Dominant T-cell Research Articles

Abstract 1516: Large scale, transcriptome-based analysis of TCR clonality reveals functional immunity in non-small cell lung cancer

Abstract Tumor-related T-cell activation and subsequent clonal expansion are essential for an effective anti-cancer immune response. Thus, quantifying the clonal distribution of T-cells in cancer tissue from patients might provide prognostic and predictive information, particularly in high immunogenic tumors like non-small cell lung cancer (NSCLC). We evaluated TCR clonality in the tumor microenvironment of NSCLC, its relation to the in situ immune phenotype, and its clinical impact in a cohort of 182 resected NSCLC patients. The α and β TCR clones were determined based on RNA sequencing data, and the Gini index was used to weight individual clonal distribution. We used matched FFPE cancer tissue for multiplex immunofluorescence cell analysis and in-situ sequencing of T-cell clones. The analysis revealed a broad spectrum of TCR clonality patterns, including tissue with high TCR diversity and high evenness (low Gini coefficient) and tissue with clonal dominance with low evenness (high Gini coefficient). Highly expended clones were detected in 15/182 patients, whereas 43/182 revealed no clonal enrichment. A low TCR clone evenness (high Gini coefficient) was significantly more frequent in normal lung areas than in the corresponding tumor areas (n=20, p=0.02). In the cancer tissue, a high Gini index further correlated with high tumor mutational burden, indicating neoantigen-induced T cell clone expansions. Correspondingly, high TCR clonality was associated with an inflamed tumor phenotype associated with PD-1+ immune cells, CD3+, CD8A+, CD163+, and CD138+ cells, as well as higher expression of genes connected to immune activation (PRF1, GZMB, GZMH) and T cell exhaustion (PD-L1, LAG3, TIGIT). Cancer tissue with dominant T-cell clones also showed higher numbers of mature tertiary lymphoid structures with germinal centers. In situ sequencing suggested a close spatial relation between specific T cell clones and tumor cells. Finally, NSCLC patients with dominant T-cell clones showed strong responses and longer survival when treated with immune checkpoint inhibitors. This suggests that TCR clonality analysis is a strong candidate for precision diagnostics. Plausibly, the dominant clones found in NSCLC are responsible for a specific but malfunctioning anti-tumor response that can be unleashed by immune checkpoint inhibition. Citation Format: Hui Yu, Anastasia Magoulopoulou, Masafumi Hori, Amanda Lindberg, Max Backman, Hans Brunström, Millaray Marincevic Zuniga, Johanna Mattsson, Akira Saito, Karin Leandersson, Mats Nilsson, Rose-Marie Amini, Patrick Micke, Carina Strell. Large scale, transcriptome-based analysis of TCR clonality reveals functional immunity in non-small cell lung cancer [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 1516.

Modified Dendritic cell-based T-cell expansion protocol and single-cell multi-omics allow for the selection of the most expanded and in vitro-effective clonotype via profiling of thousands of MAGE-A3-specific T-cells.

Adoptive cell therapy using TCR-engineered T-cells is one of the most effective strategies against tumor cells. The TCR T-cell approach has been well tested against a variety of blood neoplasms but is yet to be deeply tested against solid tumors. Among solid tumors, cancer-testis antigens are the most prominent targets for tumor-specific therapy, as they are usually found on cells that lie behind blood-tissue barriers. We have employed a novel efficient protocol for MAGE-A3-specific T-cell clonal expansion, performed single-cell multi-omic analysis of the expanded T-cells via BD Rhapsody, engineered a selected T-cell receptor into a lentiviral construct, and tested it in an in vitro LDH-cytotoxicity test. We have observed a 191-fold increase in the MAGE-A3-specific T-cell abundance, obtained a dominant T-cell receptor via single-cell multi-omic BD Rhapsody data analysis in the TCRscape bioinformatics tool, and observed potent cytotoxicity of the dominant-clonotype transduced TCR T-cells against a MAGE-A3-positive tumor. We have demonstrated the efficiency of our T-cell enrichment protocol in obtaining potent anti-tumor T-cells and their T-cell receptors, especially when paired with the modern single-cell analysis methods.

Immunosequencing applications in cutaneous T-cell lymphoma.

Immunosequencing has emerged as a newer clinical test for assessment of T-cell clonality in the blood and skin of cutaneous T-cell lymphoma (CTCL) patients. Utilization of immunosequencing, also known as high-throughput sequencing of the T-cell receptor (HTS-TCR), enables identification and quantification of the precise genetic signature of dominant T-cell clones. Although immunosequencing is more sensitive than commonly used methods such as polymerase chain reaction (PCR) paired with capillary electrophoresis or flow cytometry, it remains underutilized for CTCL management. Nonetheless, incorporation of HTS-TCR in clinical practice offers distinct advantages compared to other molecular analyses that may improve diagnostic evaluation, prognostication, and disease monitoring in CTCL. The objective of this comprehensive review is to provide a thorough explanation of the application of immunosequencing in the context of CTCL. We describe the significance of T-cell clonality and the methods used to detect it, including a detailed comparison between PCR paired with capillary electrophoresis and HTS-TCR. The utilization of immunosequencing in the blood and skin of CTCL patients is discussed in depth, specifically outlining how HTS-TCR can assist in diagnosing CTCL, predicting outcomes, and tracking disease progression. Finally, we address the potential applications of immunosequencing in clinical management and research as well as the novel challenges it presents.

The Overlap of Skin and Blood T-Cell Clones in Early-Stage Mycosis Fungoides

Introduction: Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma that initially starts in the skin but can progress to involve blood with significant mortality in late stages. Even in early stages and in the absence of blood involvement, changes in blood T-cell receptor (TCR) repertoire have been observed in MF patients. While prior work has explored the significance of dominant blood T-cell clones in the prognosis of early MF, the relationship between TCR sequences and T-cell repertoires of blood and skin in early-stage MF patients has not been characterized. Objectives: To determine the relationship of blood and skin T-cell repertories and to reassess the impact of dominant blood T-cell clones on selected outcome endpoints in early-stage MF. Materials and Methods: We used high-throughput sequencing to interrogate TCR sequences in blood and skin of MF patients without blood involvement at the Jefferson Cutaneous Lymphoma Clinic (Adaptive Biotechnologies). ImmunoSEQ Analyzer provided T-cell repertoire overlap metric (Morisita's index) and diversity measure (Simpson's clonality score). Time to systemic treatment (TTST) was calculated as the time from initial diagnosis to initiation of first systemic therapy. Results: 60 MF patients with no blood involvement were enrolled. 28% had a dominant clone in blood; of these, 18% were identical to dominant skin clones and 82% were distinct from the dominant clones identified in skin. We found that MF patients with discordant dominant T-cell clones in blood and skin had a longer TTST when compared to the identical clone cohort (P=0.0057) (Figure 1A). Furthermore, patients with discordant dominant clones had a lower degree of T-cell repertoire overlap between blood and skin (P<0.0001) and higher blood T-cell repertoire diversity scores (P=0.013) when compared to the identical clone group (Figure 1B). In all patients, the degree of T-cell overlap in blood and skin did not significantly change over a period of months to years or among skin biopsies obtained from different anatomical locations. Finally, in a subset of patients with discordant clones, the dominant skin clone did not appear to be detected in blood even at low frequencies. Conclusion: Our data highlight the importance of establishing clonal associations between dominant T-cell clones of the skin and blood in MF. We propose that early-stage MF patients with dominant clones in blood segregate into two cohorts with distinct prognoses and T-cell repertoires based on the identity of their peripheral T-cell clones. In addition, our findings suggest that not all MF patients have reservoirs of malignant T-cell clones readily available in peripheral circulation to seed new lesions in skin. Figure 1: A: Kaplan-Meier analyses of time to systemic treatment (TTST) in skin-limited mycosis fungoides (MF) patients with an identical dominant T-cell clone in blood and skin, a discordant dominant T-cell clone in blood and skin, and no dominant blood clones. NS- not significant (P =0.5274), ** (P =0.0057), *** (P <0.0003). B: Average Morisita's index for blood and skin overlap for patients in indicated groups of I: identical dominant clones in blood and skin, D: discordant dominant clones in blood and skin, and N: no dominant clone in blood. **** (P <0.0001).

Multifactorial Basis of T Cell Dysfunction in CLL: Disrupted Mitochondrial Metabolism Induces T Cell Senescence

Despite being a B-cell malignancy, dysfunction of the T-cell compartment is a key feature of chronic lymphocytic leukemia (CLL). This leads to complications in acquired immunity and low success rates in autologous T cell-based therapies in these patients, particularly chimeric antigen receptor T cells (CAR-T). Despite exhibiting features of exhaustion, T cells from CLL patients are not reinvigorated by immune checkpoint inhibitors. Although T-cell dysfunction in CLL is heterogeneous, a consistent feature among all CLL patients is T-cell subset skewing towards effector T cells at the expenses of the central memory compartment (Martens et al., Leukemia 2023) and intact ability to produce inflammatory cytokines. A common feature of senescent T cells includes this maintained ability to produce pro-inflammatory cytokines alongside dysfunctions such as cell-cycle arrest, a decline in proliferation, and reduced activation. Notably, senescence often coincides with impaired mitochondrial function which is known to play a crucial role in the development of memory phenotypes and the efficacy of CAR-T cells (Van der Windt et al., Immunity 2012; Van Bruggen et al., Blood 2019). Given these associations, our aim is to comprehensively investigate the interplay between T-cell dysfunction, senescence, and mitochondrial impairment in CLL patients through a multifaceted approach encompassing analysis of T-cell metabolism, (epi)genetics, and functional phenotyping. Metabolic profiling of T cells from CLL patients and age-matched healthy donors (HD) was performed by extracellular flux analysis, flow cytometry, metabolomics and 13C tracing. A decreased mitochondrial respiration upon T-cell receptor stimulation was observed in all CLL patients. On the other hand, glycolytic activity correlated with T-cell activation status, which varied among patients. We identified alterations in the utilization of the three main mitochondrial fuels in T cells from CLL patients: i) induction of pseudohypoxia by CLL cells, leading to a re-routing of glucose-derived pyruvate into lactate rather than into the mitochondria; ii) a reduced rate of conversion of glutamine into glutamate resulting in decreased utilization of this fuel within the mitochondria; and iii) impaired lipid uptake, along with diminished fatty acid oxidation. Accordingly, mitochondria from CLL T cells showed intrinsic features of dysfunction including depolarization and increased production of reactive oxygen species (ROS). So far, these findings strongly demonstrate a misbalance in metabolic fueling resulting in mitochondrial dysfunction, aligning more closely with senescence rather than classical T-cell exhaustion. To further reinforce this observation, we assessed the expression of cell-surface proteins that characterize senescent T cells (e.g., absence of CD27/CD28 and presence of PD1/CD57/KLRG1). Indeed, we found an increased proportion of senescent cells in the T-cell compartment of CLL patients, as compared to HD. In response to stimulation, CLL T cells showed a strikingly distinct cell-cycle pattern whereby a larger proportion of cells resided in S and G2/M phases, indicating cell-cycle arrest. Cell cycle arrest is often induced by DNA damage. Transcriptomic and epigenomic profiling of T cells of CLL patients indeed revealed a molecular profile consistent with inflammation and an increased p53/DNA damage response upon stimulation. These features are known to be induced by dysfunctional mitochondria and ROS in senescent cells, underscoring a direct link between mitochondrial metabolism and senescence. Altogether, our results strongly point towards senescence as the dominant dysfunctional T-cell state in CLL patients, with impaired mitochondrial metabolism at its basis. The distinctive mechanisms leading to T-cell senescence, as opposed to classical exhaustion, underscore the necessity for different strategies to reinvigorate T cells in the context of immunotherapy. Therefore, interventions aimed at ameliorating T-cell dysfunction in CLL should prioritize enhancement of mitochondrial metabolism to attenuate DNA damage and the induction of senescence. This would be especially beneficial to improve the fitness of autologous CAR-T cells for these patients.

The Parallel Presentation of Two Functional CTL Epitopes Derived from the O and Asia 1 Serotypes of Foot-and-Mouth Disease Virus and Swine SLA-2*HB01: Implications for Universal Vaccine Development.

Foot-and-mouth disease virus (FMDV) poses a significant threat to the livestock industry. Through their recognition of the conserved epitopes presented by the swine leukocyte antigen (SLA), T cells play a pivotal role in the antiviral immunity of pigs. Herein, based on the peptide binding motif of SLA-2*HB01, from an original SLA-2 allele, a series of functional T-cell epitopes derived from the dominant antigen VP1 of FMDV with high binding capacity to SLA-2 were identified. Two parallel peptides, Hu64 and As64, from the O and Asia I serotypes, respectively, were both crystallized with SLA-2*HB01. Compared to SLA-1 and SLA-3, the SLA-2 structures showed the flexibility of residues in the P4, P6, and P8 positions and in their potential interface with TCR. Notably, the peptides Hu64 and As64 adopted quite similar overall conformation when bound to SLA-2*HB01. Hu64 has two different conformations, a more stable 'chair' conformation and an unstable 'boat' conformation observed in the two molecules of one asymmetric unit, whereas only a single 'chair' conformation was observed for As64. Both Hu64 and As64 could induce similar dominant T-cell activities. Our interdisciplinary study establishes a basis for the in-depth interpretation of the peptide presentation of SLA-I, which can be used toward the development of universal vaccines.

Tumor Microenvironment Determinants of Immunotherapy Response Identified By Integrated Host & Viral Analysis of Post-Transplant Lymphoproliferative Disorders

Background: Post-transplant lymphoproliferative disorder (PTLD) is a major cause of morbidity in transplantation; however, some PTLD subtypes are curable using anti-CD20 immunotherapy alone or with reduction of immunosuppression (R-ISR). We hypothesized that a comprehensive integrated analysis of the tumor immune microenvironment (TME), virome, genome, transcriptome, and cell-free DNA (cfDNA) could offer biologically and clinically relevant insights into the immunobiology of PTLD. Methods: We identified 75 patients with PTLD after solid organ (n=69) or hematopoietic stem cell (n=6) transplantation. 32 cases occurred <2 years post-transplant (TTD<2, 43%) and 69% were EBV+ by clinical Epstein-Barr-encoded small RNA (EBER) ISH (Table 1). Tumor and cfDNA hybrid capture was performed as previously described, utilizing panels for 322 B-cell lymphoma genes (Kurtz JCO 2018), 180 viral species (Garofalo Blood 2019), and TCR-loci for repertoire profiling (Shukla Blood 2020). Tumor RNAseq was quantified with Salmon & analyzed by DESeq2, fgsea, CIBERSORTx, and Ecotyper. Results: Tumor microenvironment Compared to EBV-negative DLBCL-PTLD, EBV+ cases showed significantly higher expression of IFN-associated (MX1, IFI44, IFIT3) and antiviral response genes including CD30/TNFRSF8 and APOBEC3A. TME immune cell subset deconvolution revealed no difference in B-cell populations, but EBV+ cases were enriched in CD4+ memory activated T-cells, M1 macrophages, activated DCs, and eosinophils. Considering lymphoma TME ecotypes (Steen Cancer Cell 2021), the immunoreactive LE4 ecotype identified EBV+ non-GCB cases with median TTD 93 days, while the immune-poor LE8 aligned with EBV- GCB with median TTD 3686 days. TCR repertoires revealed DLBCL-PTLD tumors to have lower T-cell diversity as compared to DLBCL-NOS (p=0.007, Chao1 index), driven by dominant T-cell clones in EBV+ cases. Virome enrichment by targeted capture confirmed elevated EBV DNA in EBER+ cases, allowing stratification of clinical EBV status by sequencing. While total viral burden and anellovirus level (a marker of immunosuppression) were higher at TTD<2, CMV and HHV6 were similar for later diagnoses. Mutations & mechanisms Compared to DLBCL-NOS, DLBCL-PTLD had significantly fewer variants in the NFkB pathway genes MYD88 and PIM1 and several immune regulatory genes (TNFRSF14, B2M, and CD58). In contrast, TP53, CARD11, and TNFAIP3 were similar. Notably, 12 tumors had no detected oncogenic single-nucleotide variants: these cases were all TTD<2 & EBV+. Overall, more coding variants were seen in EBV- PTLD and TTD>2 (p<0.001 for both comparisons) (Fig 1). We compared mutational signatures within targeted genes to evaluate mechanisms driving these differences. Compared to EBV+ DLBCL-PTLD, EBV- cases were enriched in SBS84, an AID/SHM signature seen in DLBCL-NOS (p<0.001), and had higher AICDA mRNA expression (padj=0.003). Conversely, EBV+ DLBCL-PTLD was enriched in DNA mismatch repair signatures SBS6/21 (p=0.01); and SBS17 (p<0.001), a cryptic mechanism also reported in EBV+ Burkitt lymphomas (Grande Blood 2019). mRNA from EBV+ cases showed corresponding downregulation in the DNA MMR pathway (padj=0.004). Likewise, MSI quantification with mSINGS (Hause Nat Med 2016) showed more altered loci in EBV+ tumors (p=0.013), together suggesting EBV-induced inhibition of MMR as a mutational mechanism. Clinical response We compared DLBCL-PTLD patients with complete response to R-ISR to those requiring chemotherapy. Patients with response had shorter TTD but no difference in coding mutation burden. However, the TME of patients requiring chemotherapy contained more M0/non-activated macrophages and regulatory T-cells; similarly, all patients with the immune-depleted TME ecotype LE8 received chemotherapy. While no difference was seen in NFkB pathway genes, patients requiring chemotherapy showed higher expression of MYC & E2F targets. Conclusions: Through multimodal characterization of the TME and virome in the largest sequencing cohort to date, we identify distinct immunogenomic features reflecting divergent PTLD biology. These include evidence of MMR defects in EBV+ PTLD and TME depletion & MYC pathway enrichment in patients failed by R-ISR, suggesting potential markers of clinical risk. Additional analyses including non-DLBCL cases, viral, and cfDNA data are in progress to further clarify these pathogenetic mechanisms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combined chemoradiotherapy and programmed cell death-ligand 1 blockade leads to changes in the circulating T-cell receptor repertoire of patients with non-small-cell lung cancer.

Combined chemoradiotherapy (CRT) and programmed cell death-ligand 1 (PD-L1) blockade is a new care standard for unresectable stage III non-small-cell lung cancer (NSCLC). Although this consolidation therapy has improved the overall survival of patients with NSCLC, the synergistic action mechanisms of CRT and immunotherapy on T cells remain unclear. In addition, there is a paucity of reliable biomarkers to predict clinical responses to therapy. In this study, we analyzed T-cell receptor (TCR) sequences in the peripheral blood of five patients with NSCLC. T-cell receptor analysis was undertaken before treatment, after CRT, and after PD-L1 blockade. Notably, we observed the expansion and alteration of the dominant T-cell clonotypes in all cases with a complete response. In contrast, neither expansion nor alteration of the TCR repertoire was observed in cases with progressive disease. T cell expansion was initiated after CRT and was further enhanced after PD-L1 blockade. Our findings suggest the systemic effect of CRT on circulating T cells in addition to the curative effect on limited tumor sites. Dynamic changes in circulating T-cell clonotypes could have a prognostic significance for combined CRT and PD-L1 blockade.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

CD8+ T Lymphocytes in Hypopigmented Mycosis Fungoides: Malignant Cells or Reactive Clone?

Hypopigmented mycosis fungoides (HMF) is the most common type of cutaneous T-cell lymphoma in children (Amorim et al., 2018). HMF has a benign course and rarely progresses past localized disease, a striking difference from classic MF, which sometimes progresses from patches to tumors in an average of 12.4 years. On the basis of the identification of dominant clonal T-cell populations, classic MF is seen as a malignancy of CD4+ skin-resident memory cells, whereas HMF is considered to be a malignancy of CD8+ T cells (El-Shabrawi-Caelen et al., 2002).

Acute Aerobic Exercise Preferentially Mobilizes Dominant T-cell Clones To Transiently Lower Receptor Polyclonality In Circulation

Exercise mobilizes and redistributes large numbers of memory T-cells, which may contribute to enhanced immune surveillance in regular exercisers. The diversity of T-cell receptors (TCRs) is vital for the human immune system to recognize a host of foreign antigens, but how exercise affects dynamic changes in antigen specific T-cells at the clonal level is not known. PURPOSE: Determine the effect of acute exercise on T-cell clonality and diversity. METHODS: Healthy volunteers (age 21-41 years) completed an incremental continuous cycling bout consisting of 4x5-minute stages at pre-determined power outputs ranging from 50% to 80%, of VO2peak. DNA was extracted from peripheral blood mononuclear cells (PBMCs) collected at rest, during exercise at 80% VO2peak, and 1 hr post, and underwent high throughput TCR-β chain sequencing via Adaptive Biotechnologies. Data was analyzed utilizing the immunoSEQ® Analyzer online software platform. RESULTS: The top ten ranked productive clones were significantly elevated during exercise (p < 0.005) and were mobilized by a greater magnitude compared to compared to less frequent productive clones. As such, the number of unique productive clones was significantly lower at 80%-intensity exercise compared to rest (p = 0.032) and 1 hr-post (p = 0.046). Additionally, TCR clones found during exercise demonstrate a trended decrease in clonal diversity as indicated by Simpson’s D (p = 0.068) and Shannon’s Entropy (p = 0.088). CONCLUSIONS: Exercise mobilizes the most dominant T-cell clones within the repertoire resulting in a less polyclonal repertoire of T-cells in the circulation. This indicates that exercise preferentially mobilizes T-cell clones with specificity to antigens the host has previously encountered. Subsequent work will determine if exercise drives a preferential mobilization of clones that share TCR motifs and clustered networks associated with cytotoxicity, tumor-antigen specificity, and viral experience.

Using Self-Assembling ADDomer Platform to Display B and T Epitopes of Type O Foot-and-Mouth Disease Virus.

Foot-and-mouth disease virus (FMDV) is a highly contagious and devastating virus that infects cloven-hoofed livestock and various wildlife species. Vaccination is the best measure to prevent FMD. ADDomer, as a kind of non-infectious adenovirus-inspired nanoparticle, has the advantage of high thermal stability. In this study, two dominant B-cell antigen epitopes (residues 129~160 and 200~213) and a dominant T-cell antigen epitope (residues 16~44) of type O FMDV were inserted into the ADDomer variable loop (VL) and arginine–glycine–aspartic acid (RGD) loop. The 3D structure of the recombinant protein (ADDomer-RBT) was simulated by homology modeling. First, the recombinant proteins were expressed by the baculovirus expression system and detected by western blot and Q Exactive mass spectrometry. Then the formation of VLPs was observed under a transmission electron micrograph (TEM). Finally, we evaluated the immunogenicity of chimeric VLPs with a murine model. Bioinformatic software analysis preliminarily corroborated that the chosen epitopes were successfully exposed on the surface of ADDomer VLPs. The TEM assay demonstrated the structural integrity of the VLPs. After immunizing, it was found that FMDV-specific antibodies can be produced in mice to induce humoral and cellular immune responses. To sum up, the ADDomer platform can be used as an effective antigen carrier to deliver antigen epitopes. This study presents one of the candidate vaccines to prevent and control FMDV.

In rheumatoid arthritis inflamed joints share dominant patient-specific B-cell clones.

BackgroundIn patients with rheumatoid arthritis (RA) different joints were shown to share the same dominant T-cell clones, suggesting shared characteristics of the inflammatory process and indicating that strategies to selectively target the antigen receptor might be feasible. Since T- and B-lymphocytes closely interact in adaptive responses, we analysed to what extent different joints also share dominant B-cell clones.MethodsIn 11 RA patients, quantitative B-cell receptor (BCR) repertoire analysis was performed in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, from synovial fluid (SF) and peripheral blood (PB).ResultsST biopsies from different locations in the same joint showed clear overlap in the top-25 dominant BCR clones (16.7%, SD 12.5), in the same range as the overlap between ST and SF in the same joint (8.0%, SD 8.8) and the overlap between ST-ST between different joints (9.1%, SD 8.2), but clearly higher than the overlap between ST and PB (1.7%, SD 2.4; p<0.05) and SF and PB (2.7%, SD 4.1; p<0.05). Interestingly, these figures were substantially lower than the overlap observed in previous T-cell clonality studies.ConclusionsWe conclude that in RA BCR clonal responses may be more localized than TCR clonal responses, pointing to antigen-selective influx, proliferation and/or maturation of B-cells. B lineage cells in the SF may adequately represent the dominant BCR clones of the ST, which is in contrast to T-cells. Collectively, the presence of shared B- and especially T-cells in different joints from the same patient suggests that approaches might be feasible that aim to develop antigen-receptor specific targeting of lymphocyte clones in RA as an alternative to more generalized immunosuppressive strategies.

Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus

To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope. The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein. The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

PB2167: NGS CLONALITY TESTING FOR A DIFFERENTIAL DIAGNOSIS OF MYCOSIS FUNGOIDES

Background: Mycosis fungoides (MF) is a primary epidermotropic lymphoma of the skin, accounting for up to 75% of all skin lymphoproliferative diseases. Diagnosis of MF in the initial stage (Ia-Ib; IIa) or its erythrodermic forms (EMF) is a difficult task. In most cases, neither histological/immunohistochemical examination, nor PCR methods for determining clonality give a definite conclusion. It is known that in the early stages, the PCR method with fragment analysis (PCR-FA) makes it possible to detect T-cell clonality in 75% of cases. On the other hand, in non-tumor dermatoses (ND), clonal products are detected in 10-15% (false-positive result). However, next-generation sequencing (NGS) of the TCRG and TCRB genes may provide more accurate identification of the T cell clone and quantification of the clonal product at the lesion site. Aims: To compare the robustness of NGS and PCR-FA methods for T-cell clonality evaluation in MF and ND. Methods: Skin biopsy specimens and peripheral blood of patients with initial stages of MF (n-7), EMF (n-3), and ND (n-17) were included in the study. Evaluation of clonality for the TCRG and TCRB genes was carried out in parallel by the PCR-FA and NGS. Data processing and T-cell clone fraction/frequency calculation were performed according to the MiXCR protocol (https://github.com/milaboratory/mixcr). Results: PCR-FA and NGS gave the same results in all samples tested. T-cell clonality was detected in 6 out of 10 patients with MF and EMF. In these patients, the fraction of the dominant TCRG clone ranged from 5.5 to 83.4% (mean 32.3%), for TCRB - 6.1 to 47.9% (mean 20.8%). In 2/7 patients with MF and 2/3 with EMF, clonality was not detected, while the fraction of the dominant T-cell clone did not exceed 4.2%. In two patients with MF and no T-cell clonality, skin samples were examined at disease progression at 6 years and 3 years from the initial biopsy, and a dominant tumor clone was found. These dominant clones were represented by only 2.2% (TCRG) and 4.2% (TCRB) in the initial biopsy for one patient, and 0.26% for the second. In 3/7 patients with the initial stages of MF, a tumor clone was detected in the blood in the amount of 0.022-0.054%. All 3 patients with the minimal disseminated disease at the onset, rapidly progressed to the advanced stages of the disease. In the control group (ND) dominant TCRG clone fractions were 0.49-4.8% (mean 1.93%), for TCRB - 0.9-6.1% (mean 3.11%). The results are shown in Figure 1. Image:Summary/Conclusion: Despite a small sample of patients, the following trends were noted: 1 - patients with the initial stages of MF with similar clinical and histological features, are diverse in the number of tumor T-lymphocytes infiltrating the skin (from 0.26% to 83.4%); 2 - the initial stage of MF with the minimal disseminated disease, might have an unfavorable outcome; 3 - comparing to PCR-FA NGS might not provide further improvement in the specificity and sensitivity of T-cell clonality evaluation for the differential diagnosis in MF.

Association of a Dominant T-Cell Clone in Peripheral Blood With Time to Systemic Treatment in Patients With Stage IB Mycosis Fungoides

This cohort study of data from 39 patients assesses whether the presence of a dominant T-cell receptor clone in peripheral blood is associated with time to systemic treatment in patients with stage IB mycosis fungoides.

SARS-CoV-2 Omicron Variant is Expected to Retain Most of the Spike Protein Specific Dominant T-Cell Epitopes Presented by COVID-19 Vaccines - Worldwide, 2021.

What is already known about this topic?The newly emerged variant of Omicron, which carries many of the mutations found in other variants of concern (VOCs), as well as a great number of new mutations that may enhance its immune escape, has spread rapidly around the world. This has raised public concern about the effectiveness of the current coronavirus disease 2019 (COVID-19) vaccine.What is added by this report?In this study, different bioinformatic softwares were applied to predict the dominant Omicron spike (S) protein cytotoxic T lymphocyte (CTL) and T helper (Th) epitopes in representative world population and Chinese population. Compared to the original severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, limited mutations were identified within the dominant CTL and Th epitopes in Omicron variant.What are the implications for public health practice?The results of this study suggested that the current COVID-19 vaccine-induced T-cell immunity may still provide significant protection against Omicron variant infection in fully vaccinated individuals.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86–100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

Ex vivo study of IL-6 expression and function in immune cell subsets from human periapical lesions.

Even though IL-6 is a key inflammatory cytokine in periapical lesions (PLs), its function in stable periapical disease is unknown. Therefore, the aim of this study was to investigate the following: first, the ex vivo production of IL-6 by clinically different PLs; next, subsets of immune cells expressing IL-6 in PLs according to their inflammatory status and finally, modulatory effect of IL-6 on T-cell cytokine production in cell cultures. Inflammatory cells were isolated from a total of 95 human PLs. Detection of IL-6+ cells within the myeloid and lymphoid populations was performed by multicolour flow cytometry. ELISA and FlowCytomix Microbeads Assay were used to measure cytokine levels in culture supernatants. To study the role of IL-6 in PLs, mononuclear cells (MNC) from symptomatic (Sy) or asymptomatic (Asy) PLs were treated with IL-6 or Tocilizumab, an IL-6R blocking antibody. The differences between groups were tested by unpaired t-test, Mann-Whitney or Friedman tests. The levels of IL-6 in PL MNC culture supernatants were significantly higher compared with total PL cells and PL granulocytes (p<.001). MNC from Sy PLs produced significantly higher levels of IL-6 than MNC from Asy PLs (p<.001). Flow cytometry analysis showed that NKT cells, CD8+ T cells and M2 macrophages (MØ), were dominant IL-6+ cells, in contrast to CD4+ T cells. However, CD8+ and CD4+ T cells contributed the most to IL-6 production. IL-6hi producing MNC cultures had higher levels of Th1 (IFN-γ), Th17 (IL-17A), Tfh (IL-21) and RANKL, whereas Th2 (IL-4) and Tregs cytokines (IL-10, TGF-β) were lower compared with IL-6low -producing cultures. Exogenous IL-6 stimulated 17A, IL-21 and RANKL, independently of PL activation status, but decreased IFN-γ, IL-4 and IL-33 levels in IL-6hi -producing cultures. Tocilizumab increased IL-10 and TGF-β in IL-6low -producing cultures. All differences were p<.05. Most immune cells from Sy PLs expressed higher levels of IL-6 compared with Asy PLs, which correlated with IL-6 production in culture. Analysis of cytokines suggested a dominant pro-inflammatory T-cell response and osteodestructive function of IL-6 in PLs judging by Th17/Tfh cell activation, Tregs inhibition and increased RANKL/OPG ratio. Excessive IL-6 production decreased Th1/Th2 responses.

The Epidemiological and Pangenome Landscape of Staphylococcus aureus and Identification of Conserved Novel Candidate Vaccine Antigens

Background and Objective:Staphylococcus aureus (S. aureus) is a gram-positive bacterium and one of the major nosocomial pathogen. It has the ability to acquire resistance against almost all available classes of antibiotics; Methicillin-Resistant S. aureus (MRSA) is a well-known antibiotic resistance. S. aureus is a globally distributed pathogen that need in-depth epidemiological and genomic level investigation for proper treatment and prevention.Methods:To explore the genomic epidemiology of S. aureus in-silico Multi Locus Sequence Typing (MLST) was carried out for 355 complete genomes. Diversity within the species was investigated through pan-genome analysis and subtractive genomic approach was employed for identification of core immunogenic targets.Results:Epidemiological study identified 62 different sequence types (STs) of S. aureus distributed worldwide, in which ST-8, ST-5, ST-398, ST-239, and ST-30 are the most dominant STs comprising more than 50% of the isolates. The pan-genome of S. aureus is still open with 7,199 genes and there is a major contribution (80%) of MRSA strains in the S. aureus species pangenome. The core genome (2,025 genes) of S. aureus is almost stable (comprises of 72% of S. aureus genome size) while accessory and unique genes (28% of S. aureus genome size) are gradually increasing. Screening of 2,025 core genes identified putative vaccine candidates. The best scoring and dominant B-cell and T-cell epitopes were predicted out of the selected potential vaccine candidate proteins with the help of a multi-step screening procedure.Conclusion:We believe that the current study will provide insight into the genetic epidemiology and diversity of S. aureus and the predicted epitopes against the pathogen can be tested further for its immunological responses within the host and may provide both humoral and cellular immunity against the disease.