Publications
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Meriranta, Leo; Sorri, Selma; Huse, Kanutte; Liu, Xiaonan; Spasevska, Ivana & Safar, Zadia
[Show all 15 contributors for this article]
(2024).
Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma.
Blood Cancer Discovery.
ISSN 2643-3230.
5(5),
p. 331–352.
doi:
10.1158/2643-3230.BCD-23-0182.
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Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL) are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin ligase with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiologic KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the bric-a-brac tramtrack broad domain of KLHL6 disrupted its localization and heterodimerization and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL. Significance: Oncogenic BCR signaling sustains DLBCL cells. We discovered that Cullin-3-RING E3 ubiquitin ligase substrate-adapter KLHL6 targets BCR heterodimer (CD79A/CD79B) for ubiquitin-mediated degradation. Recurrent somatic mutations in the KLHL6 gene cause corrupt BCR signaling by disrupting surface BCR homeostasis. Loss of KLHL6 expression and mutant-induced phenotypes associate with targetable disease characteristics in B-cell lymphoma. See related commentary by Leveille et al. See related commentary by Corcoran et al.
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Spasevska, Ivana; Sharma, Ankush; Steen, Chloe Beate; Josefsson, Sarah Elisabet; Blaker, Yngvild Nuvin & Kolstad, Arne
[Show all 26 contributors for this article]
(2023).
Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma.
Blood Advances.
ISSN 2473-9529.
7(23),
p. 7216–7230.
doi:
10.1182/bloodadvances.2023010158.
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Abstract Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3− Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.
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Lamont, Colin Harry; Nilsson, Daniel; Huse, Kanutte; Spasevska, Ivana; Olsson, Lina M. & Porwit, Anna
[Show all 10 contributors for this article]
(2026).
Tumor cell expression of Sox11 and cyclin D1 differently impact the spatial tumor immune cell landscape in Mantle cell lymphoma- a translational Nordic MCL2/3 study.
HemaSphere.
10(S1).
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Background
Mantle cell lymphoma (MCL) is a heterogeneous B cell lymphoma with frequent relapses. Intensified first line chemoimmunotherapy with cytarabine followed by autologous stem cell transplantation (ASCT) improves outcomes in younger patients, yet high risk disease (high Ki67/MIPI B and TP53 aberrations) remains difficult to control. The tumor microenvironment (TME) contributes to this heterogeneity as immune depleted, macrophage rich MCL have been linked to adverse prognosis and resistance. However, the spatial organization of TME and associated transcriptional programs in clinically annotated cohorts are incompletely defined.
Aims
To integrate spatial proteomics with compartment resolved transcriptomics to map TME architecture in MCL and relate it to tumor biology and outcome.
Methods
We included TMA sections containing 135 diagnostic tissue cores from 98 patients treated on Nordic Lymphoma Group trials MCL2/3 with long term follow-up clinical data. Imaging mass cytometry (IMC, Hyperion) used a 31 marker panel covering tumor, T cell, myeloid, endothelial and checkpoint proteins. GeoMx™ DSP profiled 1,811 transcripts (Cancer Transcriptomic Atlas) in 190 ROIs (~2 ROIs/patient), segmented into CD20+ tumor, CD8+ cytotoxic T cells, and CD3+/CD8− helper T cells.
Results
Fourteen distinct cell types/subsets were identifed in IMC data and unsupervised analyses of these cell type frequencies identified three recurrent TME clusters: tumor rich, M2 like macrophage rich, and T cell rich. Patients with M2 rich TME had inferior progression free survival and had tumor cells with lower SOX11 and higher Ki67 and cyclin D1 protein expression. Transcriptomics analysis of tumor cell segments revealed that SOX11 low tumors (enriched in the M2 rich cluster) had upregulated proliferation and cell cycle programs (e.g., Cyclin D1/Ki67 signatures), in contrast to SOX11+ tumor cells characterized by B cell differentiation/activation program and immune regulatory genes. Exhaustion associated markers (LAG3, TIGIT) were detected in T cells within M2 rich regions, yet accompanied by higher activation marker expression relative to the other clusters. Canonical correlation analysis highlighted the inverse relationship between cyclin D1 and SOX11, and SOX11 as the strongest tumor cell feature linked to neighborhood composition: SOX11 expression correlated positively with dendritic cells and PD 1+ CD8 T cells and negatively with PD 1− CD4 and CD8 T cells and macrophage abundance.
Conclusion
MCL exhibits distinct spatial TIME architectures with prognostic relevance. A macrophage dominant, SOX11 low state couples increased tumor proliferation with altered T cell phenotypes and inferior outcome. SOX11 emerges as a central axis, connecting tumor intrinsic programs to immune neighborhood structure, supporting its use for risk stratification and for guiding microenvironment targeted strategies (e.g., macrophage reprogramming and checkpoint/exhaustion pathway combinations) in high risk MCL.
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Kongsrud, Karoline; Tvedt, Charlotte Hop; Christensen, Thea Josefine; Caulier, Benjamin William; Spasevska, Ivana & Hilden, Vera Irene
[Show all 14 contributors for this article]
(2025).
B-cell Lymphoma Patient-Derived Xenograft Models for improved preclinical in vivo testing.
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The treatment landscape for B-cell non-Hodgkin lymphoma (B-NHL) has expanded dramatically in recent years, including advanced immunotherapies and targeted drugs. Yet, early relapse is still associated with an adverse prognosis, highlighting the need for new treatment options. For identification of potent new treatment strategies and combinatorial approaches, better preclinical models are needed that can facilitate testing at larger scale. Patient-derived xenograft (PDX) models are in vivo models established by engrafting tumor fragments from cancer patients into immunodeficient mice. The PDX tumors can retain the molecular features including clonal heterogeneity of the original tumor. Our aim is to establish relevant PDX models from B-cell lymphoma tumors for in vivo and ex vivo testing of drugs and immunotherapy.
We have successfully established PDX models from diffuse large B-cell lymphoma (DLBCL, n= 3) and mantle cell lymphoma (MCL, n= 2). For phenotypical characterization, we have developed a 32-marker spectral flow cytometry panel to identify key B cell features (CD10, CD19, CD20, CD22, CD37, CD79B, and Ig heavy and Ig light chains) and other immune cells including T cell subsets and myeloid cells. In addition, targeted sequencing will be used to determine if the mutational composition is stable over time, across different PDX generations. These results will facilitate selection of the best models for future studies. As proof-of -concept, we have used one of the MCL PDX models to test the efficacy of kinase inhibitors and the efficacy of CD19 CAR T cells. These models might support the development of personalized treatment strategies.
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Spasevska, Ivana; Kongsrud, Karoline; Sharma, Ankush; Scherer, Eveline; Hoel, Hanna Julie & Vincenti, Marianna
[Show all 11 contributors for this article]
(2025).
Beyond Helpers: Unraveling the Potential of CD4+ Cytotoxic T Cells in B cell lymphoma.
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CD4+ T cells are heterogeneous and consist of several subsets. While most CD4+ T cells exhibit either helper or immunoregulatory function, emerging evidence suggest the existence of a unique subset of CD4+ T cells with direct cytotoxic potential. Recently, there has been growing interest in these CD4+ cytotoxic T lymphocytes (CTLs) due to their potential involvement in anti-tumor immune responses and their relevance in immunotherapy.
We reanalyzed our prior single-cell RNA sequencing (scRNA-seq) dataset (Spasevska et al, Blood Adv 2023) to unravel the landscape of CD4+ T cell states in diffuse large B-cell lymphoma (DLBCL, n = 3), follicular lymphoma (FL, n = 3) and non-malignant tonsils (n = 3). Among 13 clusters, one was annotated CD4 cytotoxic (CTL) due to high expression of a cytotoxic gene signature (GZMK, NKG7, CST7, GZMA, GZMB). CD4+ CTLs represented over 20 % of CD4+ T cells in DLBCL and over 10% in FL tumors but were nearly absent in non-malignant tonsils. Their tumor-specific nature was further highlighted by a shared TCR observed in more than 45% of CD4+ CTLs in DLBCL. This suggests a clonal expansion of CD4+ T cells recognizing tumor antigens. To place the CD4 CTL in the context of other cytotoxic cells (CD8 and NK cells), we performed scRNA-seq of live cells from matched tumor and PBMC from two FL patients and one healthy donor PBMC (>50 000 single cells). Clustering and analysis of cytotoxic genes confirmed high similarity between CD4 CTL, CD8 Temra, CD8Tem and NK cells.
We validated these findings using multiparameter flow cytometry, revealing that CD4+ CTLs comprised from 1-23% of intratumoral CD4+ T cells in DLBCL. The CD4+ CTLs were identified as Granzyme B+ and Perforin+ cells within the CD4+ CD8- FOXP3- CD56- population, thereby excluding CD8+ T cells, regulatory T cells and NK cells. Using a 41-marker antibody panel for spectral flow cytometry, we have further validated that CD4+ CTL share a cytotoxic signature with NK cells and CD8 T cells. We will alto test if CD4 CTLs have comparable killing capacity to CD8 T cells by employing cell sorting and functional assays.
By employing state-of-the-art technologies, we have characterized CD4+ CTLs in lymphoma and demonstrated substantial clonal expansion in DLBCL. Our findings contribute to the understanding of immune cell heterogeneity and might have implications for therapeutic strategies that leverage the potential of CD4+ CTLs in cancer immunotherapy.
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Lamont, Colin Harry; Nilsson, Daniel; Huse, Kanutte; Spasevska, Ivana; Olsson, Lina M. & Kolstad, Arne
[Show all 10 contributors for this article]
(2025).
Tumor immune cell landscape of Mantle cell lymphoma.
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Mantle cell lymphoma (MCL) is a rare form of B-cell non-Hodgkin lymphoma (B-NHL). The malignancy commonly has an aggressive clinical course but is biologically and clinically heterogeneous. MCL overexpress cyclin D1 due to the t(11;14) translocation and most cases have aberrant expression of the neural transcription factor Sox11. Whereas the genetic landscape of MCL is well characterized, an in-dept characterization of the tumor immune microenvironment (TIME) is lacking. Here, we employed spatial proteomics to map the TIME architecture and explored how gene expression in tumor cells and T cells changed dependent on the neighborhood type. The cohort was based on TMA sections containing 134 diagnostic tumor tissues from 98 MCL patients included in the Nordic clinical phase II studies MCL2/MCL3. TMA neighbor sections were analyzed by imaging mass cytometry (IMC, Hyperion) and GeoMx (Nanostring DSP) was used to collect region of interest (ROI) spatially resolved genes expression profiles (immunopanel) from CD20+ cells (tumor), CD3+CD8+ T cells and CD3+CD8- T cells. By IMC, we identified 14 different cell types and 3 main TIME subtypes: T cell rich (47%), tumor rich (41%) and M2 rich (12%). The M2 rich subtype was enriched for cases with high protein expression of cyclin D1, low or negative expression of Sox 11 and higher gene expression of genes involved in DNA replication (MCM5, PLK1), MHC class I antigen presentation (TAP1) and interferon signaling (STAT1) in tumor cells. Sox11 expression was positively correlated with the cell ratios of DC/macrophages, PD-1+/PD1- CD8 T cells and PD-1+/PD-1- CD4 T cells, suggesting more antigen presentation to T cells in Sox11high tumors. Neighborhood analysis organizing MCL TIME into tumor, margin, follicle and T cell zones revealed that more PD-1+ CD4 T cells than PD-1+ CD8 T-cells co-localized to tumor zones. Survival analysis by applying univariate or multivariate proportional hazard analysis confirmed that patients with high M2/M1 macrophages had poor outcome whereas cases with remnant follicular zones had better outcome. Unsupervised integrative analysis using multi-omics factor analysis (MOFA), which included clinical, IMC and transcriptome data identified major dimensions of disease heterogeneity in MCL. Collectively, our data identified TIME subtypes and spatial features that refine disease heterogeneity and aggressiveness of MCL.
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Kared, Hassen; Huse, Kanutte; Spasevska, Ivana; Myklebust, June Helen & Munthe, Ludvig Andre
(2025).
Mapping T Cell subsets and TME types in Prostate Cancer.
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Sharma, Ankush; Kongsrud, Karoline; Spasevska, Ivana; Lamont, Colin Harry; Steen, Chloe Beate & Lorenz, Susanne
[Show all 10 contributors for this article]
(2025).
Deciphering Tumor Microenvironment Complexity in Lymphoma Using Spatial Transcriptomics.
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The tumor microenvironment (TME) in lymphomas, such as Diffuse Large B-cell Lymphoma (DLBCL) and Follicular Lymphoma (FL), is highly heterogeneous and plays a critical role in modulating therapeutic outcomes, including responses to CD19 CAR T-cell therapy. To unravel the spatial complexity of the TME, we employed an integrative approach combining spatial transcriptomics (Visium, 10x Genomics) and spatial proteomics (imaging mass cytometry, IMC) on FFPE tissue sections from FL and DLBCL patients. Using tools such as CytoSPACE and CARD, we mapped single-cell RNA-seq data to spatial coordinates and validated cellular compositions with IMC data. We identified spatially distinct immune and stromal cell populations, including GC B cells, Tregs, macrophages, dendritic cells, and endothelial cells, across follicular and T-cell rich regions. CytoSPACE-based mapping revealed consistent enrichment of key markers (e.g., CD79B, FOXP3, GRZA) with corresponding protein expression profiles. We also developed R based ShinySpatial framework to visualize spatial data enhancing the accessibility and interpretability of complex spatial datasets. This pilot study lays the groundwork for identifying spatial biomarkers that predict therapeutic responses and resistance in lymphoma, with ongoing integration of data from patients enrolled in the JULIET, BELINDA, and ELARA trials.
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Lamont, Colin Harry; Huse, Kanutte; Spasevska, Ivana; Nilsson, Daniel; Kolstad, Arne & Jerkeman, Mats
[Show all 8 contributors for this article]
(2024).
Mapping the tumor microenvironment in mantle cell lymphoma by imaging mass cytometry.
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Background: Mantle cell lymphoma (MCL) is a rare aggressive form of B cell non-Hodgkin lymphoma, accounting for less than 7% of all cases of newly diagnosed lymphoma. MCL is characterized by specific hallmarks, including the t(11;14)(q13;q32) translocation that results in constitutive expression of Cyclin D1, and overexpression of the SOX11 transcription factor. Although the molecular profile and the genetic landscape of MCL and their impact on the disease aggressiveness have been well characterized, the tumor microenvironment (TME) and its role are still not fully understood. Here, we aimed to explore TME in MCL, determine how tumor cell features shape the TME and link TME features to clinical high-risk factors.
Methods: Diagnostic biopsies from 102 MCL patients included in the two 2 prospective trials by the Nordic Lymphoma Group, MCL2 and MCL3, were imaged by Hyperion imaging mass cytometry (IMC) using a 31-marker antibody panel. Images were segmented into single cells, batch-corrected and annotated using a novel binary clustering strategy. Using CytoMAP, the cells were divided into 30 µm-radius neighborhoods and clustered to define five types of tissue regions.
Results: We identified 13 different clusters of cells present in the MCL TME, with CD4 T cells being the most prevalent immune cell population, with a median of 10% (range 2.2-28%), followed by CD8 T cells with a median of 9% (range 1.5-22%) and CD163- macrophages 8% (range 2.1-23%). T follicular helper cells are notably absent and the follicular dendritic cell (FDC) networks are heterogenous. Neighborhood analysis reveals five types of microenvironments across all patients: tumor exclusive (22%), immune-infiltrating tumor (31%) immune-dominant tumor (19%), T cell zone like (6%), and FDC/follicle (14%). Hierarchical clustering of patients reveals that differences in the microenvironment composition are associated with architectural motifs and follicle organization.
In our dataset, 89 patients were SOX11+, and high expression of SOX11 was associated with higher CD19 (p = 0.03, two sided Mann-Whitney test) and lower Cyclin D1 (p = 2x10⁻⁴) and Ki67 (p = 1.7 x10⁻³) expression and linked to a longer time to progression (p = 0.04).
Conclusion: Our study contributes to an improved understanding of the MCL tumor microenvironment. Future analysis will determine how the observed tumor microenvironment shapes clinical outcomes.
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Kongsrud, Karoline & Spasevska, Ivana
(2024).
Beyond Helpers: Unraveling the Potential of CD4+ Cytotoxic T Cells in B cell lymphoma.
Full text in Research Archive
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Published
Nov. 16, 2018 9:11 AM
- Last modified
May 5, 2023 1:07 PM
