Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations.

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.

Alternating ventilation accelerates the mineralization and humification of food waste by optimizing the temperature-oxygen-moisture distribution in the static composting reactor.

Traditional unidirectional ventilation often leads to the loss of heat and moisture during composting, disrupting the favorable microenvironment required for aerobic microbes. This study developed a pulse alternating ventilation composting reactor and investigated the effects of alternating ventilation on composting efficiency compared with upward ventilation and downward ventilation. The results demonstrated that alternating ventilation stabilized the moisture content at approximately 60 % while reducing the temperature and oxygen concentration range within the reactor. Moreover, it extended the duration of high-temperature (>50 °C) by 31 % and 75 % compared to other two groups. It improved the microbial cooperation intensity and stimulated the core microbe (Tepidimicrobium). Seed germination index (GI) of the compost was improved (GI = 91.27 %), and the humic acid content was 1.23 times and 1.37 times higher than other two groups. These results showed that alternating ventilation can be used for efficient resource disposal of food waste.

The Expression of LDL-R in CD8+ T Cells Serves as an Early Assessment Parameter for the Production of TCR-T Cells.

BACKGROUND/AIM: The quantity and the phenotypes of desired T cell receptor engineered T (TCR-T) cells in the final cell product determine their in vivo anti-tumor efficacy. Optimization of key steps in the TCR-T cell production process, such as T cell activation, has been shown to improve cell quality. MATERIALS AND METHODS: Using a modified TCR (mTCR) derived from mice transducing PBMCs, we assessed the proportions of low-density lipoprotein receptor (LDL-R) and mTCR expressing cells under the various activation conditions of CD3/CD28-Dynabeads or OKT3 via flow cytometry. RESULTS: We demonstrate that the proportion of T cells expressing LDL-R post activation is positively correlated with the percentage of mTCR+CD8+ T cells with their less differentiated subtypes in the final product. In addition, we show that shifting the CD3/CD28-Dynabeads activation duration from a typical 48 h to 24 h can significantly increase the production of the desired mTCR+CD8+ T cells. Importantly, the percentages of TCR-T cells with less-differentiated phenotypes, namely mTCR central memory T cells (TCM), were found to be preserved with markedly higher efficiency when T cell activation was optimized. CONCLUSION: Our findings suggest that the proportion of LDL-R+ T cells may serve as an early assessment parameter for evaluating TCR-T cell quality, possibly facilitating the functional and economical improvement of current adoptive therapy.

Collective transcendence beliefs shape the sacredness of objects: The case of art.

Many objects are viewed as sacred even though few people have a strong personal connection to them. To explain this phenomenon, we used art as a case study to develop and test a theory wherein collective transcendence beliefs-beliefs that an object links the collective to something larger and more important than the self, spanning space and time-are a key determinant of the sacredness of objects. Initial inductive studies pointed to perceptions of collective spirituality, collective meaning, and historical significance to humanity as the primary collective transcendence beliefs underlying the sacredness of art (Study 1), and subsequent exploration indicated that collective meaning was a mechanism by which collective spirituality and historical significance to humanity influenced sacredness judgments (Study 2). In support of this, six experimental studies demonstrated that heightening the collective spirituality and historical significance of an artwork resulted in participants viewing the artwork as more collectively meaningful, and subsequently more sacred (Studies 3-6), worthy of protection from the profane (Studies 3c and 6), and eliciting moral outrage in the face of desecration (Study 5). In all, across these studies (N = 5,304), we found converging evidence that collective transcendence beliefs elevate various forms of art (sculpture, music, and painting) to be held as sacred, even an amateur sketch done by the first author. Our findings uncover a novel mechanism underlying sacredness judgments, theoretically advancing our understanding of the sacred while pointing to a number of important real-world implications. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants.

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

TRAP1 inhibits MIC60 ubiquitination to mitigate the injury of cardiomyocytes and protect mitochondria in extracellular acidosis.

Extracellular acidosis-induced mitochondrial damage of cardiomyocytes leads to cardiac dysfunction, but no detailed mechanism or efficient therapeutic target has been reported. Here we found that the protein levels of MIC60 were decreased in H9C2 cells and heart tissues in extracellular acidosis, which caused mitochondrial damage and cardiac dysfunction. Overexpression of MIC60 maintains H9C2 cells viability, increases ATP production and mitochondrial membrane potential, mitigates the disruptions of mitochondrial structure and cardiac injury. Mechanistically, extracellular acidosis excessively promoted MIC60 ubiquitin-dependent degradation. TRAP1 mitigated acidosis-induced mitochondrial impairments and cardiac injury by directly interacting with MIC60 to decrease its ubiquitin-dependent degradation in extracellular acidosis.

T Cell Immunity Evaluation and Immunodominant Epitope T Cell Receptor Identification of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Glycoprotein in COVID-19 Convalescent Patients.

A better understanding of the role of T cells in the immune response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is helpful not only for vaccine development but also for the treatment of COVID-19 patients. In this study, we determined the existence of SARS-CoV-2-specific T cells in the blood of COVID-19 convalescents. Meanwhile, the specific T cell response in the non-RBD region was stronger than in the RBD region. We also found that SARS-CoV-2 S-specific reactive CD4+ T cells exhibited higher frequency than CD8+ T cells in recovered COVID-19 patients, with greater number of corresponding epitopes presented. Importantly, we isolated the SARS-CoV-2-specific CD4+ T cell receptors (TCRs) and inserted the TCRs into allogenic CD4+ T cells. These TCR-T cells can be activated by SARS-CoV-2 spike peptide and produce IFN-γ in vitro. These results might provide valuable information for the development of vaccines and new therapies against COVID-19.

p38 inhibition enhances TCR-T cell function and antagonizes the immunosuppressive activity of TGF-ß.

The efficacy of adoptive cell therapy (ACT) relies on the abilities of T cells in self-expansion, survival and the secretion of effector molecules. Here, we presented an optimized method to generate T cells with improved functions by supplementing the culture medium with p38 inhibitor and the combination of IL-7 and IL-15 or IL-2 alone. The addition of p38 inhibitor, Doramapimod or SB202190, to IL-7 and IL-15 culture largely increased the capacity of T cells in the proliferation with enrichment of the naïve-like subsets and expression of CD62L. Importantly, we found this regimen has generated complete T cell resistance to TGF-ß-induced functional suppression, with sustained levels of the IFN-γ and Granzyme-B productions. Such findings were also validated in the melanoma-associated antigen recognized by T cells (MART-1) specific T cell receptor (TCR) engineered T cells, which were expanded in Doramapimod and IL-7 + IL-15 added media. In conclusion, we have established and optimized a protocol with the combination of p38 inhibitor, IL-7 and IL-15, rather than IL-2, for the generation of functionally enhanced T cells applicable for ACT.

An unusual increase of D-dimer level-pylephlebitis caused by acute appendicitis: a case report.

Acute appendicitis (AA) patients who present with a significantly increased level of D-dimer is not common. We speculated that the increase of D-dimer level was a result of pylephlebitis complication in the appendicitis patient. A 34-year-old man presented to the emergency department with sudden onset of lower quadrant abdomen pain. He was diagnosed with AA and scheduled for a laparoscopic appendectomy. He had a blood pressure of 80-90/30-40 mmHg, heart rate of 120-130/min, and his temperature was 38.3 °C. Routine blood test demonstrated a significantly elevated D-dimer (14,037 µg/L) with a negative blood gas test, normal ultrasound of the lower limbs, and normal pulmonary and abdominal computer tomography angiography (CTA) scans. Further tests showed a two-fold increase in D-dimer and abnormal hepatic function, indicating pylephlebitis, a rare but serious complication of AA. The patient was subjected to laparoscopic appendectomy, removing the cause of pylephlebitis, and received intravenous broad-spectrum antibiotics for an additional 1 week. The patient had clinical improvement with almost complete normalization of his D-dimer, white blood cell (WBC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), fibrin degradation product (FDP) and platelet (PLT) levels. The patient was fully recovered and discharged from the hospital without any complications. Pylephlebitis secondary to AA is rare and can be easily missed. The unusual increase of D-dimer level provided critical value for pylephlebitis diagnosis.

[Bioinformation analysis, eukaryotic expression and identification of ß2m-linker 2-HLA A24].

Objective To explore the structure and function of ß2 microglobulin-linker 2-human leukocyte antigen A24 (ß2m-linker 2-HLA A24) by bioinformatics, and to obtain the purified protein by eukaryotic expression. Methods The physicochemical properties of ß2m-linker 2-HLA A24 was predicted by ProtParam. The predicting software of phosphorylation site (NetPhos) and the predicting software of the secondary structure (SOPMA) were used to analyze the phosphorylation site and the secondary structure of the protein, respectively. And the predicting tool of modeling the proteins' structure (SWISS-MODEL) was used to model its tertiary structure. Then the recombinant plasmid of pcDNA3.4/ß2m-linker 2-HLA A24-His tag was constructed and transfected into Expi293F cells. The protein of interest was purified through Ni-affinity chromatography. And the purity and properties of the protein were identified by SDS-PAGE, Western blot analysis and indirect ELISA. Results The ß2m-linker 2-HLA A24 protein contained 44 phosphorylated sites. The protein was predicted to be a hydrophilic protein, of which the secondary structure mainly consists of irregular curl, and the modeling of the tertiary structure was successful. The recombinant plasmids of pcDNA3.4/ß2m-linker 2-HLA A24-His tag was successfully constructed and expressed in the Expi293F cells. Conclusion The ß2m-linker 2-HLA A24 protein has been successfully constructed. And bioinformatics results show that the protein has the function of binding to antigen peptides and activating T cells. It has established a foundation for activating antigen-specific CD8+ T cell stimulated by this protein and peptide in the subsequent experiments.