Browsing by Author "Huuhtanen, Jani"

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  • Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+ T Lymphocyte Signature
    (2020-11-19) Kelkka, Tiina; Savola, Paula; Bhattacharya, Dipabarna; Huuhtanen, Jani; Lönnberg, Tapio; Kankainen, Matti; Paalanen, Kirsi; Tyster, Mikko; Lepistö, Maija; Ellonen, Pekka; Smolander, Johannes; Eldfors, Samuli; Yadav, Bhagwan; Khan, Sofia; Koivuniemi, Riitta; Sjöwall, Christopher; Elo, Laura L.; Lähdesmäki, Harri; Maeda, Yuka; Nishikawa, Hiroyashi; Leirisalo-Repo, Marjatta; Sokka-Isler, Tuulikki; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients’ repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients.
  • EPIC-TRACE: predicting TCR binding to unseen epitopes using attention and contextualized embeddings
    (2023-12-09) Korpela, Dani; Jokinen, Emmi; Dumitrescu, Alexandru; Huuhtanen, Jani; Mustjoki, Satu; Lähdesmäki, Harri
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Motivation T cells play an essential role in adaptive immune system to fight pathogens and cancer but may also give rise to autoimmune diseases. The recognition of a peptide-MHC (pMHC) complex by a T cell receptor (TCR) is required to elicit an immune response. Many machine learning models have been developed to predict the binding, but generalizing predictions to pMHCs outside the training data remains challenging. Results We have developed a new machine learning model that utilizes information about the TCR from both α and β chains, epitope sequence, and MHC. Our method uses ProtBERT embeddings for the amino acid sequences of both chains and the epitope, as well as convolution and multi-head attention architectures. We show the importance of each input feature as well as the benefit of including epitopes with only a few TCRs to the training data. We evaluate our model on existing databases and show that it compares favorably against other state-of-the-art models.
  • Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia
    (2023-03-30) Kuusanmäki, Heikki; Dufva, Olli; Vähä-Koskela, Markus; Leppä, Aino Maija; Huuhtanen, Jani; Vänttinen, Ida; Nygren, Petra; Klievink, Jay; Bouhlal, Jonas; Pölönen, Petri; Zhang, Qi; Adnan-Awad, Shady; Mancebo-Pérez, Cristina; Saad, Joseph; Miettinen, Juho; Javarappa, Komal K.; Aakko, Sofia; Ruokoranta, Tanja; Eldfors, Samuli; Heinäniemi, Merja; Theilgaard-Mönch, Kim; Wartiovaara-Kautto, Ulla; Keränen, Mikko; Porkka, Kimmo; Konopleva, Marina; Wennerberg, Krister; Kontro, Mika; Heckman, Caroline A.; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.
  • Evolution and modulation of antigen-specific T cell responses in melanoma patients
    (2022-10-11) Huuhtanen, Jani; Chen, Liang; Jokinen, Emmi; Kasanen, Henna; Lönnberg, Tapio; Kreutzman, Anna; Peltola, Katriina; Hernberg, Micaela; Wang, Chunlin; Yee, Cassian; Lähdesmäki, Harri; Davis, Mark M.; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCRαβ-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.
  • Identification of novel STAT5B mutations and characterization of TCRβ signatures in CD4+ T-cell large granular lymphocyte leukemia
    (2022-02-24) Bhattacharya, Dipabarna; Teramo, Antonella; Gasparini, Vanessa Rebecca; Huuhtanen, Jani; Kim, Daehong; Theodoropoulos, Jason; Schiavoni, Gianluca; Barilà, Gregorio; Vicenzetto, Cristina; Calabretto, Giulia; Facco, Monica; Kawakami, Toru; Nakazawa, Hideyuki; Falini, Brunangelo; Tiacci, Enrico; Ishida, Fumihiro; Semenzato, Gianpietro; Kelkka, Tiina; Zambello, Renato; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRβ sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.
  • IFN-α with dasatinib broadens the immune repertoire in patients with chronic-phase chronic myeloid leukemia
    (2022-09-01) Huuhtanen, Jani; Ilander, Mette; Yadav, Bhagwan; Dufva, Olli M.J.; Lähteenmäki, Hanna; Kasanen, Tiina; Klievink, Jay; Olsson-Strömberg, Ulla; Stentoft, Jesper; Richter, Johan; Koskenvesa, Perttu; Höglund, Martin; Söderlund, Stina; Dreimane, Arta; Porkka, Kimmo; Gedde-Dahl, Tobias; Gjertsen, Björn T.; Stenke, Leif; Myhr-Eriksson, Kristina; Markevärn, Berit; Lübking, Anna; Dimitrijevic, Andreja; Udby, Lene; Bjerrum, Ole Weis; Hjorth-Hansen, Henrik; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRβ sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.
  • Large-scale Analysis of Immune Receptor Repertoires in RNA Sequencing Data from Autoimmune Disorders
    (2022-08-22) Theodoropoulos, Jason
     Perustieteiden korkeakoulu | Master's thesis
    The immune system protects us from harmful pathogens by various mechanisms and processes. In autoimmune diseases, the immune system is not functioning correctly and causes harmful symptoms in an individual. The exact cause of autoimmune diseases is often unclear but many of the diseases have been shown to associate with dysfunctioning T cells and risk alleles in the human leukocyte antigen (HLA) region of the major histocompatibility complex. RNA sequencing (RNA-seq) is a next-generation sequencing method commonly used to analyse differential gene expression. Modern methods in bioinformatics however can profile T-cell receptors (TCR) and HLA typing from RNA-seq data. This allows analysing the adaptive immune system from RNA-seq in different and more detailed manners. In this study we gathered nearly 2 000 publicly available RNA-seq samples from the Sequence Read Archive. These samples spanned over 20 datasets and include individuals with inflamed bowel disease (IBD) and Celiac disease. We profiled the immune receptor repertoires of the samples to study them in different conditions. Additionally, we analysed how different sequencing parameters affect this type of analysis. We used MiXCR and PHLAT to profile the TCRs and HLA typings of the samples. To further analyse the immune receptor repertoires we computed various statistics of the TCRs by VDJtools, clustered them with GLIPH2 and predicted epitope-specificities with TCRGP. Our results show that using lower quality sequencing parameters and biopsy samples correlates with profiling a lower amount of TCRs from a sample. Additionally, we see different sorting strategies affecting the V gene segment usage of the TCRs. Despite the large heterogeneity of the dataset, we show that the TCRs from different samples share similarities by which they can be clustered together. We can also see that TCRs from samples with different sorting strategies do not create systematic biases in the results of TCRGP and it could thus be used for building models of autoreactive TCRs if sufficient data would exist. We conducted internal validation to inspect the reliability of the results of HLA typing and inspected that results between multiple samples of a patient mostly agree with each other. We additionally calculated over and underexpressed HLA genes in each condition and found HLA genes that have been previously shown to be risk alleles in IBD. This thesis successfully showcases the possibilities of conducting a study of TCRs and HLAs from RNA-seq data by building a novel dataset and replicating previous results. It also works as a basis for analysing other datasets and for expanding the dataset used in the study.
  • Predicting recognition between T cell receptors and epitopes with TCRGP
    (2021-03-25) Jokinen, Emmi; Huuhtanen, Jani; Mustjoki, Satu; Heinonen, Markus; Lähdesmäki, Harri
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Adaptive immune system uses T cell receptors (TCRs) to recognize pathogens and to consequently initiate immune responses. TCRs can be sequenced from individuals and methods analyzing the specificity of the TCRs can help us better understand individuals' immune status in different disorders. For this task, we have developed TCRGP, a novel Gaussian process method that predicts if TCRs recognize specified epitopes. TCRGP can utilize the amino acid sequences of the complementarity determining regions (CDRs) from TCRα and TCRβ chains and learn which CDRs are important in recognizing different epitopes. Our comprehensive evaluation with epitope-specific TCR sequencing data shows that TCRGP achieves on average higher prediction accuracy in terms of AUROC score than existing state-of-the-art methods in epitope-specificity predictions. We also propose a novel analysis approach for combined single-cell RNA and TCRαβ (scRNA+TCRαβ) sequencing data by quantifying epitope-specific TCRs with TCRGP and identify HBV-epitope specific T cells and their transcriptomic states in hepatocellular carcinoma patients.
  • SBNO2 is a critical mediator of STAT3-driven hematological malignancies
    (2023-04-13) Brandstoetter, Tania; Schmoellerl, Johannes; Grausenburger, Reinhard; Kollmann, Sebastian; Doma, Eszter; Huuhtanen, Jani; Klampfl, Thorsten; Eder, Thomas; Grebien, Florian; Hoermann, Gregor; Zuber, Johannes; Mustjoki, Satu; Maurer, Barbara; Sexl, Veronika
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.
  • Single-cell analysis of aplastic anemia reveals a convergence of NK and NK-like CD8+ T cells with a disease-associated TCR signature
    (2025-02-26) Lundgren, Sofie; Huuhtanen, Jani; Keränen, Mikko; Feng, Xingmin; Patel, Bhavisha A.; Ryland, Georgina L.; Fox, Lucy C.; Bravo-Perez, Carlos; Clemente, Michael; Kerr, Cassandra; Walldin, Gunilla; Dufva, Olli; Zaimoku, Yoshitaka; Tuononen, Tiina; Myllymäki, Mikko; Ebeling, Freja; Jokinen, Emmi; Heinonen, Markus; Kasanen, Tiina; Klievink, Jay; Lähteenmäki, Hanna; Jaatinen, Taina; Kytölä, Sari; Siitonen, Sanna; Dulau-Florea, Alina; Braylan, Raul; Heinäniemi, Merja; Nakao, Shinji; Hellström-Lindberg, Eva; Maciejewski, Jaroslaw P.; Blombery, Piers; Young, Neal S.; Lähdesmäki, Harri; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Immune aplastic anemia (AA) is a life-threatening bone marrow failure disorder driven by an autoimmune T cell attack against hematopoietic stem and progenitor cells (HSPCs). However, the exact autoantigen targets and role of other immune cells in the pathogenesis of AA are unknown. Here, we analyzed a cohort of 218 patients with AA using single-cell RNA and T cell receptor (TCR) αβ sequencing, TCRβ sequencing, flow cytometry, and plasma cytokine profiling. We identified natural killer (NK) cells and CD8+ terminally differentiated effector T (TEMRA) cells expressing NK receptors with AA-associated TCRβ motifs as the most dysregulated immune cell populations in AA bone marrow. Functional coculture experiments using primary HSPCs and immune cells showed that NK cells cannot kill HSPCs alone but may sensitize HSPCs to CD8+ T cell–mediated killing through production of interferons. Furthermore, HSPCs induced activation of T cell clones with CD8+ TEMRA NK-like phenotype in coculture. Our results reveal a convergent phenotype of innate and adaptive immune cells that may drive AA.
  • Single-cell analysis of immune recognition in chronic myeloid leukemia patients following tyrosine kinase inhibitor discontinuation
    (2024) Huuhtanen, Jani; Adnan-Awad, Shady; Theodoropoulos, Jason; Forstén, Sofia; Warfvinge, Rebecca; Dufva, Olli; Bouhlal, Jonas; Dhapola, Parashar; Duàn, Hanna; Laajala, Essi; Kasanen, Tiina; Klievink, Jay; Ilander, Mette; Jaatinen, Taina; Olsson-Strömberg, Ulla; Hjorth-Hansen, Henrik; Burchert, Andreas; Karlsson, Göran; Kreutzman, Anna; Lähdesmäki, Harri; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαβ-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9–TIM3 and PVR–TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRβ-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.
  • Single-cell characterization of anti-LAG-3 and anti-PD-1 combination treatment in patients with melanoma
    (2023-03-15) Huuhtanen, Jani; Kasanen, Henna; Peltola, Katriina; Lönnberg, Tapio; Glumoff, Virpi; Brück, Oscar; Dufva, Olli; Peltonen, Karita; Vikkula, Johanna; Jokinen, Emmi; Ilander, Mette; Lee, Moon Hee; Mäkelä, Siru; Nyakas, Marta; Li, Bin; Hernberg, Micaela; Bono, Petri; Lähdesmäki, Harri; Kreutzman, Anna; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    BACKGROUND. Relatlimab plus nivolumab (anti-lymphocyte-activation gene 3 plus anti-programmed death 1 [anti-LAG-3+anti-PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown. METHODS. We evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy-refractory patients with metastatic melanoma treated with anti-LAG-3+anti-PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-Seq) combined with other multiomics profiling. RESULTS. The highest LAG3 expression was noted in NK cells, Tregs, and CD8+ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3+ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8+ T cell clones gained a more cytotoxic and NK-like phenotype. CONCLUSION. Anti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells. TRIAL REGISTRATION. ClinicalTrials.gov (NCT01968109) FUNDING. Cancer Foundation Finland, Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Relander Foundation, State funding for university-level health research in Finland, a Helsinki Institute of Life Sciences Fellow grant, Academy of Finland (grant numbers 314442, 311081, 335432, and 335436), and an investigator-initiated research grant from BMS.
  • Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8+ T-cell large granular lymphocytic leukemia
    (2022-04-11) Huuhtanen, Jani; Bhattacharya, Dipabarna; Lönnberg, Tapio; Kankainen, Matti; Kerr, Cassandra; Theodoropoulos, Jason; Rajala, Hanna; Gurnari, Carmelo; Kasanen, Tiina; Braun, Till; Teramo, Antonella; Zambello, Renato; Herling, Marco; Ishida, Fumihiro; Kawakami, Toru; Salmi, Marko; Loughran, Thomas; Maciejewski, Jaroslaw P.; Lähdesmäki, Harri; Kelkka, Tiina; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    T cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of mature, clonally expanded T cells, where somatic-activating STAT3 mutations are common. Although T-LGLL has been described as a chronic T cell response to an antigen, the function of the non-leukemic immune system in this response is largely uncharacterized. Here, by utilizing single-cell RNA and T cell receptor profiling (scRNA+TCRαβ-seq), we show that irrespective of STAT3 mutation status, T-LGLL clonotypes are more cytotoxic and exhausted than healthy reactive clonotypes. In addition, T-LGLL clonotypes show more active cell communication than reactive clones with non-leukemic immune cells via costimulatory cell–cell interactions, monocyte-secreted proinflammatory cytokines, and T-LGLL-clone-secreted IFNγ. Besides the leukemic repertoire, the non-leukemic T cell repertoire in T-LGLL is also more mature, cytotoxic, and clonally restricted than in other cancers and autoimmune disorders. Finally, 72% of the leukemic T-LGLL clonotypes share T cell receptor similarities with their non-leukemic repertoire, linking the leukemic and non-leukemic repertoires together via possible common target antigens. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clonotype.
  • Single-cell functional genomics reveals determinants of sensitivity and resistance to natural killer cells in blood cancers
    (2023-12-12) Dufva, Olli; Gandolfi, Sara; Huuhtanen, Jani; Dashevsky, Olga; Duàn, Hanna; Saeed, Khalid; Klievink, Jay; Nygren, Petra; Bouhlal, Jonas; Lahtela, Jenni; Näätänen, Anna; Ghimire, Bishwa R.; Hannunen, Tiina; Ellonen, Pekka; Lähteenmäki, Hanna; Rumm, Pauliina; Theodoropoulos, Jason; Laajala, Essi; Härkönen, Jouni; Pölönen, Petri; Heinäniemi, Merja; Hollmén, Maija; Yamano, Shizuka; Shirasaki, Ryosuke; Barbie, David A.; Roth, Jennifer A.; Romee, Rizwan; Sheffer, Michal; Lähdesmäki, Harri; Lee, Dean A.; De Matos Simoes, Ricardo; Kankainen, Matti; Mitsiades, Constantine S.; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.
  • Somaattisten mutaatioiden tunnistaminen yksisolusekvensointidatasta
    (2022-09-13) Marttila, Maija
     Sähkötekniikan korkeakoulu | Bachelor's thesis
  • Somatic mutations and T-cell clonality in patients with immunodeficiency
    (2020-12) Savola, Paula; Martelius, Timi; Kankainen, Matti; Huuhtanen, Jani; Lundgren, Sofie; Koski, Yrjö; Eldfors, Samuli; Kelkka, Tiina; Keränen, Mikko A.I.; Ellonen, Pekka; Kovanen, Panu E.; Kytölä, Soili; Saarela, Janna; Lähdesmäki, Harri; Seppänen, Mikko R.J.; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Common variable immunodeficiency (CVID) and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is, however, implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset CVID and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2,533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor b-sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in CVID, and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (2 patients), C5AR1 (2 patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, CVID patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.
  • Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia
    (2021-05) Lundgren, Sofie; Keränen, Mikko A.I.; Kankainen, Matti; Huuhtanen, Jani; Walldin, Gunilla; Kerr, Cassandra M.; Clemente, Michael; Ebeling, Freja; Rajala, Hanna; Brück, Oscar; Lähdesmäki, Harri; Hannula, Sari; Hannunen, Tiina; Ellonen, Pekka; Young, Neal S.; Ogawa, Seishi; Maciejewski, Jaroslaw P.; Hellström-Lindberg, Eva; Mustjoki, Satu
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients’ CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.
  • The spectrum of somatic mutations in large granular lymphocyte leukemia, rheumatoid arthritis, and Felty's syndrome
    (2022-07) Savola, Paula; Bhattacharya, Dipabarna; Huuhtanen, Jani
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    T cell large granular lymphocyte leukemia (T-LGLL) is an interesting case at the intersection of autoimmunity and cancer. In T-LGLL, T cells with somatic pathogenic mutations (mainly in STAT3) are linked to rheumatoid arthritis (RA) and neutropenia. A rare subtype of RA, Felty's syndrome, exhibits overlapping clinical features and comparable frequencies of activating STAT3 mutations in T cells as T-LGLL, which hints at a potential T-LGLL–Felty's syndrome–RA axis. Somatic mutations could shed light on the unexplained pathologies of these disorders. However, the causality of somatic mutations–do somatic mutations in immune cells cause inflammation, or does prolonged inflammation predispose to mutagenesis–remains unanswered. This review will focus on the recent advances in understanding somatic mutations in T-LGLL and related autoimmune conditions as a master regulatory network that sustains lymphoproliferation and inflammation.
  • TCRconv: predicting recognition between T cell receptors and epitopes using contextualized motifs
    (2023-01-01) Jokinen, Emmi; Dumitrescu, Alexandru; Huuhtanen, Jani; Gligorijevic, Vladimir; Mustjoki, Satu; Bonneau, Richard; Heinonen, Markus; Lähdesmäki, Harri
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Motivation: T cells use T cell receptors (TCRs) to recognize small parts of antigens, called epitopes, presented by major histocompatibility complexes. Once an epitope is recognized, an immune response is initiated and T cell activation and proliferation by clonal expansion begin. Clonal populations of T cells with identical TCRs can remain in the body for years, thus forming immunological memory and potentially mappable immunological signatures, which could have implications in clinical applications including infectious diseases, autoimmunity and tumor immunology.Results: We introduce TCRconv, a deep learning model for predicting recognition between TCRs and epitopes. TCRconv uses a deep protein language model and convolutions to extract contextualized motifs and provides state-of-the-art TCR-epitope prediction accuracy. Using TCR repertoires from COVID-19 patients, we demonstrate that TCRconv can provide insight into T cell dynamics and phenotypes during the disease.
  • Using single-cell sequencing in determining responses to cancer treatments
    (2023-05-12) Kreivi, Nea
     Sähkötekniikan korkeakoulu | Bachelor's thesis