Biological and clinical significance of tryptophan-catabolizing enzymes in cutaneous T-cell lymphomas

Indoleamine 2,3-deoxygenase 1 (IDO1) induces immune tolerance in the tumor microenvironment (TME) and is recognized as a potential therapeutic target. We studied the expression of both IDO1 and the related tryptophan 2,3-dioxygenase (TDO) in several different subtypes of cutaneous T-cell lymphoma (CTCL), and evaluated the kynurenine (KYN) pathway in the local TME and in patient sera. Specimens from the total of 90 CTCL patients, including mycosis fungoides (MF, n = 37), lymphomatoid papulosis (LyP, n = 36), primary cutaneous anaplastic large cell lymphoma (pcALCL, n = 4), subcutaneous panniculitis-like T-cell lymphoma (SPTCL n = 13), and 10 patients with inflammatory lichen ruber planus (LRP), were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), quantitative PCR, and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three CTCL cell lines also were studied. Expression of both IDO1 and TDO was upregulated in CTCL. In MF specimens and in the MF cell line MyLa2000, IDO1 expression exceeded that of TDO, whereas the opposite was true for LyP, ALCL, and corresponding Mac1/2A cell lines. The spectrum of IDO1-expressing cell types differed among CTCL subtypes and was reflected in the clinical behavior. In MF, SPTCL, and LyP, IDO1 was expressed by malignant cells and by CD33(+) myeloid-derived suppressor cells, whereas in SPTCL CD163(+) tumor-associated macrophages also expressed IDO1. Significantly elevated serum KYN/Trp ratios were found in patients with advanced stages of MF. Epacadostat, an IDO1 inhibitor, induced a clear decrease in KYN concentration in cell culture. These results show the importance of IDO1/TDO-induced immunosuppression in CTCL and emphasize its role as a new therapeutic target.Peer reviewe

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion

Errata: 10.1016/j.jcyt.2017.06.003Background. Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Methods. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. Results. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T-SCM, CD95(+)CD45RO(-)CD45RA(+)CD27(+)) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CART cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CART cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10 fold cell expansion and the cells were functionally potent. Discussion. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost:effective T-cell manufacturing.Peer reviewe

Molecular characterization of subcutaneous panniculitis-like T-cell lymphoma reveals upregulation of immunosuppression- and autoimmunity-associated genes

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RUNX1mutations in blast-phase chronic myeloid leukemia associate with distinct phenotypes, transcriptional profiles, and drug responses

Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations,RUNX1mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate thatPHF6andBCORL1mutations,IKZF1deletions, and AID/RAG-mediated rearrangements are enriched inRUNX1(mut)BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primaryRUNX1(mut)CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity ofRUNX1(mut)blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells fromRUNX1(mut)patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygousRUNX1(-/-)and heterozygousRUNX1(-/mut)BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role ofRUNX1mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treatingRUNX1(mut)BP-CML patients.Peer reviewe

Expression of Human Endogenous Retrovirus-W Including Syncytin-1 in Cutaneous T-Cell Lymphoma

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Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG’s crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided.Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells.Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1β, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control.Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing

Novel factors in the pathogenesis of cutaneous T cell lymphomas

This thesis concentrates on exploring novel agents in the pathogenesis of cutaneous T cell lymphomas (CTCL) and reveals potential new biomarkers and therapeutic targets to improve diagnostics. CTCLs are also known as a heterogeneous group of non-Hodgkin lymphomas. The clinical behavior of these lymphomas varies from benign forms of lymphoproliferative diseases such as lymphomatoid papulosis (LyP) to non-progressive mycosis fungoides (MF) and further to rapidly-progressing leukaemic Sézary syndrome (SS). The incidence of the disease is growing, particularly in Western countries. The mechanisms underlying the disease are still largely unknown and thus far no curative therapy exists. Regarding pathogenesis, it has been suggested that persistent antigen stimulation leads to continuous stimulation of T cells and chronic inflammation, eventually causing the development of a malignant T cell clone. Another hypothesis suggests that a specific viral agent could serve as a triggering factor. The first part of this thesis explores the activity of human endogenous retroviruses (HERVs) in CTCLs. HERVs are retroviral sequences comprising about 8% of the human genome and are derived from ancient retrovirus infections of germ line cells. HERVs have their own promoter areas and genes, some of which encode functional proteins. HERVs may also affect the function of neighbouring genes and influence chromosomal instability (e.g. due to random positioning in the genome). However, HERVs are usually epigenetically silenced, but are reactivated in situtations such as cancer. Healthy tissues also show HERV activity. Moreover, tissue-specificity is typical feature of HERVs. We show that a skin-specific HERV pattern is actively transcribed in MF. We also found that the HERV-W-derived viral protein Syncytin-1 is expressed in morphologically malignant lymphocytes. The second part of this thesis is a unique molecular study on a rare subtype of CTCL. Known as subcutaneous panniculitis-like T cell lymphoma (SPTL), this subtype primarily affects young individuals. The diagnosis of SPTL is challenging and SPTL is usually confused with other panniculitis-related inflammatory diseases. The most important finding of the gene expression profiling was the high expression of indoleamine-2,3-dioxygenase (IDO-1), which catabolises the essential amino acid tryptophan (Trp) into kynurenine (Kyn) metabolites. The enzymatic activity of IDO-1 results in the depletion of Trp in the local microenvironment. Because T cells are particularly sensitive to low Trp levels, the process ultimately leads to immunosuppression and subsequent inhibition of T cell responses. Our results suggest that autoimmune inflammation is underlying the development the disease with the support of an immunosuppressive microenvironment. The last part of this thesis is focused on further studying IDO-1 expression in other CTCL subtypes and lymphoproliferative diseases and also how the tumor microenvironment could be involved in malignant transformation. In this study, we showed that distinct subpopulations of CTCL can be recognized based on IDO1-expressing cells. Especially interesting was the finding that IDO-1 is more extensively expressed in LyP rather than in MF, bearing in mind that LyP is a benign disease. Analyzing the Kyn/Trp ratio from patient sera also showed significant differences between MF and LyP. The Kyn/Trp ratio may therefore serve as a potential prognostic biomarker for evaluating disease activity. Taken together, this study revealed completely new information on gene and protein expression in CTCL. The results will likely be most useful in improving future diagnostics as well as patient care. The results may also be further applied in finding new therapeutic targets and developing novel therapies.Tämän väitöskirjatutkimuksen tarkoituksena on selventää ihon T-solulymfooman (CTCL) eri alamuotojen syntymekanismeja, etsiä lääkeainekohteita, ja tunnistaa uusia biomarkkereita diagnostiikan helpottamiseksi. Ihon T-solulymfoomat luokitellaan osaksi monikirjoista non-Hodgkin-lymfoomien tautiryhmää. Vuosikymmeniä kestäneestä tutkimuksesta huolimatta tarkkaa syntymekanismia ei ole kyetty selvittämään. Syyksi on kuitenkin ehdotettu niin sanottua pitkäaikaista antigeenistimulaatiota, joka johtaa jatkuvan T-soluaktivaation ja kroonisen tulehduksen kautta malignin T-solukloonin kehittymiseen. Laukaisevaksi tekijäksi on ehdotettu muun muassa virusperäistä antigeeniä. Antigeenillä tarkoitetaan mitä tahansa molekyyliä, joka aiheuttaa elimistössä immuunivasteen. Ensimmäisessä osatyössä tarkastelemme ihmisen endogeenisten retrovirusten (HERV) mahdollista roolia CTCL:n yleisimmän alaryhmän, mycosis fungoideksen (MF) syntymekanismissa. HERV:t ovat toistuvia sekvenssialueita ihmisen genomissa (noin 8%) ja ne ovat peräisin miljoonien vuosien takaisista retrovirusinfektioista. Ne sisältävät omat säätelyalueensa ja voivat vaikuttaa viereisten geenien toimintaan. Lisäksi ne sisältävät omia geenejä ja osa jopa kykenee koodaamaan omia proteiineja. Useimmiten HERVit ovatkin epigeneettisesti hiljennettyjä. Tästä huolimatta, ne voivat aktivoitua uudelleen mm. ympäristön vaikutuksesta. Useissa syövissä niiden on osoitettu ilmentyvän poikkeavasti. Myös normaali, terve kudos voi ilmentää HERV:a ja niille onkin tyypillistä kudosspesifinen ilmentyminen. Koska HERV:t voivat "liikkua" genomissa paikasta toiseen, niillä saattaa olla merkitystä myös kromosomipoikkeamien muodostumisessa. Ensimmäisen osatyön merkittävin löydos oli retroviraalisen Synsytin-1 proteiinin esiintyminen morfologisesti tunnistetuissa syöpäsoluissa. Toisessa osatyössä selvitimme ensimmäistä kertaa geenien ilmentymisprofiilin CTCL:n harvinaisemmalle, ihonalaisen rasvakudoksen T-solulymfoomalle (SPTL). SPTL:n tutkiminen on tärkeää, koska sitä esiintyy erityisesti nuorilla potilailla ja sen diagnosointi on haastavaa. SPTL sekoittuukin usein muihin rasvakudoksen tulehdukselliin sairauksiin (ns. pannikuliitteihin). Havaitsimme erityisesti IDO-1 geenin yli-ilmentymisen. IDO-1 proteiini on entsyymi (indoleamiini-2,3-dioksygenaasi), joka pilkkoo elimistössä tryptofaania (Tryp) kynureniinin (Kyn) aineenvaihduntatuotteiksi. Sitä ilmentävät yleensä dendriittisolut ja magrofagit ja IDO:n yli-ilmentymisen epäillään aiheuttavan solujen mikroympäristössä syöpäsolujen lisääntymistä edesauttavia muutoksia. Tuloksemme osoittivat, että SPTL:n taustalla voi olla autoimmuunityyppinen tulehdus, jonka rinnalla malignit solut kehittyvät immuunivastetta heikentävän mikroympäristön suojissa. Kolmannessa osatyössä tutkimme laajemmin IDO-1 ilmentymistä muissa ihon T-solulymfooman alaryhmissä ja lymfoproliferatiivisissä sairauksissa sekä millä tavoin pahanlaatuista solukkoa ympäröivän kudoksen tuottamat tekijät voivat vaikuttavaa itse lymfooman kasvuun. Havaitsimme IDO-1:n ilmentyminen jakavan lymfomatoidi populosis (LyP) ja MF populaatiot voimakkaasti ja heikosti ilmentäviin alaryhmiin, ja koska LyP ei vielä kliinisesti ole tunnustettu maligniksi taudiksi, pidimme erityisen mielenkiitoisena sitä, että IDO-1 ilmentymä oli yleisesti ottaen voimakkaampaa LyP:ssä kuin MF:ssä. Toisaalta havaitsimme myös tieteellisesti merkitsevän eron kynureniinin ja tryptofaani-aminohapon konsentraatioiden suhdetta kuvaavassa Kyn/Tryp-arvossa, kun niiden konsentraatiot mitattiin MF-potilaiden ja terveiden kontrollien veren seerumista. Tämä väitöskirjatutkimus kokonaisuudessaan tarjoaa täysin uutta tietoa geenien ja proteiinien ilmenemisestä ihon primaareissa T-solulymfoomissa. Tuloksemme auttavat parantamaan tulevaisuuden diagnostiikkaa erityisesti vaikeasti diagnosoitavien, ja harvinaisten sairauksien osalta ja näin myös nopeuttavat oikean hoitomuodon valintaa. Tutkimustuloksemme edistävät uusien hoidollisten kohdemolekyylien löytymistä ja ovat perustana uusien terapiamuotojen kehittämiselle

Tumour Suppressor Neuron Navigator 3 and Matrix Metalloproteinase 14 are Co-expressed in Most Melanomas but Downregulated in Thick Tumours

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Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors

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Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity

Funding Information: The authors thank Laura Turunen, Jani Saarela, Katja Suomi, and Maria Nurmi of the High-Throughput Biomedicine Unit and the personnel of the Sequencing Laboratory at the Institute of Molecular Medicine Finland. The authors also thank Caroline Heckman, Sirpa Lepp?, and Olli Lohi for collaboration by providing the cell lines used in the study. The Biomedicum Helsinki Flow Cytometry Core Unit and the Biomedicum Virus Core are acknowledged for their services. The primary B-ALL samples were provided by the Finnish Hematology Registry and Clinical Biobank (FHRB). We thank all the patients for their generous participation. The FHRB biobank is supported by the Finnish Association of Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. This study was supported by the Cancer Foundation Finland, the Sigrid Juselius Foundation, the Relander Foundation, state funding for university-level health research in Finland, and HiLife fellow funds from the University of Helsinki. O.D. was supported by grants from the Biomedicum Helsinki Foundation and the Finnish Medical Society. J. Koski was supported by grants from the Finnish Pediatric Cancer Foundation, the V?re Pediatric Research Foundation, the Cancer Foundation Finland, the Orion Research Foundation, and state funding for university-level health research in Finland. Funding Information: This study was supported by the Cancer Foundation Finland, the Sigrid Juselius Foundation, the Relander Foundation, state funding for university-level health research in Finland, and HiLife fellow funds from the University of Helsinki. O.D. was supported by grants from the Biomedicum Helsinki Foundation and the Finnish Medical Society. J. Koski was supported by grants from the Finnish Pediatric Cancer Foundation, the Väre Pediatric Research Foundation, the Cancer Foundation Finland, the Orion Research Foundation, and state funding for university-level health research in Finland. Funding Information: The authors thank Laura Turunen, Jani Saarela, Katja Suomi, and Maria Nurmi of the High-Throughput Biomedicine Unit and the personnel of the Sequencing Laboratory at the Institute of Molecular Medicine Finland. The authors also thank Caroline Heckman, Sirpa Leppä, and Olli Lohi for collaboration by providing the cell lines used in the study. The Biomedicum Helsinki Flow Cytometry Core Unit and the Biomedicum Virus Core are acknowledged for their services. The primary B-ALL samples were provided by the Finnish Hematology Registry and Clinical Biobank (FHRB). We thank all the patients for their generous participation. The FHRB biobank is supported by the Finnish Association of Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. Publisher Copyright: © 2020 American Society of HematologyChimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics.Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity. Key Points: • Survey of immunomodulatory effects of >500 drugs identifies SMAC mimetics as sensitizers to CAR T-cell cytotoxicity. • Genome-scale CRISPR screen reveals essentiality of death receptor signaling for CAR T-cell cytotoxicity.Peer reviewe