Clonal structure and the specificity of vaccine-induced T cell response to SARS-CoV-2 Spike protein.

Adenovirus vaccines, particularly the COVID-19 Ad5-nCoV adenovirus vaccine, have emerged as promising tools in the fight against infectious diseases. In this study, we investigated the structure of the T cell response to the Spike protein of the SARS-CoV-2 virus used in the COVID-19 Ad5-nCoV adenoviral vaccine in a phase 3 clinical trial (NCT04540419). In 69 participants, we collected peripheral blood samples at four time points after vaccination or placebo injection. Sequencing of T cell receptor repertoires from Spike-stimulated T cell cultures at day 14 from 17 vaccinated revealed a more diverse CD4+ T cell repertoire compared to CD8+. Nevertheless, CD8+ clonotypes accounted for more than half of the Spike-specific repertoire. Our longitudinal analysis showed a peak T cell response at day 14, followed by a decline until month 6. Remarkably, multiple T cell clonotypes persisted for at least 6 months after vaccination, as demonstrated by ex vivo stimulation. Examination of CDR3 regions revealed homologous sequences in both CD4+ and CD8+ clonotypes, with major CD8+ clonotypes sharing high similarity with annotated sequences specific for the NYNYLYRLF peptide, suggesting potential immunodominance. In conclusion, our study demonstrates the immunogenicity of the Ad5-nCoV adenoviral vaccine and highlights its ability to induce robust and durable T cell responses. These findings provide valuable insight into the efficacy of the vaccine against COVID-19 and provide critical information for ongoing efforts to control infectious diseases.

Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study.

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient's peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19+ cells from the patient's peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo.

Interplay between Immune Cell Infiltration and Tumor Histological Subtype: A Case of Adrenocortical Cancer.

The analysis of the tumor microenvironment, especially tumor-infiltrated immune cells, is essential for predicting tumor prognosis, clinical outcomes, and therapy strategies. Adrenocortical cancer is a rare nonimmunogenic malignancy in which the importance of the presence of immune cells is not well understood. In our study, we made the first attempt to understand the interplay between the histology of adrenocortical cancer and its immune landscape using cases from The Cancer Genome Atlas database and the Endocrinology Research Centre collection (Moscow, Russia). We showed that the oncocytic variant of adrenocortical cancer is characterized by intensive immune infiltration and better survival, and it is crucial to analyze the effect of immune infiltration independently for each histological variant.

Multi-dimensional immunoproteomics coupled with in vitro recapitulation of oncogenic NRASQ61R identifies diagnostically relevant autoantibody biomarkers in thyroid neoplasia.

Tumor-associated antigen (TAA)-specific autoantibodies have been widely implicated in cancer diagnosis. However, cancer cell lines that are typically exploited as candidate TAA sources in immunoproteomic studies may fail to accurately represent the autoantigen-ome of lower-grade neoplasms. Here, we established an integrated strategy for the identification of disease-relevant TAAs in thyroid neoplasia, which combined NRASQ61R oncogene expression in non-tumorous thyroid Nthy-ori 3-1 cells with a multi-dimensional proteomic technique DISER that consisted of profiling NRASQ61R-induced proteins using 2-dimensional difference gel electrophoresis (2D-DIGE) coupled with serological proteome analysis (SERPA) of the TAA repertoire of patients with thyroid encapsulated follicular-patterned/RAS-like phenotype (EFP/RLP) tumors. We identified several candidate cell-based (nicotinamide phosphoribosyltransferase NAMPT, glutamate dehydrogenase GLUD1, and glutathione S-transferase omega-1 GSTO1) and autoantibody (fumarate hydratase FH, calponin-3 CNN3, and pyruvate kinase PKM autoantibodies) biomarkers, including NRASQ61R-induced TAA phosphoglycerate kinase 1 PGK1. Meta-profiling of the reactivity of the identified autoantibodies across an independent SERPA series implicated the PKM autoantibody as a histological phenotype-independent biomarker of thyroid malignancy (11/38 (29%) patients with overtly malignant and uncertain malignant potential (UMP) tumors vs 0/22 (p = 0.0046) and 0/20 (p = 0.011) patients with non-invasive EFP/RLP tumors and healthy controls, respectively). PGK1 and CNN3 autoantibodies were identified as EFP/RLP-specific biomarkers, potentially suitable for further discriminating tumors with different malignant potential (PGK1: 7/22 (32%) patients with non-invasive EFP/RLP tumors vs 0/38 (p = 0.00044) and 0/20 (p = 0.0092) patients with other tumors and healthy controls, respectively; СNN3: 9/29 (31%) patients with malignant and borderline EFP/RLP tumors vs 0/31 (p = 0.00068) and 0/20 (p = 0.0067) patients with other tumors and healthy controls, respectively). The combined use of PKM, CNN3, and PGK1 autoantibodies allowed the reclassification of malignant/UMP tumor risk in 19/41 (46%) of EFP/RLP tumor patients. Taken together, we established an experimental pipeline DISER for the concurrent identification of cell-based and TAA biomarkers. The combination of DISER with in vitro oncogene expression allows further targeted identification of oncogene-induced TAAs. Using this integrated approach, we identified candidate autoantibody biomarkers that might be of value for differential diagnostic purposes in thyroid neoplasia.

Serum Immunoproteomics Combined With Pathological Reassessment of Surgical Specimens Identifies TCP-1ζ Autoantibody as a Potential Biomarker in Thyroid Neoplasia.

CONTEXT: Current methods of preoperative diagnostics frequently fail to discriminate between benign and malignant thyroid neoplasms. In encapsulated follicular-patterned tumors (EnFPT), this discrimination is challenging even using histopathological analysis. Autoantibody response against tumor-associated antigens is a well-documented phenomenon with prominent diagnostic potential; however, autoantigenicity of thyroid tumors remains poorly explored. OBJECTIVES: Objectives were exploration of tumor-associated antigen repertoire of thyroid tumors and identification of candidate autoantibody biomarkers capable of discrimination between benign and malignant thyroid neoplasms. DESIGN, SETTING, AND PATIENTS: Proteins isolated from FTC-133 cells were subjected to two-dimensional Western blotting using pooled serum samples of patients originally diagnosed with either papillary thyroid carcinoma (PTC) or EnFPT represented by apparently benign follicular thyroid adenomas, as well as healthy individuals. Immunoreactive proteins were identified using liquid chromatography-tandem mass-spectrometry. Pathological reassessment of EnFPT was performed applying nonconservative criteria for capsular invasion and significance of focal PTC nuclear changes (PTC-NCs). Recombinant T-complex protein 1 subunitζ (TCP-1ζ) was used to examine an expanded serum sample set of patients with various thyroid neoplasms (n = 89) for TCP-1ζ autoantibodies. All patients were included in tertiary referral centers. RESULTS: A protein demonstrating a distinct pattern of EnFPT-specific seroreactivity was identified as TCP-1ζ protein. A subsequent search for clinicopathological correlates of TCP-1ζ seroreactivity revealed nonclassical capsular invasion or focal PTC-NC in all TCP-1ζ antibody-positive cases. Further studies in an expanded sample set confirmed the specificity of TCP-1ζ autoantibodies to malignant EnFPT. CONCLUSIONS: We identified TCP-1ζ autoantibodies as a potential biomarker for presurgical discrimination between benign and malignant encapsulated follicular-patterned thyroid tumors. Our results suggest the use of nonconservative morphological criteria for diagnosis of malignant EnFPT in biomarker identification studies and provide a peculiar example of uncovering the diagnostic potential of a candidate biomarker using incorporation of pathological reassessment in the pipeline of immunoproteomic research.