In Situ Characterization of Follicular Helper CD4 T Cells Using Multiplexed Imaging.

Follicular helper CD4 T (Tfh) cells play an essential role in the formation of germinal centers (GCs), where mature B cells proliferate, differentiate, and provide long-term protective humoral responses. Despite the extensive phenotypic characterization and identification of human Tfh cell subsets, their spatial positioning at tissue level is not well understood. Here, we describe a quantitative multiplexed immunofluorescence approach allowing for the comprehensive in situ characterization of Tfh cells in human tonsils and lymph nodes (LNs) from individuals with angioimmunoblastic T-cell lymphoma (AITL). We have developed eight multiplexed panels comprising a spectrum of Tfh cell markers, like PD-1, CXCR5, and ICOS, along with transcription factors (Bcl6, Tbet, GATA3), to assess their expression, frequencies, spatial distribution and co-localization in a quantitative manner. Combined analysis of relevant markers revealed the presence of several Tfh cell subsets at tissue level based on the differential expression of surface receptors, nuclear factors as well as their distinct localization within the follicular areas. Interestingly, we found a considerable amount of tonsillar Tfh cells expressing high levels of the Th2 regulator GATA3. The co-expression of GATA3, CXCR5, and BCL6, points to an important role of GATA3 for the generation of effector human Tfh cells. Furthermore, our data revealed significantly different Tfh cell profile signatures between health and disease. Therefore, our imaging platform generates meaningful information for the in situ characterization of human Tfh cells and could provide the base for future studies aiming to a comprehensive understanding of Tfh cell tissue heterogeneity

The prognostic significance of peritumoral tertiary lymphoid structures in breast cancer

Tumors and their surrounding area represent spatially organized “ecosystems”, where tumor cells and the immune contextures of the different compartments are in a dynamic interplay, with potential clinical impact. Here, we aimed to investigate the prognostic significance of peritumoral tertiary lymphoid structures (TLS) either alone or jointly with the intratumoral densities and spatial distribution of CD8 + and CD163 + cells in breast cancer (BCa) patients. TLS were identified peritumorally, within the area distancing up to 5 mm from the infiltrative tumor border, counted and further characterized as adjacent or distal, in formalin-fixed, paraffin-embedded tumor tissue samples from a cohort of 167 patients, with histologically confirmed invasive ductal BCa. TLS and tumor-infiltrating immune cells were determined by H&E and immunohistochemistry. Clinical follow-up was available for 112 of these patients. Patients with peritumoral TLS exhibited worse disease-free survival (DFS) and overall survival (OS) as compared to patients lacking TLS. Moreover, the density of peritumoral TLS was found to be crucial for prognosis, since patients with abundant TLS exhibited the worst DFS and OS. By combining the density of adjacent TLS (aTLS) with our recently published intratumoral signatures based on the differential distribution of CD8 + and CD163 + in the tumor center and invasive margin, we created two improved immune signatures with superior prognostic strength and higher patient population coverage. Our observations strengthen the notion for the fundamental role of the dynamic interplay between the immune cells within the tumor microenvironment (center/invasive margin) and the tumor surrounding area (peritumoral TLS) on the clinical outcome of BCa patients. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature