PD-L2 glycosylation promotes immune evasion and predicts anti-EGFR efficacy

BackgroundCombination therapy has been explored for advanced head and neck squamous cell carcinoma (HNSCC) owing to the limited efficacy of anti-epidermal growth factor receptor (EGFR) therapy. Increased expression and glycosylation of immune checkpoint molecules in tumors are responsible for cetuximab therapy refractoriness. The role of programmed death ligand 2 (PD-L2), a ligand of PD-1, in the immune function is unclear. Here, we examined the regulatory mechanism of PD-L2 glycosylation and its role in antitumor immunity and cetuximab therapy.MethodsSingle-cell RNA sequencing and immunohistochemical staining were used to investigate PD-L2 expression in cetuximab-resistant/sensitive HNSCC tissues. The mechanism of PD-L2 glycosylation regulation was explored in vitro. The effects of PD-L2 glycosylation on immune evasion and cetuximab efficacy were verified in vitro and using mice bearing orthotopic SCC7 tumors.ResultsThe PD-L2 levels were elevated and N-glycosylated in patients with cetuximab-resistant HNSCC. Glycosylated PD-L2 formed a complex with EGFR, which resulted in the activation of EGFR/signal transducer and activator of transcription 3 (STAT3) signaling and decreased the cetuximab binding affinity to EGFR. The N-glycosyltransferase fucosyltransferase (FUT8), a transcriptional target of STAT3, was required for PD-L2 glycosylation. Moreover, glycosylation modification stabilized PD-L2 by blocking ubiquitin-dependent lysosomal degradation, which consequently promoted its binding to PD-1 and immune evasion. Inhibition of PD-L2 glycosylation using Stattic, a specific STAT3 inhibitor, or PD-L2 mutation blocking its binding to FUT8, increased cytotoxic T lymphocyte activity and augmented response to cetuximab.ConclusionsIncreased expression and glycosylation of PD-L2 in tumors are an important mechanism for cetuximab therapy refractoriness. Thus, the combination of PD-L2 glycosylation inhibition and cetuximab is a potential therapeutic strategy for cancer.

Effects of Selenium Supplement on B Lymphocyte Activity in Experimental Autoimmune Thyroiditis Rats

Background and Objective. Thyroid is the organ with the highest selenium content in mammals, and selenium is an essential micronutrient that has close relationship with thyroid autoimmunity. However, the mechanism of how selenium modulates autoimmune thyroiditis remains to be elucidated. Thyroglobulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb), which are produced by B lymphocytes, play a crucial role in autoimmune thyroiditis. The present study was aimed to investigate the effects of selenium supplement on thyroid autoimmunity and B lymphocyte activity in experimental autoimmune thyroiditis (EAT) model rats. Methods. 45 healthy and adult female SD rats were randomly divided into three groups: normal control group, EAT model group, and selenium yeast supplement EAT group. The EAT model rats were induced by subcutaneous injection of porcine thyroglobulin and fed with high iodine water. The concentrations of serum thyroid-stimulating hormone (TSH), TGAb, TPOAb, and B cell activating factor (BAFF) were detected in each group by enzyme-linked immunosorbent assay (ELISA), and the expression of interleukin-10 (IL-10) in thyroid tissue was detected by immunohistochemistry. B cells and regulatory B cells (Bregs) ratios in the spleen of rats were analyzed by flow cytometry. Results. In contrast with the EAT model group, the levels of serum TSH, TGAB, TPOAb, and BAFF were decreased, while IL-10 expression was increased in thyroid tissue, and Bregs ratio was upregulated in the spleen (all p < 0.05 ) in the selenium yeast supplement EAT group. Conclusion. Selenium yeast supplement could partially attenuate immune imbalance in EAT rats, which may be related to the mechanism of modulating B lymphocyte activity.

A Murine CD8+ T Cell Epitope Identified in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has posed a devastating threat worldwide. The receptor-binding domain (RBD) of the spike protein is one of the most important antigens for SARS-CoV-2 vaccines, while the analysis of CD8 cytotoxic T lymphocyte activity in preclinical studies using mouse models is critical for evaluating vaccine efficacy. Here, we immunized C57BL/6 wild-type mice and transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) with the SARS-CoV-2 RBD protein to evaluate the IFN-γ-producing T cells in the splenocytes of the immunized mice using an overlapping peptide pool by an enzyme-linked immunospot assay and flow cytometry. We identified SARS-CoV-2 S395–404 as a major histocompatibility complex (MHC) class I-restricted epitope for the RBD-specific CD8 T cell responses in C57BL/6 mice.

Activin a suppresses peripheral CD8+ T lymphocyte activity in acute-phase Kawasaki disease

Background Kawasaki disease is an autoimmune disease characterized by systemic vasculitis of unknown aetiology and most commonly occurs in children under 5 years old. Previous studies have found that the over-activation of lymphocytes is an important mechanism of Kawasaki disease. Activin A, also known as immunosuppressive factor P, is a multifunctional growth and transforming factor. However, whether activin A is involved in the regulation of peripheral lymphocytes activity in Kawasaki disease is unclear. Thus, we aimed to investigate the effect of activin A on the activity of peripheral lymphocytes in acute-phase Kawasaki disease. Methods Seven patients with Kawasaki disease and seven healthy controls were studied. Peripheral blood lymphocytes were isolated by Ficoll density gradient centrifugation. The activation of CD4+ and CD8+ T cells and CD19+ B cells was investigated by flow cytometry. The expression of activin type IIA receptors was investigated by flow cytometry. Results Immune imbalance in CD4 and CD8 lymphocytes were detected in acute-phase Kawasaki disease. The expression of activin type IIA receptors on CD8+ T cells and CD19+ B cells was increased in acute-phase Kawasaki disease and decreased following treatment with activin A. Activin A suppressed the expression of CD25 and CD69 on CD8+ T cells and the expression of CD69 on CD19+ B cells. Conclusions The expression of activin type IIA receptor was increased on CD8+ T cells and CD19+ B cells in Kawasaki disease. Activin A suppressed the expression of CD25, CD69 and activin type IIA receptors on peripheral CD8+ T lymphocyte. Activin A plays different roles in different lymphocyte subsets and suppresses peripheral CD8+ T lymphocyte activity in acute-phase Kawasaki disease.

Vasculature-Associated Lymphoid Tissue: A Unique Tertiary Lymphoid Tissue Correlates With Renal Lesions in Lupus Nephritis Mouse Model

Lupus nephritis (LN) is a common complication in young patients and the most predominant cause of glomerulonephritis. Infiltrating immune cells and presence of immunocomplexes in the kidney are hallmarks of LN, which is closely associated with renal lesions (RLs). However, their regulatory mechanism in the kidney remains unclear, which is valuable for prevention of RL development. Here, we show the development of vasculature-associated lymphoid tissue (VALT) in LN, which is related to renal inflammatory cytokines, indicating that VALT is a unique tertiary lymphoid tissue. Transcriptomic analysis revealed different chemokines and costimulatory molecules for VALT induction and organization. Vascular and perivascular structures showed lymphoid tissue organization through lymphorganogenic chemokine production. Transcriptional profile and intracellular interaction also demonstrated antigen presentation, lymphocyte activity, clonal expansion, follicular, and germinal center activity in VALT. Importantly, VALT size was correlated with infiltrating immune cells in kidney and RLs, indicating its direct correlation with the development of RLs. In addition, dexamethasone administration reduced VALT size. Therefore, inhibition of VALT formation would be a novel therapeutic strategy against LN.

Immune checkpoint proteins CTLA-4, PD-1 and PD-L1 in melanoma as predictive biomarkers to guide clinical decisions

Background CTLA-4, PD-1 and PD-L1 are potential targets in cancer immunotherapy. However, to predict patients who are likely to respond to treatment with PD-1/PD-L1/CTLA-4 blockers still remains a challenge. Unfortunately, it is often ignored that CTLA-4 exists also in cells other than lymphocytes. This study aimed to assess the expression and diagnostic significance of predictive biomarkers for melanoma. Methods To address this issue, we performed an immunophenotyping of CTLA4/PD1/PD-L1-positive cells in melanoma employing multiple immunofluorescent immunolabeling. For immunolabeling, we used primary antibodies to PD-1 and PD-L1, a panel of anti–CTLA-4 antibodies of different clones and a panel of CD antibodies against diverse cell types (CD1a, CD3, CD8 and CD68). Results In contrast to the currently accepted view, immunolabeling with anti-CTLA-4 antibodies of different clones permitted to reveal CTLA-4 in cells other than lymphocytes. CTLA-4 and PD-L1 are both expressed in the tumor microenvironment cells including antigen-presenting cells ÷ macrophages and dendritic cells. PD-1 is expressed solely in T-lymphocytes. CTLA-4 and PD-L1 in melanoma are not expressed in malignant cells. Conclusion Accordingly, PD-L1 expression by tumor cells cannot serve as a biomarker of clinical response to checkpoint blockade in melanoma immunotherapy, but PD-L1 and CTLA-4 expressed in the tumor microenvironment cells are valuable biomarkers to guide clinical decisions. The expression of CTLA-4 also in cells other than T-lymphocytes suggests the involvement of this molecule in alternative CTLA-4 signaling pathways together with the well-known classical scheme for the regulation of T-lymphocyte activity.