C, Abraham A, Abdel-Azim H, Dandoy C and others. Outcomes following autologous hematopoietic stem cell transplant for patients with relapsed Wilms’ tumor: a CIBMTR retrospective analysis. Bone Marrow Transplant 2017;52(11):1549–1555. Heczey A, Louis CU. Advances in chimeric antigen receptor immunotherapy for neuroblastoma. Discov Med 2013;16(90):287–94. Park JR, Digiusto DL, Slovak M, Wright C, Naranjo A, Wagner J, Meechoovet HB, Bautista C, Chang WC, Ostberg JR and others. Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Mol Ther 2007;15(4):825–33. Lamers CH, Sleijfer S, van Steenbergen S, van Elzakker P, van Krimpen B, Groot C, Vulto A, den Bakker M, Oosterwijk E, Debets R and others. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Ther 2013;21(4):904–12. Hont AB, Cruz CR, Ulrey R, O’Brien B, Stanojevic M, Datar A, Albihani S, Saunders D, Hanajiri R, Panchapakesan K and others. Immunotherapy of relapsed and refractory solid tumors with ex vivo expanded multiantigen-associated specific cytotoxic T lymphocytes: A Phase I Study. J Clin Oncol 2019:JCO1900177.

Background & Aim: T-cell responses to SARS-CoV-2 have been described in convalescent patients;and adoptive immunotherapy in immunocompromised individuals may be a viable therapeutic strategy to treat or prevent COVID-19 related morbidity and mortality. However, sparse data exists regarding the ideal cytokines to use during viral specific T-cell (VST) expansion in general, and coronavirus specific T-cells (CST) in particular, to maximize specificity and cell count. Here, we demonstrate that polyfunctional coronavirus-specific T cells (CSTs) can be generated rapidly from convalescent donors, and that CSTs can be optimized by varying cytokine expansion cocktails. Methods, Results & Conclusion: PBMCs from SARS-CoV-2 convalescent donors were stimulated with overlapping peptide pools encompassing membrane, spike, envelope and nucleocapsid proteins, and expanded in 96-well plates for 10 days. Cells were expanded either with IL4 (400IU/mL) +IL7 (10ng/mL), IL15 (5ng/mL), IL15+4, IL15+6 (100ng/mL), or IL15+7. After expansion, T-cell phenotype and functionality were assessed using ELISpot and intracellular flow cytometry. Cytokine combinations with the best performance on micro-expansion were retested using GMP-applicable methods. [Figure presented] [Figure presented] CST expansion was maximized using IL15+7 (total CD3+8.2E4±2.6E4 cells) and IL 4+7 (total CD3+ 8.4E4±2.2E4 cells) as compared with IL15 (total CD3+6.1E4±1.0E4 cells), IL4+15 (total CD3+6.6E4 cells±1.4E4 cells), or IL15+6 (total CD3+ 6.1E4±1.7E4 cells). CD4+ specificity was maximized using IL4+7, IL15+6, and IL15+7 (Fig. 1), while CD8+ specificity was maximized using IL15+7, IL15+6, and IL15 (Fig.e 2). T-cell phenotype was similar across cytokine conditions (median 6.9±0.2% naive, 8.3±0.8% central memory, 79±0.9% effector memory, 4.5±0.2% terminal effector). NK cell expansion was increased in the setting of IL15 and its combinations (median 10.4±12% CD3-/CD56+ versus 1.0±1% without IL-15 p

Hippocampal neural stem cells (H-NSCs) self-renewal and neurogenesis decrease with aging, but the intrinsic mechanism is unclear. In the current study, we detected the expression level of 8 conserved long intergenic noncoding RNAs (lincRNAs) in H-NSCs during aging, and investigated the function and mechanism of lincRNAs in regulating of H-NSCs. We found the proliferation and neuronal-differentiation capacities of H-NSCs reduced with aging and that this effect was accompanied by an increase in linc-FOXD3. Linc-FOXD3 knockdown improved H-NSCs proliferation and neuronal-differentiation capacities. Further mechanistic studies revealed that the effect of linc-FOXD3 knockdown on H-NSCs phenotypes was partially mediated by the up-regulation of Wnt/β-catenin pathway. Thus, our study provides evidence that linc-FOXD3 could be a promising therapeutic target for the recovery of H-NSCs function during aging.

Background: Patients with hematologic malignancies relapsing after allogeneic blood or marrow transplantation (BMT) have limited response to conventional salvage therapies with an expected 1-year overall survival (OS) of Methods: Lymphocytes obtained from the BMT donor were manufactured to target TAAs; WT1, PRAME and survivin, which are over-expressed and immunogenic in most hematologic malignancies. Patients received TAA-T infusions at doses of 0.5-4x10 7/m 2. Patients were evaluated for acute infusion-related toxicity, cytokine release syndrome (CRS), neurotoxicity, GVHD and monitored for clinical response post-infusion. T-cell receptor sequencing was conducted on the TAA-T product and the recipients' peripheral blood prior to and post-infusion to monitor persistence and expansion of the product. Results: Twenty-three BMT recipients with relapsed/refractory (n=11) and/or high-risk (n=12) acute myeloid leukemia (n=20) and acute lymphoblastic leukemia (n=3) were infused post-transplant. No patient developed CRS or neurotoxicity, and only one patient developed grade III GVHD. Of the patients who relapsed post-BMT and received bridging therapy, the majority (n=9/11) achieved complete hematologic remission before receiving TAA-T. Relapsed patients exhibited a 1-year OS of 36% and 1-year leukemia-free survival of 27.3% post-TAA-T (Figure 1A and B). The poorest prognosis patients (relapsed less than 6 months after transplant) exhibited a 1-year OS of 42.8% post-relapse (n=7) (Figure 1C). Median survival was not reached for high-risk patients who received pre-emptive TAA-T post-transplant (n=12) with eight of the nine (88.9%) evaluable patients at 1-year post-infusion remaining alive (Figure 1B). Of those, five (62.5%) were alive and in continued remission at the time of submission. Evaluation of unique T-cell receptor clonotypes (present in the product but not the recipient prior to infusion) demonstrated expansion and persistence in patients up to 1-year post-infusion (Figure 1D). Conclusions: Although as a Phase-I study, concomitant anti-leukemic therapy was allowed, TAA-T cell therapy was shown to be safe and well-tolerated with sustained remissions observed in high-risk and relapsed patients. Moreover, adoptively transferred TAA-T expanded in vivo as detected by T-cell receptor V-beta (TCRVb) sequencing persisted up to at least 1-year post-infusion (Figure 1D). (ClinicalTrials.gov numbers, NCT002203902) Figure 1 Figure 1. Disclosures Cruz: Mana Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Catamaran Bio: Consultancy, Patents & Royalties, Research Funding. Hanley: Mana Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellevolve: Consultancy; Maxcyte: Membership on an entity's Board of Directors or advisory committees. Bollard: SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Catamaran Bio: Membership on an entity's Board of Directors or advisory committees; Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees; ROCHE: Consultancy, Honoraria; Repertoire Immune Medicines: Current equity holder in publicly-traded company; Neximmune: Current equity holder in publicly-traded company; Mana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Background Patients with relapsed CNS malignancies or DIPG face terrible prognoses. We hypothesized that T cells specific for 3 tumor-associated antigens (TAA), WT1, PRAME and survivin, would be safe and elicit anti-tumor immunity. Methods Patients (n=18) received autologous tumor antigen-associated T cells (TAAT) (up to 8x107/m2) for newly diagnosed DIPG (Group A) or recurrent CNS malignancies (Group B) on a Phase I dose-escalation study (NCT03652545) and were monitored for safety and response. Results/Discussion 16/18 patients who received TAAT completed the 45-day safety monitoring phase with no dose-limiting toxicities. Adverse events were minimal despite multiple pretreatments in Group B. Infused cells were predominantly CD3+ T cells (median 96%; range: 87–99%), with CD4+ and CD8+ comprising 16% (range: 5–87%) and 40% (range: 4–67%) respectively. Specificity for 1–3 TAAs was demonstrated in 11/18 TAAT by a-IFN-γ ELISPOT. Dose escalation is complete, and clinical and immunologic response assessments are ongoing. Plasma cytokine and proteomic analyses demonstrated dynamic post-infusion immune cytokine and protein responses. Consistent with an infusion-mediated immune response all patients in Grp A showed increased T cell effector, inflammatory and immune-stimulatory cytokines IFN-γ, TNF-α, IL-2, IL-5, IL-7, IL-1β, IL-6, IL-8, IL-12p70, IL-17A and GM-CSF at Weeks 1 and 2 post-infusion (n = 6). Of 9 patients who have been tested, 29/92 plasma proteins showed significant differences between dose levels 1 and 2, including increased IL-7 (p

Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating the biological functions and underlying molecular mechanisms of glioma. Here, we investigated the expression level and biological function of linc-OIP5 in glioma. In the current study, we used quantitative real-time polymerase chain reaction (qRT-PCR) to determine the expression of linc-OIP5 in glioma tissues and in adjacent normal tissues. Level of linc-OIP5 was up-regulated in glioma tissues and significantly correlated with the advanced tumor stage (III/IV). Subsequently, the efficacy of knockdown of linc-OIP5 by linc-OIP5-small interfering RNA (siRNA) was evaluated in vitro, and we found that knockdown of linc-OIP5 can inhibit glioma cells proliferation, migration in vitro and tumor formation in vivo. Further mechanistic studies revealed the effect of linc-OIP5 knockdown on glioma cell phenotype at least partially through down-regulation of YAP and inhibition of Notch signaling pathway activity. Thus, our study provides evidence that linc-OIP5 is a potential therapeutic target and novel molecular biomarker for glioma.

Traumatic brain injury (TBI) is one of the leading causes of death and disability in children, and progressive hemorrhagic injury (PHI) post TBI is associated with poor outcomes. Therefore, the objective of this study was to develop and validate a prognostic model that uses the information available at admission to determine the likelihood of PHI occurrence after TBI in children. The identified demographic data, cause of injury, clinical predictors on admission, computed tomography scan characteristics, and routine laboratory parameters were collected and used to develop a PHI prognostic model with logistic regression analysis, and the prediction model was validated in 68 children. Eight independent prognostic factors were identified: lower Glasgow coma scale score (3 ~ 8) (6 points), intra-axial bleeding/brain contusion (4 points), midline shift ≥5 mm (9 points), platelets 14 s (6 points), international normalized ratio >1.25 (7 points), D-dimer ≥5 mg/L (14 points), and glucose ≧10 mmol/L (11 points). We calculated risk scores for each child and defined three risk groups: low risk (0–16 points), intermediate risk (17–36 points), and high risk (37–68 points). In the development cohort, the PHI rates after TBI for the low-, intermediate-, and high-risk groups were 10.1, 47.9, and 84.2%, respectively. In the validation cohort, the corresponding PHI rates were 10.9, 47.5, and 85.4%, respectively. The C-statistic for the point system was 0.873 (p = 0.586 by the Hosmer-Lemeshow test) in the development cohort and 0.877 (p = 0.524 by the Hosmer-Lemeshow test) in the validation cohort. Conclusion: Using admission predictors, we developed a relatively simple risk score that accurately predicted the risk of PHI after TBI in children.

Angiogenesis is a key event in the progression of gliomas. Exosomes, as signaling extracellular organelles, modulate the tumor microenvironment and promote angiogenesis and tumor progression. We previously demonstrated that long intergenic non-coding RNA CCAT2 (linc-CCAT2) was overexpressed in glioma tissues and functioned to promote glioma progression. Therefore, this study aimed to explore an underlying mechanism of glioma cell-affected angiogenesis. First, qRT-PCR was used to determine the expression level of linc-CCAT2 in 4 glioma cell lines and 293T cells, and the results revealed that the U87-MG cells exhibited the highest expression level. Subsequently, the pro-angiogenesis function of exosomes that were derived from negative control shRNA-treated U87-MG cells (ncU87-Exo) and linc-CCAT2 shRNA-treated U87-MG cells (shU87-Exo) was evaluated in vitro and in vivo. We found that ncU87-Exo, which was enriched in linc-CCAT2, could be taken up by HUVECs. ncU87-Exo improved the linc-CCAT2 expression level in HUVECs and more strongly promoted HUVEC migration, proliferation, tubular-like structure formation in vitro and arteriole formation in vivo as well as inhibited HUVEC apoptosis induced by hypoxia. Further mechanistic studies revealed that ncU87-Exo could upregulate VEGFA and TGFβ expression in HUVECs as well as promote Bcl-2 expression and inhibit Bax and caspase-3 expression. Finally, gain-/loss-of-function studies revealed that the overexpression of linc-CCAT2 in HUVECs activated VEGFA and TGFβ, promoted angiogenesis, promoted Bcl-2 expression and inhibited Bax and caspase-3 expression, thus decreasing apoptosis. Downregulation of linc-CCAT2 revealed the opposite effect. Thus, our results revealed a new exosome‑mediated mechanism by which glioma cells could promote angiogenesis through the transfer of linc-CCAT2 by exosomes to endothelial cells. Moreover, we suggest that exosomes and linc-CCAT2 are putative therapeutic targets in glioma.

Background Adoptive immunotherapy using banked virus-specific T-lymphocytes (VSTs) derived from partially HLA-matched donors has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT), although broad availability of this therapy is currently limited, and best methods for donor-recipient matching remain unclear. Objective To determine the feasibility and efficacy of third-party VSTs for the treatment of immunocompromised pediatric patients with refractory viral infections in a multicenter phase I/II consortium study. Design/Methods There are two arms to this study. Arm A are pediatric subjects who had infections with CMV, EBV, and/or adenovirus after hematopoietic stem cell transplant (HSCT) and had failed antiviral therapy, or who had toxicities related to anti-viral drug-related. Arm B enrolls patients with primary immunodeficiency disorders with refractory viral infection prior to HSCT. A VST bank containing products generated from 40 donors at Children's National was utilized, and antiviral HLA mapping was applied to identify the most suitable product for each patient referred. Patients received a dose of 2 × 10^7 VSTs/m2, with optional additional doses given 28 days after the initial VST infusion. Viral PCRs were monitored for efficacy, and VST persistence and expansion in vivo was measured via IFN-g ELISpot and flow cytometry. Results Twenty-three patients were enrolled from 11 study sites to date. Twenty-two were treated in Arm A (after HSCT), at a median time of 95 days post-HSCT (range 16-476). Fourteen patients were treated for adenovirus and 9 for CMV. Patients received a median of 1 infusion (range 1-3). Infused VSTs were matched at a median of 3/10 HLA alleles (range 2-5), and 22 had confirmation of antiviral activity through at least one shared HLA allele. Of 18 evaluable patients, 14 had antiviral responses at 1-month post-infusion (4/5 with CMV, and 10/13 with adenovirus, Figure 1), including 7 who had complete resolution of viremia. No GVHD was observed after infusion, and 17 of 23 (74%) are virus free up to 15 months post-infusion. Virus-specific T-cell activity was higher by day 45 (mean 177 IFN-g SPW) post-infusion than at baseline (mean 13 IFN-g SPW, p=0.045). VSTs remained detectable for up to 3 months via flow cytometry staining of discordant HLA alleles (Figure 2). Conclusions Third-party VSTs utilized from a single bank can be distributed and administered in a multicenter consortium setting, and are safe and feasible for the treatment of refractory viral infections in pediatric patients. Preliminary efficacy data shows high rates of response and long-term survival. Studies of targeting strategies and characterization of T-cell epitopes are ongoing.

PURPOSE Tumor-associated antigen cytotoxic T cells (TAA-Ts) represent a new, potentially effective and nontoxic therapeutic approach for patients with relapsed or refractory solid tumors. In this first-in-human trial, we investigated the safety of administering TAA-Ts that target Wilms tumor gene 1, preferentially expressed antigen of melanoma, and survivin to patients with relapsed/refractory solid tumors. MATERIALS AND METHODS TAA-T products were generated from autologous peripheral blood and infused over three dose levels: 1, 2, and 4 × 107 cells/m2. Patients were eligible for up to eight infusions administered 4 to 7 weeks apart. We assessed dose limiting toxicity during the first 45 days after infusion. Disease response was determined within the context of a phase I trial. RESULTS There were no dose-limiting toxicities. Of 15 evaluable patients, 11 (73%) with stable disease or better at day 45 postinfusion were defined as responders. Six responders remain without progression at a median of 13.9 months (range, 4.1 to 19.9 months) after initial TAA-Ts. Patients who were treated at the highest dose level showed the best clinical outcomes, with a 6-month progression-free survival of 73% after TAA-T infusion compared with a 38% 6-month progression-free survival with prior therapy. Antigen spreading and a reduction in circulating tumor-associated antigens using digital droplet polymerase chain reaction was observed in patients after TAA-T infusion. CONCLUSION TAA-Ts safely induced disease stabilization, prolonged time to progression, and were associated with antigen spreading and a reduction in circulating tumor-associated antigen DNA levels in patients with relapsed/refractory solid tumors without lymphodepleting chemotherapy before infusion. TAA-Ts are a promising new treatment approach for patients with solid tumors.

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (P = 0·0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT.

BACKGROUND Patients with relapsed CNS malignancies or DIPG face terrible prognoses. We hypothesized that T cells specific for 3 tumor-associated antigens (TAA), WT1, PRAME and survivin, would be safe and elicit anti-tumor immunity. METHODS Patients (n=9) have received autologous tumor antigen-associated T cells (TAAT) (up to 4x107/m2) for newly diagnosed DIPG (Group A) or recurrent CNS malignancies (Group B) on a Phase I dose-escalation study (NCT03652545) and were monitored for safety and response. RESULTS/ DISCUSSION 9/9 patients who received TAAT completed the 45-day safety monitoring phase with no dose-limiting toxicities. Infused cells were predominantly CD3+ T cells (median 96%; range: 87–99%), with CD4+ and CD8+ comprising 16% (range: 5–87%) and 40% (range: 4–67%) of the CD3+ cells, respectively. TAAT with specificity for 1–3 TAAs, at varying frequencies, was demonstrated in 8/9 TAAT by anti-IFN-γ ELISPOT. Plasma cytokine profiles demonstrated infusion-related immune cytokine responses. In summary, TAAT are safe and may elicit anti-tumor responses in vivo. To confirm TAAT-driven effects, we are evaluating plasma proteomic profiles for immune-response signatures and assessing unique T cell receptor rearrangements of infused TAAT. Response assessment and dose escalation are ongoing.

A stellate ganglion block (SGB) is a clinical sympathetic block which can inhibit the body systemic inflammatory response. However, whether and how SGB can attenuate the sepsis-induced acute lung injury remains unclear. Here, we evaluated the effect of SGB on sepsis-induced acute lung injury in rats. Ninety healthy Sprague Dawley (SD) male rats were divided into three groups: the sham operation group (S group), sepsis group (Sep group), and SGB group. The sepsis model rats were produced by cecum ligation and puncture (CLP), and blood samples were taken from the abdominal aorta of the rats at different time points for evaluating the concentration of TNF-α, IL-6, and IL-10 by enzyme-linked immunosorbent assay (ELISA). The rats were sacrificed, and lungs were collected to measure the wet/dry (W/D) lung tissue weight ratio, score the lung tissue pathological damage by microscopic examination, determine the myeloperoxidase (MPO) activity by spectrophotometry, and measure nuclear factor-kappa B (NF-κB) p65 expression by Western blot. The concentration of serum TNF-α, IL-6, and IL-10, lung tissue W/D ratio, pathological injury score, MPO activity, and expression of NF-κB p65 were higher in the Sep group compared with the S group at T1-4. Furthermore, the concentration of serum TNF-α and IL-6, lung tissue W/D ratio, pathological damage score, MPO activity, and expression of NF-κB p65 were reduced and the concentration of IL-10 was increased in the SGB group compared with the Sep group at T1-4. The successful sepsis model rats were induced by CLP, and SGB attenuated the sepsis-induced acute lung injury in rats.

Viral infections can be life threatening in patients with severe combined immunodeficiency (SCID) and other forms of profound primary immunodeficiency disorders both before and after hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with virus-specific T cells (VSTs) has been utilized in many patients in the setting of HSCT, but has very rarely been attempted for treatment of viral infections before HSCT. Here we describe the use of VSTs in an infant with RAG1 SCID who had developed disseminated adenovirus which failed to improve on cidofovir. Adenovirus cleared following 2 doses of VSTs and marrow infusion from a matched unrelated donor, without incidence of graft versus host disease. T cell receptor-b sequencing demonstrated expansion of adenovirus-specific T cell fraction of the VSTs, suggesting that infusion facilitated viral clearance. This report suggests that VSTs are likely safe in the pre-HSCT period, and may be a useful bridge therapy for infants with SCID and persistent viral infections.

[This corrects the article DOI: 10.1371/journal.pone.0186603.].

Background & Aim Viral infections are a significant cause of morbidity and mortality in patients with primary immunodeficiency disorders and following hematopoietic stem cell transplantation. Adoptive immunotherapy using virus specific T-cells (VSTs) has been shown to prevent and treat viral infections in immunocompromised hosts. Human Parainfluenza Virus-3 (HPIV3) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies and has not previously been used as a target for T cell therapeutics. The primary aim was to determine whether donor derived hexaviral specific T-cells are effective in preventing and treating CMV, EBV, AdV, BK virus, HHV-6, and HPIV3. Methods, Results & Conclusion Patients and Methods This was a “first in man” study where we studied the antiviral effect in 8 patients who received hexa-valent VSTs after stem cell transplant on a Phase I trial. Assessment of virus-specific immunity was measured by IFN-gamma ELIspot. Viral loads for the primary targeted viruses were measured at specific time points post VST infusion. Results Three patients were treated for active CMV and had resolution of viremia. Two patients treated for active BK virus had complete resolution of symptoms and viremia, while one had resolution of hemorrhagic cystitis but fluctuating viral loads in the blood and urine. Two patients were treated prophylactically. One patient did not develop any infections, while the other developed EBV viremia requiring rituximab. Two patients received VSTs under expanded access for emergency treatment – 1 patient was treated for disseminated adenoviremia and the second patient was treated for HPIV3 pneumonia. These critically ill patients demonstrated partial clinical improvements, but both died (one of progressive adenovirus, and the other of gram negative sepsis). VST persistence was likely hindered by concomitant steroid use which resulted in incomplete antiviral responses. ELISpot showed evidence of antiviral T-cell activity in 3 of 4 evaluable patients by 3 months post-infusion, with in vivoVST expansion detectable in 2 patients. Conclusions Preliminary results show that hexaviral specific VSTs are safe and may be effective in preventing and treating multiple viral infections. Further studies are warranted to determine if VSTs are effective against active HPIV3 infections.

Background Adoptive immunotherapy using virus-specific T-lymphocytes (VSTs) have been successful in treating viral infections in immunocompromised patients, and use of VSTs from healthy third-party donors have improved the accessibility and timeliness of this therapy. However, best methods of donor/recipient matching of HLA-mismatched VST products remain unclear. Objective To determine the factors influencing the chances of antiviral responses following VST therapy between HLA-mismatched donor/recipient pairs. Design/Methods Patients who received HLA-mismatched VST infusions for active infections with CMV, Epstein-Barr virus, or adenovirus were analyzed for response. Antiviral response was defined as >1 log decrease in viral load within 3 months of VST infusion. Degree of high and low resolution HLA match was determined, and presence of confirmed antiviral HLA restrictions in the utilized VST products was recorded. Unpaired, 2-tailed T-tests were performed for group comparisons. Results Twenty one patients received 30 VST infusions with 27 T-cell products (20 from third-party donors, and 7 from mismatched related donors). Eleven patients were treated for CMV, 8 for adenovirus, and 2 for EBV infection. All patients had persistent viral infections in spite of standard antiviral therapy prior to study enrollment. Seven patients were admitted to intensive care units due to severe viral disease and received VSTs under expanded access for emergency treatment. Overall, of this poor prognosis patient cohort, 12 achieved antiviral responses within 3 months of infusion (2 of 7 patients who received VST derived from MMRD, and 10 of 20 patients who received third-party VSTs). Of 25 evaluable infusions, chances of antiviral response following VST infusion were greater when utilizing a VST product with a confirmed antiviral HLA restriction that is shared at high resolution with the recipient (p=0.02). There was no difference in overall degree of HLA match between responders and non-responders (p=0.83). Conclusions VST matching based on sharing of high resolution HLA alleles that mediate target antiviral restrictions is more successful than overall degree of HLA match. Further testing of this methodology for VST therapy is ongoing in a current phase II trial (PBMTC SUP1701).

Background Viral infections are a significant cause of morbidity and mortality in patients awaiting immune reconstitution post-transplant. Adoptive immunotherapy using virus specific T-cells (VSTs) has been shown to prevent and treat viral infections in immunocompromised hosts. Human Parainfluenza Virus-3 (HPIV3) is a common cause of respiratory illness in these patients and has no approved antiviral therapies. Objective The primary aim was to determine whether donor derived hexaviral specific T-cells are effective in preventing and treating CMV, EBV, AdV, BK virus, HHV-6, and HPIV3. Patients and Methods We studied 7 patients who received hexaviral VSTs after stem cell transplant on a Phase I trial. Virus-specific immunity was measured by IFN-g ELIspot. Viral loads for the targeted viruses were measured at specific time points post VST infusion. Results Three patients were treated for active CMV with resolution of viremia. Two patients treated for BK virus had complete resolution of symptoms and viremia, while one had resolution of hemorrhagic cystitis but intermittent viremia and viruria. Two patients were treated prophylactically. One patient remained virus-free, while the other developed EBV viremia requiring a dose of rituximab. Two patients received VSTs under expanded access for emergency treatment – one for disseminated adenoviremia, and the second for HPIV3 pneumonia. These critically ill patients had partial clinical improvements, but VST persistence was likely hindered by steroid use which resulted in incomplete antiviral responses. ELISpot showed evidence of antiviral T-cell activity in 3 of 4 evaluable patients by 3 months post-infusion. Conclusions Preliminary results show that hexaviral specific VSTs are safe and may be an effective therapy against multiple viral infections. Further studies are warranted to determine if VSTs are effective against active HPIV3 infections.