Multi-omics analysis of overexpressed tumor-associated proteins: gene expression, immunopeptide presentation, and antibody response in oropharyngeal squamous cell carcinoma, with a focus on cancer-testis antigens.

Introduction: The human leukocyte antigen complex (HLA) is essential for inducing specific immune responses to cancer by presenting tumor-associated peptides (TAP) to T cells. Overexpressed tumor associated antigens, mainly cancer-testis antigens (CTA), are outlined as essential targets for immunotherapy in oropharyngeal squamous cell carcinoma (OPSCC). This study assessed the degree to which presentation, gene expression, and antibody response (AR) of TAP, mainly CTA, are correlated in OPSCC patients to evaluate their potential as immunotherapy targets. Materials and methods: Snap-frozen tumor (NLigand/RNA=40), healthy mucosa (NRNA=6), and healthy tonsils (NLigand=5) samples were obtained. RNA-Seq was performed using Illumina HiSeq 2500/NovaSeq 6000 and whole exome sequencing (WES) utilizing NextSeq500. HLA ligands were isolated from tumor tissue using immunoaffinity purification, UHPLC, and analyzed by tandem MS. Antibodies were measured in serum (NAb=27) utilizing the KREX™ CT262 protein array. Data analysis focused on 312 proteins (KREX™ CT262 panel + overexpressed self-proteins). Results: 183 and 94 of HLA class I and II TAP were identified by comparative profiling with healthy tonsils. Genes from 26 TAP were overexpressed in tumors compared to healthy mucosa (LFC>1; FDR<0.05). Low concordance (r=0.25; p<0.0001) was found between upregulated mRNA and class I TAP. The specific mode of correlation of TAP was found to be dependent on clinical parameters. A lack of correlation was observed both between mRNA and class II TAP, as well as between class II tumor-unique TAP (TAP-U) presentation and antibody response (AR) levels. Discussion: This study demonstrates that focusing exclusively on gene transcript levels fails to capture the full extent of TAP presentation in OPSCC. Furthermore, our findings reveal that although CTA are presented at relatively low levels, a few CTA TAP-U show potential as targets for immunotherapy.

The Role of the Transcriptional Coactivator BOB.1/OBF.1 in Adaptive Immunity.

BOB.1/OBF.1 is a transcriptional coactivator involved in octamer-dependent transcription. Thereby, BOB.1/OBF.1 is involved in the transcriptional regulation of genes important for lymphocyte physiology. BOB.1/OBF.1-deficient mice reveal multiple B- and T-cell developmental defects. The most prominent defect of these mice is the complete absence of germinal centers (GCs) resulting in severely impaired T-cell-dependent immune responses. In humans, BOB.1/OBF.1 is associated with several autoimmune and inflammatory diseases but also linked to liquid and solid tumors. Although its role for B-cell development is relatively well understood, its exact role for the GC reaction and T-cell biology has long been unclear. Here, the contribution of BOB.1/OBF.1 for B-cell maturation is summarized, and recent findings regarding its function in GC B- as well as in various T-cell populations are discussed. Finally, a detailed perspective on how BOB.1/OBF.1 contributes to different pathologies is provided.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with oropharyngeal squamous cell carcinoma (OPSCC) caused by human papilloma virus (HPV) exhibit a better prognosis than those with HPV-negative OPSCC. This study investigated the distinct molecular pathways that delineate HPV-negative from HPV-positive OPSCC to identify biologically relevant therapeutic targets. Bulk mRNA from 23 HPV-negative and 39 HPV-positive OPSCC tumors (n = 62) was sequenced to uncover the transcriptomic profiles. Differential expression followed by gene set enrichment analysis was performed to outline the top enriched biological process in the HPV-negative compared with HPV-positive entity. INHBA, the highest overexpressed gene in the HPV-negative tumor, was knocked down. Functional assays (migration, proliferation, cell death, stemness) were conducted to confirm the target's oncogenic role. Correlation analyses to reveal its impact on the tumor microenvironment were performed. We revealed that epithelial-to-mesenchymal transition (EMT) is the most enriched process in HPV-negative compared with HPV-positive OPSCC, with INHBA (inhibin beta A subunit) being the top upregulated gene. INHBA knockdown downregulated the expression of EMT transcription factors and attenuated migration, proliferation, stemness, and cell death resistance of OPSCC cells. We uncovered that INHBA associates with a pro-tumor microenvironment by negatively correlating with antitumor CD8+ T and B cells while positively correlating with pro-tumor M1 macrophages. We identified three miRNAs that are putatively involved in repressing INHBA expression. Our results indicate that the upregulation of INHBA is tumor-promoting. We propose INHBA as an attractive therapeutic target for the treatment of INHBA-enriched tumors in patients with HPV-negative OPSCC to ameliorate prognosis. SIGNIFICANCE: Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

Corrigendum: Exploring the dual role of B cells in solid tumors: implications for head and neck squamous cell carcinoma.

[This corrects the article DOI: 10.3389/fimmu.2023.1233085.].

Exploring the dual role of B cells in solid tumors: implications for head and neck squamous cell carcinoma.

In the tumor milieu of head and neck squamous cell carcinoma (HNSCC), distinct B cell subpopulations are present, which exert either pro- or anti-tumor activities. Multiple factors, including hypoxia, cytokines, interactions with tumor cells, and other immune infiltrating lymphocytes (TILs), alter the equilibrium between the dual roles of B cells leading to cancerogenesis. Certain B cell subsets in the tumor microenvironment (TME) exhibit immunosuppressive function. These cells are known as regulatory B (Breg) cells. Breg cells suppress immune responses by secreting a series of immunosuppressive cytokines, including IL-10, IL-35, TGF-ß, granzyme B, and adenosine or dampen effector TILs by intercellular contacts. Multiple Breg phenotypes have been discovered in human and mouse cancer models. However, when compartmentalized within a tertiary lymphoid structure (TLS), B cells predominantly play anti-tumor effects. A mature TLS contains a CD20+ B cell zone with several important types of B cells, including germinal-center like B cells, antibody-secreting plasma cells, and memory B cells. They kill tumor cells via antibody-dependent cytotoxicity and phagocytosis, and local complement activation effects. TLSs are also privileged sites for local T and B cell coordination and activation. Nonetheless, in some cases, TLSs may serve as a niche for hidden tumor cells and indicate a bad prognosis. Thus, TIL-B cells exhibit bidirectional immune-modulatory activity and are responsive to a variety of immunotherapies. In this review, we discuss the functional distinctions between immunosuppressive Breg cells and immunogenic effector B cells that mature within TLSs with the focus on tumors of HNSCC patients. Additionally, we review contemporary immunotherapies that aim to target TIL-B cells. For the development of innovative therapeutic approaches to complement T-cell-based immunotherapy, a full understanding of either effector B cells or Breg cells is necessary.

Influence of Bruton's Tyrosine Kinase (BTK) on Epithelial-Mesenchymal Transition (EMT) Processes and Cancer Stem Cell (CSC) Enrichment in Head and Neck Squamous Cell Carcinoma (HNSCC).

Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.

The transcriptional program during germinal center reaction - a close view at GC B cells, Tfh cells and Tfr cells.

The germinal center (GC) reaction is a key process during an adaptive immune response to T cell specific antigens. GCs are specialized structures within secondary lymphoid organs, in which B cell proliferation, somatic hypermutation and antibody affinity maturation occur. As a result, high affinity antibody secreting plasma cells and memory B cells are generated. An effective GC response needs interaction between multiple cell types. Besides reticular cells and follicular dendritic cells, particularly B cells, T follicular helper (Tfh) cells as well as T follicular regulatory (Tfr) cells are a key player during the GC reaction. Whereas Tfh cells provide help to GC B cells in selection processes, Tfr cells, a specialized subset of regulatory T cells (Tregs), are able to suppress the GC reaction maintaining the balance between immune activation and tolerance. The formation and function of GCs is regulated by a complex network of signals and molecules at multiple levels. In this review, we highlight recent developments in GC biology by focusing on the transcriptional program regulating the GC reaction. This review focuses on the transcriptional co-activator BOB.1/OBF.1, whose important role for GC B, Tfh and Tfr cell differentiation became increasingly clear in recent years. Moreover, we outline how deregulation of the GC transcriptional program can drive lymphomagenesis.

BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

T Cell Specific BOB.1/OBF.1 Expression Promotes Germinal Center Response and T Helper Cell Differentiation.

The transcriptional co-activator BOB.1/OBF.1 is expressed in both B and T cells. The main characteristic of conventional BOB.1/OBF.1 deficient mice is the complete absence of germinal centers (GCs). This defect was mainly attributed to the defective B cell compartment. However, it is unknown whether and how BOB.1/OBF.1 expression in T cells contributes to the GC reaction. To finally clarify this question, we studied the in vivo function of BOB.1/OBF.1 in CD4+ T and follicular T helper (TFH) cell subpopulations by conditional mutagenesis, in the presence of immunocompetent B lymphocytes. BOB.1/OBF.1 deletion in CD4+ T as well as TFH cells resulted in impaired GC formation demonstrating that the impaired GC reaction described for conventional BOB.1/OBF.1-deficient mice cannot exclusively be traced back to the B cell compartment. Furthermore, we show a requirement of BOB.1/OBF.1 for T helper (TH) cell subsets, particularly for TFH cell differentiation.

Differential Requirement of Vav Proteins for Btk-dependent and -Independent Signaling During B Cell Development.

Btk and Vav proteins are all components of the signalosome that builds upon B cell receptor (BCR) activation. However, the role of Vav proteins within the signalosome is quite complex and not yet fully understood. Until now, studies of these have focused predominantly on a deficiency of Vav proteins alone or in combination with other Vav protein family members. Since a physical association of Btk with Vav was shown previously, we asked whether these molecules lie in the same or independent signaling pathways. By analyzing Vav1 and Vav3 single knock-out mice and generating double-knock-out animals deficient for either Vav1 or Vav3 and Btk, we observed, in line with previous publications, no severe B cell developmental defects when either Vav1 or Vav3 alone are not expressed. However, a simultaneous deficiency of Btk together with either Vav1 or Vav3 leads to a severe reduction of splenic B cells, which exhibit an immature phenotype. B cell developmental defects of Btk/Vav1-double deficient mice in the periphery were more severe than those observed in Btk-single-deficient animals. Additionally, morphological changes in splenic microarchitecture were observed in double- but also in single-knock-out mutants. These observations were accompanied by reduced BCR-induced Ca2+ mobilization, proliferation, germinal center formation and immunoglobulin secretion. Although deletion of Btk alone impaired Ca2+ mobilization upon BCR activation, the defect was even more severe when Vav1 or Vav3 were also mutated, indicating that Btk and the Vav proteins act in separate pathways that converge on Ca2+ signaling. In vitro ASC differentiation suggests that both B and T cells contribute to the observed phenotype of a Btk/Vav-double deficiency. Our results show that Vav proteins and Btk are both components of the BCR-activated signalosome but control separate signaling pathways important for B cell development.

BOB.1/OBF.1 is required during B-cell ontogeny for B-cell differentiation and germinal center function.

BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.

Impaired Peyer's patch development in BOB.1/OBF.1-deficient mice.

BOB.1/OBF.1 expression regulates the transcription of direct and indirect target genes. We propose that BOB.1/OBF.1 affects CXCL13-CXCR5 signaling of LTinducer and LTorganizer cells during embryonic Peyer's patch organogenesis as well as of B cells and follicular dendritic cells during lymphocyte homing at postnatal stages of secondary lymphoid organ development.

NF-κB and Its Role in Checkpoint Control.

Nuclear factor-κB (NF-κB) has been described as one of the most important molecules linking inflammation to cancer. More recently, it has become clear that NF-κB is also involved in the regulation of immune checkpoint expression. Therapeutic approaches targeting immune checkpoint molecules, enabling the immune system to initiate immune responses against tumor cells, constitute a key breakthrough in cancer treatment. This review discusses recent evidence for an association of NF-κB and immune checkpoint expression and examines the therapeutic potential of inhibitors targeting either NF-κB directly or molecules involved in NF-κB regulation in combination with immune checkpoint blockade.