T2 Cells Research Articles

Abstract LB442: Novel antibodies against a KK-LC-1-derived peptide presented on HLA-A*01 on tumor cells

Abstract The prognostic benefit of immune checkpoint inhibitors (CPIs) is mainly restricted to specific cancers, such as melanoma and lung adenocarcinoma. For patients with cancers which are noninflamed (“cold”), or CPI-resistant, therapeutic options are currently limited. Recently, the US FDA approved the first bispecific molecule targeting a tumor peptide MHC. This highlights the potential of targeting MHC-presented tumor antigens to empower T cells to specifically recognize and eliminate solid tumors. While there is a growing number of agents targeting pMHCs, most of them are restricted to HLA-A*02. There is an urgent need to identify effective pMHC targeted therapies for solid tumors, especially on other alleles to increase the proportion of eligible patients. Kita-Kyushu lung cancer antigen-1 (KK-LC-1/CT83) peptide presented on HLA-A*01 is a promising tumor target as its expression is restricted to tumors and it is broadly expressed in many cancers including gastric, lung, breast, and cervical cancer. However, targeting the KK-LC-1/HLA-A*01 complex is challenging due to the high levels of similar off-target peptides presented in healthy tissues. Through in silico analysis we identified peptides within the human genome that share a high sequence identity to the target. These peptides were found in a database containing >1 million mass spectrometry (MS)-identified peptides presented by healthy tissues, cancer cell lines, and tumor tissue samples. A peptide derived from 6-pyruvoyltetrahydropterin synthase (PTS) was found to be abundantly presented in healthy tissues. MS confirmed that this peptide occurs at extremely high natural levels (620 - 3500 copies per cell) on HLA-A*01 cell lines and healthy tissue. Here, we describe a set of pMHC T-cell engager (TCE) antibodies with high specificity towards HLA-A*01 restricted KK-LC-1 epitope. ScFv phage display libraries were rigorously screened for binding to the pMHC target and counter-screened against predicted off-target peptides including PTS. Specific scFv hits were reformatted into TCEs using an anti-CD3 Fab effector arm. The TCEs showed efficient killing of KK-LC-1/HLA-A*01-positive human cell lines NCI-H1703 and EKVX in co-cultures with human PBMCs, while KK-LC-1-negative/HLA-A*01-positive cell lines SK-MEL-30 and PC-3 (known to present the risk peptides on the surface) were minimally affected. TCE-dependent T cell activation measured by IFNγ was significantly higher in presence of TAP-deficient, (empty) HLA-A*01-expressing T2 cells pulsed with the target peptide compared to the risk peptides. Alanine scans of isolated binders confirmed that at least 6 amino acids of the KK-LC-1 peptide are required for binding. Overall, the data show that these antibodies have promising anti-tumor activity and specificity for KK-LC-1/HLA-A*01 positive tumors. Future activities will focus on lead optimization for therapeutic development. Citation Format: Fabian Scheifele, Stephanie Jungmichel, Nagjie Alijaj, Athanasia Dasargyri, Romina Doerig, Philip Knobel, Hannes Merten, Philipp Richle, Thomas Schleier, Anna Sobieraj, Alessio Vantellini, Tim Fugmann, Rom Leidner, Swethajit Biswas, Leonardo Borras. Novel antibodies against a KK-LC-1-derived peptide presented on HLA-A*01 on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB442.

Early Serum Markers for Immune Checkpoint Inhibitor Induced Hypophysitis in Melanoma Patients.

Immune checkpoint inhibitors (ICIs) have shown promising anti-tumor activities and are widely used for the treatment of advanced cancers. However, they may lead to immune-related adverse events (irAEs) and some of them, such as hypophysitis, can be life-threatening. Here, early diagnosis is critical. We retrospectively analyzed 40 melanoma patients who developed hypophysitis during ICI treatment with either ipilimumab and/or anti-PD1 therapy and compared them to 40 control patients who did not develop hypophysitis during the ICI treatment, matched for age, gender, type of immunotherapy, and stage. Clinical data and blood values such as LDH, CRP, TSH, T3, T4, and absolute immune cell counts were retrieved from the medical records. Patient characteristics, laboratory values, progression-free survival, and overall survival were compared between the two groups. Patients with ir-hypophysitis had a median age of 59 years, and most of them were male. Clinically, frequent symptoms were fatigue, headache, dizziness, and gastrointestinal symptoms such as nausea or abdominal pain. The onset of ir-hypophysitis differed much between ipilimumab- (median 8 weeks) and anti-PD1 (median 40 weeks)-induced hypophysitis (p < 0.001). At baseline, besides a slightly increased CRP level (p = 0.06), no differences were observed in patients who later developed hypophysitis compared to the control. After treatment started, hypophysitis patients showed a constant and significant decline in T4 levels from the start of therapy until diagnosis (p < 0.05), independent of the ICI treatment regime. However, a decline in T3 and TSH was only noted in patients with ipilimumab-induced ir-hypophysitis. Furthermore, serum sodium levels declined rapidly at the diagnosis of hypophysitis (p < 0.001). In addition, there was a constant increase in the absolute counts of eosinophils and lymphocytes from baseline in hypophysitis patients (p < 0.05). Ir-hypophysitis reveals different clinical pictures and onset times depending on the ICI regime used. Whereas a drop in T4 levels was indicative of developing hypophysitis independent of the ICI regime, TSH levels only declined in patients under ipilimumab-based ICI regimes. To best monitor our patients, it is important to recognize these differences.

Analysis and Development of Antigen-specific T Cells Derived from Peripheral Blood Mononuclear Cells of Healthy Donors.

Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.

Abstract 3194: Discovery of T cell receptor mimic antibody against MAGE-A4 with single B-cell cloning platform

Abstract Antibody therapeutics has been proven clinically against various cancer types and have achieved great success. Majority of the therapeutic antibodies target cell surface or extracellular proteins. Most of the tumor-specific antigens that control cell growth, proliferation, and death are intracellular, and traditional antibody therapies fail to recognize intracellular antigen targets. Such tumor specific intracellular targets consist of overexpressed self antigens, oncofetal protein, cancer testis antigens, viral protein and neo-antigens of mutant proteins. Recently, an emerging approach to target these neo-epitopes has been developed called T-cell receptor-like/TCR-mimic antibodies as they recognize similar epitopes to those of T cell receptors which target specific peptide-MHC complexes(pMHC). TCR mimic antibodies open a new avenue for targeting intracellular antigens yet there are lots of challenges for generating highly selective and potent TCR mimic antibodies. MAGE-A4, melanoma-associated antigen A4, is a member of the MAGE protein family of cancer-testis antigen (CTA). In healthy adult, MAGE-A4 expression is restricted to immune-privileged sites. But MAGE-A4 is widely expressed on many cancers such as lung cancer, head and neck squamous cell cancer, synovial sarcoma(SS), ovarian cancer, urothelial cancer and melanoma. The decapeptide GVYDGREHTV(amino acids 230-239) derived from MAGE-A4, can be presented by HLA-A*02:01 molecule, elicits specific cytotoxicity T cell response. Afami-cel, a genetically modified autologous T cell therapy against this pMHC complex, showed promising potency for patients with metastatic SS. So MAGE-A4/HLA-A*02:01 may be a good target for TCR mimic antibodies and cancer immunotherapy. In the present study, we utilized a newly established single cell cloning platform LyTARS to generate TCR mimic antibodies against HLA restricted MAGE-A4 peptide. Recombinant HLA-A*02:01 MAGE-A4 protein was used to immunize mice and plasma B cells from immunized mice were sorted and loaded onto LyTARS system. Clones that showed reactivity to recombinant HLA-A*02:01 MAGE-A4 but not to irrelevant pMHC (WT-1) were exported and sequenced. Purified antibodies were extensively characterized by the specificity, affinity, and potency by ADCC. Results demonstrated that the TCR mimic antibodies identified have sub-nano molar affinity by protein based ELISA, bind to peptide pulsed T2 cells that endogenously expressing HLA with nano-Molar apparent affinity. TCR mimic antibodies also demonstrated antibody dependent cell killing with the potency of sub-nanomolar. In summary, we have generated and extensively characterized a panel of high affinity and selective TCR mimic antibodies against HLA-A*02:01 MAGE-A4 using a newly established single cell cloning platform. Citation Format: Hao Cheng, You Wu, Tianfeng Shi, Yang Yu, Min Wang, Chenjun Jia, Wenfang Xin, Hu Liu, Teddy Yang, Danmei Yao. Discovery of T cell receptor mimic antibody against MAGE-A4 with single B-cell cloning platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3194.

Cross-reactive CD8+ T cell responses to tumor-associated antigens (TAAs) and homologous microbiota-derived antigens (MoAs)

BackgroundWe have recently shown extensive sequence and conformational homology between tumor-associated antigens (TAAs) and antigens derived from microorganisms (MoAs). The present study aimed to assess the breadth of T-cell recognition specific to MoAs and the corresponding TAAs in healthy subjects (HS) and patients with cancer (CP).MethodA library of > 100 peptide-MHC (pMHC) combinations was used to generate DNA-barcode labelled multimers. Homologous peptides were selected from the Cancer Antigenic Peptide Database, as well as Bacteroidetes/Firmicutes-derived peptides. They were incubated with CD8 + T cells from the peripheral blood of HLA-A*02:01 healthy individuals (n = 10) and cancer patients (n = 16). T cell recognition was identified using tetramer-staining analysis. Cytotoxicity assay was performed using as target cells TAP-deficient T2 cells loaded with MoA or the paired TuA.ResultsA total of 66 unique pMHC recognized by CD8+ T cells across all groups were identified. Of these, 21 epitopes from microbiota were identified as novel immunological targets. Reactivity against selected TAAs was observed for both HS and CP. pMHC tetramer staining confirmed CD8+ T cell populations cross-reacting with CTA SSX2 and paired microbiota epitopes. Moreover, PBMCs activated with the MoA where shown to release IFNγ as well as to exert cytotoxic activity against cells presenting the paired TuA.ConclusionsSeveral predicted microbiota-derived MoAs are recognized by T cells in HS and CP. Reactivity against TAAs was observed also in HS, primed by the homologous bacterial antigens. CD8+ T cells cross-reacting with MAGE-A1 and paired microbiota epitopes were identified in three subjects. Therefore, the microbiota can elicit an extensive repertoire of natural memory T cells to TAAs, possibly able to control tumor growth (“natural anti-cancer vaccination”). In addition, non-self MoAs can be included in preventive/therapeutic off-the-shelf cancer vaccines with more potent anti-tumor efficacy than those based on TAAs.

Zn-MOF as a tumor responsive carrier for combination therapy

Combination therapy is considered as a useful therapeutic strategy for tumor. Here, Zn-based MOF Zn-TPN was prepared for combination with chemotherapy and chemodynamic therapy. Co-precipitation method was used to obtain the antitumor system DOX&Mn@Zn-TPN, which incorporated DOX (9.37 wt%) and Mn2+ (5.75 wt%). The Fenton-like reaction of Mn2+ provided positive chemodynamic therapy, and drug release exhibited responses to pH, temperature and glutathione (GSH), enabling specific therapeutic effects in the tumor environment. According to the Korsmeyer-Peppas model, DOX release was a result of drug diffusion. In vitro cell experiments confirmed that the system exhibited significant lethality against 4T1 cells while maintaining biocompatibility with L929 cells.

Modulation of macrophage polarity with carboxymethyl chitin gated hollow mesoporous silica nanoparticles for elevating anti-tumor chemotherapy

The weak immunity of tumors after chemotherapy could cause tumor metastasis and progression. Therefore, to overcome the dilemma of obvious immune deficiency caused by chemotherapy, a nanosystem (N-IL-12/DOX/α-TOS) consisted of thioketal (TK) bonds linked-hollow mesoporous silica nanoparticles (HMSNs) coated with carboxymethyl chitin (CMCH) by electrostatic interaction, and surface-functionalized glucose-regulated protein 78 binding peptide was prepared for loading doxorubicin (DOX), IL-12 and α-tocopheryl succinate (α-TOS). N-IL-12/DOX/α-TOS displayed a mean size of 275 nm after encapsulated DOX, IL-12 and α-TOS with loading contents of 2.04 × 10−4, 4.01 × 10−2 and 7.12 × 10−2, respectively. The drug-free nanoparticles (NPs) showed good biocompatibility to both 4 T1 cells and RAW264.7 macrophages. N-IL-12/DOX/α-TOS could achieve localized release of IL-12, DOX and α-TOS by pH and H2O2 trigger in the tumor microenvironment (TME). Moreover, the combined therapy by N-IL-12/DOX/α-TOS remarkably elevated the anti-tumor therapeutic efficacy, enhanced immune responses via promoting tumor-associated macrophage (TAM) polarization into tumoricidal M1 phenotypes, and decreased lung metastasis with reduced side effects. N-IL-12/DOX/α-TOS exhibited as a promising strategy for combining chemotherapy and local macrophage modulation-immunotherapy for anti-tumor therapy.

Development of injectable in situ hydrogels based on hyaluronic acid via Diels-Alder reaction for their antitumor activities studies

The objective of this study was to develop an injectable hydrogel based on furfuryl amine-conjugated hyaluronic acid (FA-conj-HA) and evaluate the in vivo anti-4 T1 tumor activity of doxorubicin-loaded hydrogel (DOX@FA-conj-HAgel). The cargo-free hydrogel (FA-conj-HAgel) was fabricated through a Diels-Alder reaction at 37 °C with FA-conj-HA as a gel material and four armed poly(ethylene glycol)2000-maleimide (4-arm-PEG2000-Mal) as a cross-linker. The bio-safety of FA-conj-HAgel were assessed, and the in vivo antitumor activity of DOX@FA-conj-HAgel was also investigated. Many 3D network structures were observed from scanning electron microscope (SEM) photograph, confirming the successful preparation of FA-conj-HAgel. The absence of cytotoxicity from FA-conj-HAgel was proved by the high viability of 4 T1 cells. In vivo bio-safety studies suggested that the obtained FA-conj-HAgel did not induce acute toxicity or other lesions in treated mice, confirming its high bio-safety. The reduced tumor volumes, hematoxylin-eosin staining (H&E), and TdT-mediated dUTP-biotin nick end labeling (TUNEL) analysis indicated the potent in vivo anti-4 T1 tumor effects of DOX@FA-conj-HAgel. In conclusion, the favorable bio-safety and potent antitumor activity of DOX@FA-conj-HAgel highlighted its potential application in oncological therapy.

Assessment of the anti-cancer potential of Ephedra foeminea leaf extract on MDA-MB-231, MCF-7, 4 T1, and MCF-10 breast cancer cell lines: Cytotoxic, apoptotic and oxidative assays

Ephedra foeminea is traditionally used to treat breast cancer in several Arab countries. Scientific studies have reported different effects of this plant on some cancer cell lines. The current study determined the anti-cancer potential of the methanolic extract of Ephedra foeminea against four different types of breast cancer cell lines in-vitro. The extract was prepared by maceration and phytoconstituents were identified by LC-MS analysis. The IC50 value was determined against MDA-MB-231, MCF-7, 4 T1, and MCF-10 cell lines using the MTT assay. Further investigations were carried out using IC50 concentration of the extract (40.09 µg/ml) to determine live/dead cells by acridine orange/ethidium bromide staining. The effect on the expression of reactive oxygen species (ROS) was evaluated by flow cytometry. The results were analyzed using one-way ANOVA followed by Tukey’s test. The LC-MS analysis revealed the presence of 34 and 30 phytoconstituents in positive and negative modes respectively. The Ephedra foeminea extract was most effective against 4 T1 cells in a dose-dependent manner (P < 0.001) with an IC50 value of 40.09 µg/ml and showed negligible effect against MCF-10 cells. It increased apoptosis in 77.84 % of 4 T1 cells, as determined by acridine orange/ethidium bromide staining. The extract also increased the ROS expression in the 39.57 % of 4 T1 cells. The study results showed that Ephedra foeminea extract possesses an anti-cancer effect against 4 T1 cells by increasing the expression of ROS and inducing apoptosis in the 4 T1 cells. The result suggests Ephedra foemenia methanolic extract possesses a reasonable anti-cancer effect due to its effect on apoptosis and oxidative pathways. The results confirm the traditional belief that Ephedra is effective against breast cancerز

Preparation and antitumor study of intelligent injectable hydrogel: Carboxymethyl chitosan–aldehyde gum Arabic composite graphene oxide hydrogel

In this study, we used polyaldehyde gum Arabic (OGA) and carboxymethyl chitosan (CMCS) as a gel matrix to form an injectable self-healing hydrogel by Schiff-base bonding. Further, graphene oxide (GO) was loaded with doxorubicin (DOX) to the hydrogel, which resulted in a CMCS-OGA/GO@DOX hydrogel. We achieved a DOX drug loading capacity of 43.80 ± 1.13 %. Rheological studies showed that GO hydrogels have improved mechanical properties. The in vitro release profile showed pH responsiveness with 88.21 % DOX release at pH 5.5. Biocompatibility studies showed that the hydrogel composition had good cytocompatibility with L929 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a cell survival rate of 93.88 % within 48 h. The DOX-loaded hydrogel exhibited higher cell mortality in breast cancer cells (4 T1), with an inhibition rate of 79.4 % at 48 h. Acridine orange/ethidium bromide staining experiments on 4 T1 cells showed that when loaded with the same DOX concentration, the hydrogel significantly reduced the toxic effects on normal cells, whereas it had significant cytotoxic effects on cancer cells. This result indicates that the prepared GO hydrogel drug delivery system can serve as a novel approach for localized breast cancer treatment.

T4 apoptosis in the acute phase of SARS-CoV-2 infection predicts long COVID.

As about 10% of patients with COVID-19 present sequelae, it is important to better understand the physiopathology of so-called long COVID. To this aim, we recruited 29 patients hospitalized for SARS-CoV-2 infection and, by Luminex®, quantified 19 soluble factors in their plasma and in the supernatant of their peripheral blood mononuclear cells, including inflammatory and anti-inflammatory cytokines and chemokines, Th1/Th2/Th17 cytokines, and endothelium activation markers. We also measured their T4, T8 and NK differentiation, activation, exhaustion and senescence, T cell apoptosis, and monocyte subpopulations by flow cytometry. We compared these markers between participants who developed long COVID or not one year later. None of these markers was predictive for sequelae, except programmed T4 cell death. T4 lymphocytes from participants who later presented long COVID were more apoptotic in culture than those of sequelae-free participants at Month 12 (36.9 ± 14.7 vs. 24.2 ± 9.0%, p= 0.016). Our observation raises the hypothesis that T4 cell death during the acute phase of SARS-CoV-2 infection might pave the way for long COVID. Mechanistically, T4 lymphopenia might favor phenomena that could cause sequelae, including SARS-CoV-2 persistence, reactivation of other viruses, autoimmunity and immune dysregulation. In this scenario, inhibiting T cell apoptosis, for instance, by caspase inhibitors, could prevent long COVID.

Tumor nano-lysate activates dendritic cells to evoke a preventative immune response

A tumor nano-lysate “TNL” vaccine comprised of sonicated 4 T1 cells was developed, characterized and implemented for the prevention of triple-negative breast cancer. This study aims to gain a better understanding of the immune response behind the success of the vaccine in vivo, through use of ex vivo and in vivo assays. Here, we analyze the activation of various immune cells isolated from healthy mouse spleens and find that antigen-presenting cells (APCs) such as dendritic cells (DCs) are being activated following 24 h incubation with 1:10 mg TNL/mg splenocytes. These cells were further explored to determine the pathway by which activation is occurring, and it was observed that TNL are phagocytosed by DCs to activate NF-kB and c-Fos pathways, resulting in enhanced cytokine release after 24 h. An in vivo temporal analysis was performed in mice to understand the immune response at 1, 3, 7 and 10 days after one 100 μL dose of TNL consisting of 105 sonicated 4 T1 cells via cardiac puncture and splenocyte and peripheral blood mononuclear cell (PBMC) analysis. Changes were observed for up to one week. A multiple dose study was performed comparing mice that were vaccinated with one dose of TNL administered every ten days for 3 doses total, as well as a PBS vehicle control. Survival for TNL-vaccinated mice was enhanced compared to the PBS control, and there was an average delay of 10 days in the onset of metastasis. The differences between the groups at the end of the study demonstrate the potential for TNL as a preventative therapeutic.

Prediction and identification of HLA-A*0201-restricted epitopes from cancer testis antigen CT23

ABSTRACT Cancer-testis antigen CT23 is a class of tumor-associated antigens (TAA) characterized by restricted expression in male germ cells and a variety of tumor tissues. Numerous studies have shown that CT23 is closely related to tumor cell viability, proliferation, metastasis and invasion. CT23 is immunogenic and can cause specific immune response in tumor patients. Therefore, it is considered to be one of the best target antigens for designing therapeutic tumor vaccines and T-cell-mediated tumor immunotherapy. In this study, we initially obtained seven HLA-A*0201-restricted CT23 epitope candidate peptides through the T cell epitope prediction program. Subsequently, a T2 cell binding assay revealed the potential binding of all candidate peptides with HLA-A2 molecules. Notably, peptide P7 (ALLVLCYSI) exhibited the highest affinity, as evidenced by a fluorescence index (FI) of 2.19. Dendritic cells (DCs) loaded with CT23 candidate peptide can stimulate CD8+T cell activation and proliferation, and compared with other candidate peptides, candidate peptide P7 is superior. The cytotoxic T lymphocytes (CTLs) stimulated by the peptide P7 had killing effect on tumor cells (HLA-A*0201+, CT23+), but no killing effect on tumor cells (HLA-A*0201−, CT23+). The CTLs induced by the peptide P7 also had a specific killing effect on T2 cells bearing the peptide P7. In summary, our findings suggest that the CT23 peptide P7 (ALLVLCYSI) can induce immune responses and holds potential for tumor-specific CTL therapy.

T-Cell Receptor-Engineered T Cells Targeting FLT3-D835 Mutation-Derived Neoantigens in Acute Myeloid Leukemia

The adoptive transfer of T-cell receptor (TCR)-engineered T cells has demonstrated a remarkable anti-leukemic potential in mice models and human research for acute myeloid leukemia (AML). Neoantigens arising from somatic mutations are ideal targets for immunotherapy due to their high immunogenicity and harmless to normal cells. Therefore, neoantigen-specific TCR-based immunotherapy may have great prospects for AML. Activating mutations in FMS-like tyrosine kinase 3 ( FLT3) represent the most frequent genomic alterations of AML, including internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain, mostly at the activation loop residue D835. A number of FLT3 inhibitors have been clinically employed for FLT3-mutated AML. However, a durable response is limited by resistance-conferring secondary kinase domain mutations, notably point mutations at D835. Therefore, there is a critical need for novel and potent treatments to eliminate AML cells harboring FLT3-D835 mutations. Here, we show the construction of TCR-engineered T cells targeting FLT3-D835 mutants, presented in the context of HLA-A *02:01. We first identified neoepitopes derived from D835Y/H/V/F/I with strong affinities to HLA-A *02:01 using NetMHCpan 4.1. These neoepitopes could form the stable complex with HLA-A *02:01 on the cell surface. Furthermore, CD8 + T cells specific for D835H were detected in the peripheral blood of AML patients carrying D835H mutant at different stages of the illness, most of which were memory T cells. Moreover, one neoepitope (Neo-D835H) induced IFN-γ secretion when co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from AML patients with D835H mutant. Neo-D835H-specific T cells were effectively generated through the rapid stimulation of CD8 + T cells isolated from an HLA-A *02:01-positive healthy donor with Neo-D835H-loaded autologous dendritic cells. These T cells were recognized by Neo-D835H-HLA-A2-Tetramer (0.37%) and consequently isolated by flow cytometry to perform TCRα and TCRβ sequencing. Both the TCRA and TCRB clonotypes showed dominant sequences that accounted for more than 50% of the total, suggesting that T cells with these dominant TCRA and TCRB-paired sequences were likely Neo-D835H-specific. We thus next generated TCR-engineered T cells with these TCRA- and TCRB-paired sequences for further functional analysis. These TCR-engineered T cells recognized Neo-D835H and showed cytotoxic activity against Neo-D835H-pulsed T2 cells in an antigen dose-dependent manner, while showed no responses to the T2 cells pulsed with the corresponding wild-type peptide. Taken together, we present novel neoantigens with promise as immunotherapy targets for AML and other hematologic malignancies with FLT3-D835 mutations. Future studies will evaluate the efficacy of Neo-D835H-specific TCR-engineered T-cell-based immunotherapy in vivo in patient-derived xenograft murine model and TCR-based therapeutic approaches targeting other FLT3-TKD mutations.

Different spectral sensitivities of ON- and OFF-motion pathways enhance the detection of approaching color objects in Drosophila

Color and motion are used by many species to identify salient objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. Here, we demonstrate an unexpected, additional contribution of color to motion vision in Drosophila. We show that behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and we identify cellular pathways connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, this contribution of color circuitry to motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and we show how this could generalize for systems with ON- and OFF-motion pathways. Our results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects.

The immunostimulant effects of the rice ragged stunt virus genome on the growth and metastasis of breast cancer in mouse model

There are multiple treatment strategies that have been reported for breast cancer, while new and effective therapies against it are still necessary. Stimulating the immune system and its components against cancer cells is one of the unique treatment strategies of immunotherapy and long dsRNAs are immunostimulant in this regard. Based on bioinformatics approaches, a fragment of the Rice ragged stunt RNA virus genome was selected and synthesized according to its immunogenicity. Based on the in vitro transcription technique, dsRNA was synthesized and its binding ability to the PEI/PEI-Ac Polyethylenimine (PEI) or Acetylated polyethylenimine (PEI-Ac) was verified by the gel retardation assay. Then, the PEI-Ac was synthesized by adding acetyl groups to the PEI, and the results of the 1H NMR method indicated its successful synthesis. After cancer induction by 4 T1 cells in Balb/C mice, intraperitoneal (IP) and intratumoral (IT) treatment by the PEI/PEI-Ac-dsRNA were performed and the tumor growth inhibition was evaluated. Results demonstrated that PEI/PEI-Ac-dsRNA can lead to a decrease in tumor weight and volume in both the IP and IT routes. Also, by using macro-metastatic nodule counting and hematoxylin and eosin (H&E) staining we showed that PEI/PEI-Ac-dsRNA can prevent micro and macro-metastasis in the lung. Therefore, the PEI/PEI-Ac-dsRNA acts as an effective inhibitor of growth and metastasis of the breast cancer models. We showed that viral dsRNA can exert its antitumor properties by stimulating TNF-α and IFN-γ. In general, our results revealed that dsRNA derived from the plant virus genome stimulates the intrinsic immune system and can be a potential immune stimulant drug for cancer treatment.

Low levels of peripheral blood activated and senescent T cells characterize people with HIV-1-associated neurocognitive disorders.

HIV infection induces a 75% increase in the risk of developing neurocognitive impairment (NCI), which has been linked to immune activation. We therefore looked for immune activation markers correlating with NCI. Sixty-five people aged 55-70 years living with controlled HIV-1 infection were enrolled in the study and their neurocognitive ability was assessed according to the Frascati criteria. Fifty-nine markers of T4 cell, T8 cell, NK cell, and monocyte activation, inflammation and endothelial activation were measured in their peripheral blood. White matter hyperintensities (WMH) were identified by magnetic resonance imaging. Double hierarchical clustering was performed for the activation markers and 240 patients including the 65 whose neurocognitive performance had been evaluated. Thirty-eight percent of volunteers presented NCI. Twenty-four percent of them were asymptomatic and fourteen percent had a mild disorder. Strikingly, activated (HLA-DR+) as well as senescent (CD57+CD28-CD27±) T4 cells and T8 cells were less prevalent in the peripheral blood of participants with NCI than in participants without the disorder. Accordingly, the percentage of HLA-DR+ T4 cells was lower in volunteers with periventricular and deep WMH. The double hierarchical clustering unveiled six different immune activation profiles. The neurocognitive performances of participants with two of these six profiles were poor. Here again, these two profiles were characterized by a low level of T4 and T8 cell activation and senescence. Our observation of low circulating levels of activated and senescent T cells in HIV-1 patients with NCI raises the interesting hypothesis that these lymphocytes may be recruited into the central nervous system.

Site-specific phosphorylation of tau impacts mitochondrial function and response to stressors.

Phosphorylation of tau at sites associated with Alzheimer's disease (AD) likely plays a role in the disease progression. Mitochondrial impairment, correlating with increased presence of phosphorylated tau, has been identified as a contributing factor to neurodegenerative processes in AD. However, how tau phosphorylated at specific sites impacts mitochondrial function has not been fully defined. We examined how AD-relevant phosphomimetics of tau impact selected aspects of mitochondrial biology. To mimic phosphorylation at AD-associated sites, the serine/threonine (Ser/Thr) sites in wild-type green fluorescent protein (GFP)-tagged tau (T4) were converted to glutamic acid (E) to make pseudo-phosphorylated GFP-tagged Ser-396/404 (2EC) and GFP-tagged Thr-231/Ser-235 (2EM) constructs. These constructs were expressed in immortalized mouse hippocampal neuronal cell lines, and their impact on specific mitochondrial functions and responses to stressors were measured. Phosphomimetic tau altered mitochondrial distribution. Specifically, mitochondria accumulated in the soma of cells expressing either 2EC or 2EM and neurite-like extensions in 2EC cells were shorter. Additionally, adenosine triphosphate levels were reduced in both 2EC- and 2EM-expressing cells, and reactive oxygen species (ROS) production increased in 2EC cells during oxidation of succinate when compared to T4-expressing cells. Thapsigargin reduced mitochondrial membrane potential and increased ROS production in both 2EC and 2EM cells relative to T4 cells, with no significant difference in the effects of rotenone. These results show that tau phosphorylation at specific AD-relevant epitopes negatively affects mitochondria, with the extent of dysfunction and stress response varying according to the sites of phosphorylation. Altogether, these findings show that phosphorylated tau increases mitochondrial susceptibility to stressors and extend our understanding of potential mechanisms whereby phosphorylated tau promotes mitochondria dysfunction in tauopathies, including AD.

The participation of vacuoles and the regulation of various metabolic pathways under acid stress promote the differentiation of chlamydospore in Trichoderma harzianum T4.

Chlamydospores are a special, differentiated type with high environmental resistance. Consequently, the chlamydospores of Trichoderma harzianum T4 can used to industrialize the latter. This study aimed to investigate the key factors affecting the sporulation type of T. harzianum T4 and the mechanisms underlying this effect. In the liquid fermentation of T. harzianum T4, ammonium sulfate (AS) inhibited conidia formation and chlamydospore production. Fermentation tests revealed that acid stress induced sporulation type alteration. Transcriptomic analysis was used to evaluate the adaptation strategy and mechanism underlying spore type alteration under acid stress. The fermentation experiments involving the addition of amino acids revealed that branched-chain amino acids benefited conidia production, whereas β-alanine benefited chlamydospore production. Confocal microscope fluorescence imaging and chloroquine intervention demonstrated that vacuole function was closely related to chlamydospore production. The sporulation type of T. harzianum T4 can be controlled by adjusting the fermentation pH. T. harzianum T4 cells employ various self-protection measures against strong acid stress, including regulating their metabolism to produce a large number of chlamydospores for survival.

Circulating extracellular vesicles derived from tumor endothelial cells hijack the local and systemic anti-tumor immune response: Role of mTOR/G-CSF pathway

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model.4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.