Single-Cell Transcriptome Analysis of Chronic Antibody-Mediated Rejection After Renal Transplantation

Renal transplantation is currently the most effective treatment for end-stage renal disease. However, chronic antibody-mediated rejection (cABMR) remains a serious obstacle for the long-term survival of patients with renal transplantation and a problem to be solved. At present, the role and mechanism underlying immune factors such as T- and B- cell subsets in cABMR after renal transplantation remain unclear. In this study, single-cell RNA sequencing (scRNA-seq) of peripheral blood monocytes (PBMCs) from cABMR and control subjects was performed to define the transcriptomic landscape at single-cell resolution. A comprehensive scRNA-seq analysis was performed. The results indicated that most cell types in the cABMR patients exhibited an intense interferon response and release of proinflammatory cytokines. In addition, we found that the expression of MT-ND6, CXCL8, NFKBIA, NFKBIZ, and other genes were up-regulated in T- and B-cells and these genes were associated with pro-inflammatory response and immune regulation. Western blot and qRT-PCR experiments also confirmed the up-regulated expression of these genes in cABMR. GO and KEGG enrichment analyses indicated that the overexpressed genes in T- and B-cells were mainly enriched in inflammatory pathways, including the TNF, IL-17, and Toll-like receptor signaling pathways. Additionally, MAPK and NF-κB signaling pathways were also involved in the occurrence and development of cABMR. This is consistent with the experimental results of Western blot. Trajectory analysis assembled the T-cell subsets into three differentiation paths with distinctive phenotypic and functional prog rams. CD8 effector T cells and γδ T cells showed three different differentiation trajectories, while CD8_MAI T cells and naive T cells primarily had two differentiation trajectories. Cell-cell interaction analysis revealed strong T/B cells and neutrophils activation in cABMR. Thus, the study offers new insight into pathogenesis and may have implications for the identification of novel therapeutic targets for cABMR.

Hyaluronidase treatment of synovial fluid is required for accurate detection of inflammatory cells and soluble mediators

Background Synovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex composition and especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. In this study, we investigated the importance of homogenization of the total SF sample before subsequent analysis. Methods SF was obtained from the knee of 29 arthritis patients (26 rheumatoid arthritis, 2 osteoarthritis, and 1 juvenile idiopathic arthritis patient) as part of standard clinical care. Synovial fluid was either treated with hyaluronidase as a whole or after aliquoting to determine whether the concentration of soluble mediators is evenly distributed in the viscous synovial fluid. Cytokine and IgG levels were measured by ELISA or Luminex and a total of seven fatty acid and oxylipin levels were determined using LC-MS/MS in all aliquots. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry. Results In all patients, there was less variation in IgG, 17-HDHA, leukotriene B4 (LTB4), and prostaglandin E2 (PGE2) levels when homogenization was performed before aliquoting the SF sample. There was no difference in variation for cytokines, 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Between 0.8 and 70% of immune cells (median 5%) remained in suspension and were missing in subsequent analyses when the cells were isolated from untreated SF. This percentage was higher for T and B cells: 7–85% (median 22%) and 7–88% (median 23 %), respectively. Conclusions Homogenization of the entire SF sample leads to less variability in IgG and oxylipin levels and prevents erroneous conclusions based on incomplete isolation of synovial fluid cells.

Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination

Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.

Immune Metabolism of IL-4-Activated B Cells and Th2 Cells in the Context of Allergic Diseases

Over the last decades, the frequency of allergic disorders has steadily increased. Immunologically, allergies are caused by abnormal immune responses directed against otherwise harmless antigens derived from our environment. Two of the main cell types driving allergic sensitization and inflammation are IgE-producing plasma cells and Th2 cells. The acute activation of T and B cells, their differentiation into effector cells, as well as the formation of immunological memory are paralleled by distinct changes in cellular metabolism. Understanding the functional consequences of these metabolic changes is the focus of a new research field termed “immune metabolism”. Currently, the contribution of metabolic changes in T and B cells to either the development or maintenance of allergies is not completely understood. Therefore, this mini review will introduce the fundamentals of energy metabolism, its connection to immune metabolism, and subsequently focus on the metabolic phenotypes of IL-4-activated B cells and Th2 cells.

T-Cell Phenotype Varies in Distinct Tumor Microenvironments and CD57 + T FH Cells Are Associated with Disease Progression and Inferior Survival in Follicular Lymphoma

The tumor microenvironment plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed single cell suspensions created from pre-treatment biopsies in a cohort of 82 FL patients and retrospectively extracted patient clinical parameters. Hierarchical analysis of the tumor microenvironment composition stratified these patients into 4 groups: group 1 represents patients with high percentage of monocyte/macrophages/NK cells; patients from group 2 and 3 are rich of intratumoral T and B cells, respectively. Patients with intermediate number of T and B cells are classed into group 4. Intratumoral T cells from these 4 groups exhibited distinct phenotypical characteristics. Group 1 was enriched with KLRG1 +CD8 + T cells, while group 4 showed increased number of CXCR3 - T EM and CD4 + T N cells. Patients from group 2 and 3 featured with increased number of ICOS + T reg cells and CD57 + T FH cells, respectively. We observed that higher numbers of T cells significantly correlated with early disease stage, lack of B-symptoms, event-free survival at 24 months (EFS24) and a favorable overall and EFS survival in patients with histological grade 1 and 2. However, this association was not seen in patients with histological grade 3a and b, suggesting that T cell-mediated anti-tumor immunity is preserved in low grade patients, but overridden in higher grade patients. CITRUS analysis revealed that a cluster that is CD57 + and PD-1 high was significantly more abundant in patients who had disease progression than patients who had a complete response to therapy, suggesting a role of CD57 + T FH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that higher number of CD57 + T FH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57 + T FH cells exhibited a substantially different transcriptome when compared to CD57 - T FH cells. Genes that were differentially upregulated in CD57 + T FH cells when compared to CD57 - T FH cells included genes involved in cell survival, compromised inflammatory response, and metabolism activation including GZMK, CCL4, CST7, DUSP2, LGALS3, CYTOR, CHI3L2, SYNE2, CXCL13, CD27 and FABP5. Taken together, our results indicate different tumor microenvironments among FL patient groups that is associated with variable T-cell phenotype. We found that CD57 + T FH cells play an important role in predicting disease progression and patient prognosis in FL. Figure 1 Figure 1. Disclosures Novak: Celgene/BMS: Research Funding. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding.

The Role of Cell Organelles in Rheumatoid Arthritis with Focus on Exosomes

Auto-immune diseases involved at least 25% of the population in wealthy countries. Several factors including genetic, epigenetic, and environmental elements are implicated in development of Rheumatoid Arthritis as an autoimmune disease. Autoantibodies cause synovial inflammation and arthritis, if left untreated or being under continual external stimulation, could result in chronic inflammation, joint injury, and disability. T- and B-cells, signaling molecules, proinflammatory mediators, and synovium-specific targets are among the new therapeutic targets. Exosomes could be employed as therapeutic vectors in the treatment of autoimmune diseases. Herein, the role of cell organelle particularly exosomes in Rheumatoid Arthritis had discussed and some therapeutic applications of exosome highlighted.

Cellular and Antibody Immunity after COVID-19 Vaccination at >4-Month Follow Up in Immunocompetent and Immunocompromised Subjects

We evaluated post-vaccination immunity after COVID-19 vaccination with serial changes in cellular and antibody responses to the spike protein S, its S2 component which is conserved between SARS-CoV-2 and human coronaviruses, and the S1 component, which is specific to SARS-CoV-2 and also contains its receptor binding domain (RBD). In 21 healthy immunocompetent subjects all of whom demonstrated circulating IgG antibodies 4 months after mRNA1273 or BNT162b vaccination, a) the strength of S-IgG was stable while RBD-IgG declined, b) S2-reactive B-cell frequencies increased progressively (p=0.002) c) S1-reactive CD8+T-cells and CD19+B-cells were undetectable after a transient increase, and d) monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSC, PMN-MDSC) increased after the first vaccine dose. Compared with 4-month measurements from immunocompetent subjects, single samples from 20 vaccinated immunocompromised (IC) subjects revealed a) circulating S-IgG and RBD-IgG in 13 (65%) and 9 (45%) subjects, respectively, b) no differences in S2-reactive T- and B-cells, c) undetectable S1-reactive T- and B-cells, and d) fewer S-reactive CD8+T-cells and CD19+B-cells (p<0.05). Among 11 IC recipients who failed to make RBD-IgG, frequencies of PMN-MDSC were significantly higher (p<0.0004) compared with IC or immunocompetent subjects with RBD-IgG. COVID-19 vaccination induces stable antibodies to the spike protein and expands circulating B-cells reactive to the conserved spike protein sequence in immunocompetent subjects. MDSC which are known to suppress T- and B-cells, and which increase after vaccination, may limit post-vaccination responses especially among immunocompromised subjects. Antibody and cellular responses to SARS-CoV-2-specific spike antigenic sequences appear to be less durable.

Pleiotropic Effects of Glucocorticoids on the Immune System in Circadian Rhythm and Stress

Glucocorticoids (GCs) are a class of steroid hormones secreted from the adrenal cortex. Their production is controlled by circadian rhythm and stress, the latter of which includes physical restraint, hunger, and inflammation. Importantly, GCs have various effects on immunity, metabolism, and cognition, including pleiotropic effects on the immune system. In general, GCs have strong anti-inflammatory and immunosuppressive effects. Indeed, they suppress inflammatory cytokine expression and cell-mediated immunity, leading to increased risks of some infections. However, recent studies have shown that endogenous GCs induced by the diurnal cycle and dietary restriction enhance immune responses against some infections by promoting the survival, redistribution, and response of T and B cells via cytokine and chemokine receptors. Furthermore, although GCs are reported to reduce expression of Th2 cytokines, GCs enhance type 2 immunity and IL-17-associated immunity in some stress conditions. Taken together, GCs have both immunoenhancing and immunosuppressive effects on the immune system.