Spatial dynamics of tertiary lymphoid aggregates in head and neck cancer: insights into immunotherapy response.

BACKGROUND: Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) generally has a poor prognosis for patients with limited treatment options. While incorporating immune checkpoint inhibitors (ICIs) has now become the standard of care, the efficacy is variable, with only a subset of patients responding. The complexity of the tumor microenvironment (TME) and the role of tertiary lymphoid structures (TLS) have emerged as critical determinants for immunotherapeutic response. METHODS: In this study, we analyzed two independently collected R/M HNSCC patient tissue cohorts to better understand the role of TLS in response to ICIs. Utilizing a multi-omics approach, we first performed targeted proteomic profiling using the Nanostring GeoMx Digital Spatial Profiler to quantify immune-related protein expression with spatial resolution. This was further characterized by spatially resolved whole transcriptome profiling of TLSs and germinal centers (GCs). Deeper single-cell resolved proteomic profiling of the TLSs was performed using the Akoya Biosciences Phenocycler Fusion platform. RESULTS: Our proteomic analysis revealed the presence of T lymphocyte markers, including CD3, CD45, and CD8, expressing cells and upregulation of immune checkpoint marker PD-L1 within tumor compartments of patients responsive to ICIs, indicative of 'hot tumor' phenotypes. We also observed the presence of antigen-presenting cells marked by expression of CD40, CD68, CD11c, and CD163 with upregulation of antigen-presentation marker HLA-DR, in patients responding to ICIs. Transcriptome analysis of TLS and GCs uncovered a marked elevation in the expression of genes related to immune modulation, diverse immune cell recruitment, and a potent interferon response within the TLS structure. Notably, the distribution of TLS-tumor distance was found to be significantly different across response groups (H = 9.28, p = 0.026). The proximity of TLSs to tumor cells was found to be a critical indicator of ICI response, implying that patients with TLSs located further from tumor cells have worse outcomes. CONCLUSION: The study underscores the multifaceted role of TLSs in modulating the immunogenic landscape of the TME in R/M HNSCC, likely influencing the efficacy of ICIs. Spatially resolved multi-omics approaches offer valuable insights into potential biomarkers for ICI response and highlight the importance of profiling the TME complexity when developing therapeutic strategies and patient stratification.

Spatial insights into immunotherapy response in non-small cell lung cancer (NSCLC) by multiplexed tissue imaging.

The spatial localisation of immune cells within tumours are key to understand the intercellular communications that can dictate clinical outcomes. Here, we demonstrate an analysis pipeline for highly multiplexed CODEX data to phenotype and profile spatial features and interactions in NSCLC patients that subsequently received PD1 axis immunotherapy. We found that regulatory T cells (Tregs) are enriched in non-responding patients and this was consistent with their localization within stromal and peripheral tumour-margins. Proximity-based interactions between Tregs and both monocytes (p = 0.009) and CD8+ T cells (p = 0.009) were more frequently found in non-responding patients, while macrophages were more frequently located in proximity to HLADR+ tumour cells (p = 0.01) within responding patients. Cellular neighbourhoods analysis indicated that both macrophages (p = 0.003) and effector CD4+ T cells (p = 0.01) in mixed tumour neighbourhoods, as well as CD8+ T cells (p = 0.03) in HLADR+ tumour neighbourhoods were associated with favorable clinical response. Evaluation of the inferred regulatory functions between immune cells relative to the tumour suggested that macrophages exhibit an immunosuppressive phenotype against both CD4+ and CD8+ T cells, and that this association scores more highly in ICI refractory patients. These spatial patterns are associated with overall survival in addition to ICI response and may thus indicate features for the functional understanding of the tumour microenvironment.

Heparan sulfate regulates IL-21 bioavailability and signal strength that control germinal center B cell selection and differentiation.

In antibody responses, mutated germinal center B (BGC) cells are positively selected for reentry or differentiation. As the products from GCs, memory B cells and antibody-secreting cells (ASCs) support high-affinity and long-lasting immunity. Positive selection of BGC cells is controlled by signals received through the B cell receptor (BCR) and follicular helper T (TFH) cell-derived signals, in particular costimulation through CD40. Here, we demonstrate that the TFH cell effector cytokine interleukin-21 (IL-21) joins BCR and CD40 in supporting BGC selection and reveal that strong IL-21 signaling prioritizes ASC differentiation in vivo. BGC cells, compared with non-BGC cells, show significantly reduced IL-21 binding and attenuated signaling, which is mediated by low cellular heparan sulfate (HS) sulfation. Mechanistically, N-deacetylase and N-sulfotransferase 1 (Ndst1)-mediated N-sulfation of HS in B cells promotes IL-21 binding and signal strength. Ndst1 is down-regulated in BGC cells and up-regulated in ASC precursors, suggesting selective desensitization to IL-21 in BGC cells. Thus, specialized biochemical regulation of IL-21 bioavailability and signal strength sets a balance between the stringency and efficiency of GC selection.

CD23 expression on switched memory B cells bridges T-B cell interaction in allergic rhinitis.

BACKGROUND: The contribution of B-cell subsets and T-B cell interaction to the pathogenesis of allergic rhinitis (AR) and mechanisms of allergen immunotherapy (AIT) remain poorly understood. This study aimed to outline circulating B-cell signature, the underlying mechanism, and its association with clinical response to AIT in patients with AR. METHODS: IgD/CD27 and CD24/CD38 core gating systems were used to determine frequencies and phenotypes of B cells. Correlations between B cells, T cells, antigen-specific IgE, and disease severity in AR patients were investigated. Switched memory B cells were co-cultured with type 2 follicular helper T (Tfh2) cells and follicular regulatory T (Tfr) cells. Associations between B-cell subsets and clinical benefits of AIT were analyzed. RESULTS: Frequencies and absolute numbers of circulating memory B cells were increased in AR patients. CD23 expression on CD19+ CD20+ CD27+ IgD- switched memory B cells was significantly enhanced and positively correlated with antigen-specific IgE levels, symptom scores, and Tfh2/Tfr cell ratio in AR patients. Compared with those from healthy controls, Tfh2 cells from AR patients had a greater capacity to induce CD23 expression on switched memory B cells via IL-4, which was unable to be sufficiently suppressed by AR-associated Tfr cells with defective IL-10 expression. CD23 expression on switched memory B cells was downregulated after 12-month AIT, which positively associated with disease remission in AR patients. CONCLUSION: T-B cell interaction, bridged by CD23 expression particularly on switched memory B cells, may be involved in the disease pathogenesis and mechanism of AIT in patients with AR.

Murine Cytomegalovirus Glycoprotein O Promotes Epithelial Cell Infection In Vivo.

Cytomegaloviruses (CMVs) establish systemic infections across diverse cell types. Glycoproteins that alter tropism can potentially guide their spread. Glycoprotein O (gO) is a nonessential fusion complex component of both human CMV (HCMV) and murine CMV (MCMV). We tested its contribution to MCMV spread from the respiratory tract. In vitro, MCMV lacking gO poorly infected fibroblasts and epithelial cells. Cell binding was intact, but penetration was delayed. In contrast, myeloid infection was preserved, and in the lungs, where myeloid and type 2 alveolar epithelial cells are the main viral targets, MCMV lacking gO showed a marked preference for myeloid infection. Its poor epithelial cell infection was associated with poor primary virus production and reduced virulence. Systemic spread, which proceeds via infected CD11c+ myeloid cells, was initially intact but then diminished, because less epithelial infection led ultimately to less myeloid infection. Thus, the tight linkage between peripheral and systemic MCMV infections gave gO-dependent infection a central role in host colonization.IMPORTANCE Human cytomegalovirus is a leading cause of congenital disease. This reflects its capacity for systemic spread. A vaccine is needed, but the best viral targets are unclear. Attention has focused on the virion membrane fusion complex. It has 2 forms, so we need to know what each contributes to host colonization. One includes the virion glycoprotein O. We used murine cytomegalovirus, which has equivalent fusion complexes, to determine the importance of glycoprotein O after mucosal infection. We show that it drives local virus replication in epithelial cells. It was not required to infect myeloid cells, which establish systemic infection, but poor local replication reduced systemic spread as a secondary effect. Therefore, targeting glycoprotein O of human cytomegalovirus has the potential to reduce both local and systemic infections.