Session 2 – Clinical Applications
Thursday, 11.9.2025, 10:30 am – 11:45 am
Chair: Bertram Bengsch
This session will discus novel developments in translational cytometry. A focus will be on high-parametric single-cell analysis with high-throughput approaches (e.g. highly multiplexed cytometry) with genome-wide methods (e.g., single-cell RNASeq) and spatial single-cell analysis suitable for clinical application.
Enrico Lugli
Laboratory of Translational Immunology
Head, Humanitas Flow Cytometry Core
Humanitas Clinical and Research Center
Pieve Emanuele, Italy
Biosketch
Enrico Lugli is group leader and head of facility at Humanitas Research Hospital, where he leads the Translational Immunology Lab and the Flow Cytometry core facility.In his Postdoc at ethe moved to the Vaccine Research Center at the National Institutes of Health in Bethesda, MD, USA, he joined the ImmunoTechnology Section directed by Dr. Mario Roederer, where in collaboration with Nicholas Restifo, he identified stem-like memory T cells, thus defining a novel paradigm for the development of more effective adoptive cell transfer immunotherapies and was instrumental in moving Interleukin (IL)-15, a cytokine capable of activating anti-tumor effector cells, from the preclinical stage to the first-in-human phase I clinical trial in patients with solid cancers. He is now a group leader at Humanitas Research Hospital, where he studies how T cell responses mediate human tumor regression and how these are hindered by the presence of immunosuppressive populations, especially CD4+ regulatory T cells (Treg).
Leonard Fiebig
Leonard Fiebig1, Heike Hirseland1, Axel R. Schulz1, Lisa-Marie Diekmann1, Antonia Niedobitek1, Simon Reinke2, Sebastian Hardt3, Schayan Yousefian4, Simon Haas4, Hyun-Dong Chang1 and Henrik E. Mei1
1 German Rheumatology Research Center (DRFZ) Berlin, a Leibniz Institute, Berlin, Germany,
2 Cell Harvesting Core, Berlin Institute of Health, Berlin, Germany
3 Center for Musculoskeletal Surgery, Charité Universitätsmedizin Berlin, Berlin, Germany
4 Max Delbrück Center – Berlin Institute for Medical Systems Biology
Surface Marker Dynamics and Trajectory Analysis Reveal Insights into Longevity-Associated Plasma Cell Subsets in Human Bone Marrow
Mature antibody-secreting plasma cells (PCs) in the bone marrow (BM) are essential for maintaining humoral immunity and memory, yet the factors shaping their phenotypic diversity remain unclear. Significant heterogeneity among human BM plasma cells (BMPCs) has been observed, including the loss of canonical B cell markers e.g. CD19, CD45 and the acquisition of non-canonical markers like CCR2, CD56 or CD86, a phenotype shared with aberrant plasma cells in multiple myeloma.
To explore potential transcriptomic drivers of BMPC phenotypes, we performed targeted single-cell profiling combining a 62-marker AbSeq panel, 399-gene transcriptomic profiling, and B cell receptor sequencing in four donors. In parallel, we conducted antigen-specific phenotyping of BMPC targeting SARS-CoV-2, tetanus toxoid, and Epstein-Barr virus (EBV) using 21-parameter spectral flow cytometry in 31 donors, enabling a detailed investigation of BMPC heterogeneity in relation to antigen encounter history.
We detected antigen-specific BMPC predominantly expressed IgG, with distinct phenotypic patterns. Tetanus- and EBV-specific BMPC, likely generated early in life, were significantly enriched in BMPC subsets lacking CD19 and CD45 while expressing CD56, suggesting that this non-canonical subset is associated with long-term humoral immunity. Supporting this, longitudinal bone marrow sampling in a single donor over two years revealed a reduction of CD45 among SARS-CoV-2-specific BMPC.
Analysis of IgVH properties and clonal lineage tracing indicates remodeling of surface markers over time within BMPC originating from the same progenitor, reflecting patterns observed in antigen-specific compartments. Transcriptomic profiling of e.g. CD19⁻CD56⁺ BMPCs revealed distinct gene expression signatures enriched for programs related to cell adhesion and survival, further supporting their BM retention and long-term survival.
Our study reveals that antigen-specific BMPC involved in long-term humoral immunity exhibit distinct phenotypical setups linked to their antigen encounter history. Notably, phenotypes associated with longevity are enriched among BMPC presumably formed during early-life immune reactions. The convergence of flow cytometry and single-cell transcriptomic data underscores the robustness of these findings and provides new insights into the regulation of human BMPC heterogeneity and longevity.
Claudia Peitzsch
Daria Klusa2,3, Fabian Lohaus4, Tobias Hölscher4, and Claudia Peitzsch1
1 Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany
2 National Center for Tumor Diseases (NCT), Dresden, Germany
3 OncoRay–National Center for Radiation Research in Oncology, Dresden, Germany
4 Department of Radiotherapy and Radiooncology, Medical Faculty Carl Gustav Carus, Dresden, Germany
Deep immune phenotyping of patients with oligometastatic prostate cancer treated with metastasis-directed radiotherapy
Oligometastatic prostate cancer (PCa) classifies a heterogeneous subgroup of patients with ≤5 metastatic lesions. This transitional disease state is routinely identified with advanced imaging modalities such as PSMA-PET/CT. Local and systemic treatment options with curative intent are available and include metastasis-directed, stereotactic radiotherapy in combination with androgen-deprivation therapy. Local treatment of the primary tumor can also lead to a reduction of the tumor burden and improves the disease-specific survival. This so-called abscopal effect enhances immune surveillance and the antitumor immune response, and may explain the indolent course and delayed metastatic progression observed in some patients with oligometastatic PCa. In addition, metastasis-directed radiotherapy enhances this effect by inducing an immunogenic cell death and immune cell priming.
Deep immune cell phenotyping of peripheral blood mononuclear cells (PBMCs) isolated from metastatic PCa patients with progressive disease (n=16, retrospective exploratory study) and patients with oligo-progressive, castration-resistant PCa (prospective phase II study, n=43) was performed using a 41-parameter mass cytometry panel (CyTOF2, Standard BioTools). Blood samples were collected longitudinal before and 3-month after metastatic-directed radiotherapy as well as at time of relapse. The immune phenotypes were further correlated with the amount of EpCAM+, panCK+CXCR4+ and CLDN4+ circulating tumor cells (CTCs) detected with imaging cytometry (ImageStream X Mk II, Amnis/Cytek) as surrogate of metastatic spread.
We found CCL2 plasma concentrations, proinflammatory HLA-DR+CD16+CD14− monocytes and panCK+CXCR4+ CTCs with prognostic relevance in the blood of oligometastatic PCa patients which correlated with biochemical progression-free survival.
Alexandra Emilia Schlaak
Alexandra Emilia Schlaak¹, Antonio D’Alessio², Ira Godbole¹, Patricia Otto-Mora¹, Jürgen Beck¹, Günter Päth¹, Claudia A. M. Fulgenzi²˒⁵, Madhava Pai³, Duncan Spalding³, B. Scheiner², J. Korolewicz², Robert D. Goldin⁴, Caroline Ward², Georgios Nteliopoulos², Heng Cai², Chloe John², Vincent Yip⁶, Sarah Slater⁷, Ayse U Akarca⁸, Mikael Sodergren³, Paul Tait⁹, Nagy Habib³, Robert Thomas⁹, Alessio Cortellini², Teresa Marafioti⁸, Julian R. Marchesi¹⁰, Robert Thimme¹, Rohini Sharma², David J. Pinato²˒¹¹, Bertram Bengsch¹˒¹²
A CD38+ reinvorigated CD8 T cell subset serves as an immune correlate of response to immune-checkpoint therapy in early-stage hepatocellular carcinoma
Introduction:
CD8⁺ T cells are key players in antitumour immunity and response to immunotherapy in hepatocellular carcinoma (HCC). While immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in some HCC patients, a substantial proportion of patients still do not achieve durable clinical responses. Understanding the mechanisms that govern response to ICI is therefore crucial to personalise immunotherapy in HCC. In the prospective PRIME-HCC trial, we investigated the dynamic remodelling of peripheral and tumour-infiltrating CD8⁺ T cells under neoadjuvant ICI in early-stage HCC.
Methods:
30 patients with radiologically or histologically confirmed HCC therapy were included. Ipilimumab was given once on Day 1 (1mg/kg BW), and nivolumab on Day 1 and Day 22 (3mg/kg BW) over two 21-day cycles (6 weeks) prior to liver resection. Blood samples were collected at Cycle 1 and at the first follow-up, 3 weeks post-treatment. Peripheral blood mononuclear cells (PBMCs) were analysed using mass cytometry by time of flight (CyTOF). Tumour biopsies and resection tissue were analysed by Imaging Mass Cytometry (IMC). Radiological response was evaluated using RECIST v1.1.
Results:
Immune profiling revealed a robust remodelling of peripheral CD8⁺ T cells following ICI therapy. We identified a distinct CD38⁺PD-1⁺ CD8⁺ T cell subset, co-expressing proliferative (Ki-67), cytotoxic (Granzyme K), and exhaustion-associated markers (TOX, TIGIT), consistent with a reinvigorated exhausted T cell phenotype.
Spatial analysis confirmed that this CD38⁺ population was enriched pre-treatment in the tumour microenvironment of responders. Following ICI, this subset expanded in both the peripheral blood and tumour tissue of responders, whereas no such expansion was observed in non-responders.
Conclusion:
Our data identifies CD38⁺PD-1⁺ reinvigorated CD8⁺ T cells as immune correlates of clinical response to ICI in early-stage HCC.