Identification of novel STAT5B mutations and characterization of TCR beta signatures in CD4+T-cell large granular lymphocyte leukemia

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. TCR beta 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.Peer reviewe

Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8(+) T-cell large granular lymphocytic leukemia

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+TCR alpha beta-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 IFN gamma. 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. T cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder involving clonally expanded T cell clones and is not fully understood. Here the authors show that the rest of the immune repertoire is interconnected with the T-LGLL clonotype(s) and is more mature, cytotoxic and clonally restricted than in other cancers and autoimmune disorders.Peer reviewe

Single-cell characterization of leukemic and non-leukemic immune repertoires in CD8+ T-cell large granular lymphocytic leukemia

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 IFN gamma. 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.</p

Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+T Lymphocyte Signature

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 (TCR beta) 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 TCR beta 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

Clonally expanded T cells in T cell large granular lymphocytic leukaemia and immune-mediated arthritis

T cell large granular lymphocytic leukaemia (T-LGLL) is a rare type of leukaemia. It is a lymphoproliferative disorder that leads to clonal expansion of cytotoxic T cells in the blood, bone marrow and spleen. Activating somatic STAT3 mutations are found in 40% of patients with CD8+ T-LGLL, plausibly conferring a growth advantage to leukaemic clones in these patients. The incidence of autoimmune disease rheumatoid arthritis (RA) is also significantly common in STAT3-mutated T-LGLL patients (up to 50% of patients). For the more indolent CD4+ T-LGLL, somatic mutations have been reported in STAT5B in upto55% of patients. Existing studies hypothesise that leukaemic T cell clones in T-LGLL patients expand as a result of immune response directed towards antigens that have not been discovered yet. RA is the most common form of immune-mediated arthritis (IMA) affecting up to 1% of the population worldwide. Enrichment of CD4+ memory T cell subsets with local clonal expansions in the RA synovial tissue (ST) has been extensively reported in the literature but little is known about whether these populations represent autoantigen-experienced migratory T cells or locally proliferated populations. In this thesis, we aimed to characterise T cells based on their somatic mutation profiles and to further investigate the functional consequences of aberrant T cells in T-LGLL and IMA. In Study I, we discovered and functionally characterised novel STA5B mutations in CD4+ T-LGLL in the largest patient cohort studied in CD4+ T-LGLL thus far (n=35). Deep sequencing of STAT5B led to the discovery of six novel STAT5B variants (Q220H, E433K, T628S, P658R, P702A and V712E). Two of these variants, E433K and V712E, were found to be highly activating in downstream functional studies. Additionally, using deep TCRβ sequencing, we examined the leukaemic and non-leukaemic TCR repertoires to detect a potential bias in the TCR gene usage in T-LGLL. We noticed that although CD4+ T-LGLL clones are predominantly private to patients, leukaemic clones display a bias towards expressing the Vβ13.1 family of TCR genes. In Study II, we studied CD45+ lymphocytes in CD8+ T-LGLL at a single cell resolution using single cell RNA and TCR αβ sequencing (scRNA+TCRαβ-seq). Following a data-driven approach supplemented with functional studies, we successfully separated leukaemic clones from other T cell clones in T-LGLL. We discovered that leukaemic hyper-expanded T-LGLL cells differ from normal hyper-expanded T cell clones and are more cytotoxic (GZMA+ GZMB+ PRF1+) and exhausted (LAG3+ and TIGIT+). Furthermore, we found that STAT3-mutated and STAT3 wild-type leukaemic cells are transcriptomically different with distinct maturation endpoints. Specifically, wild-type cells are more cytotoxic (GZMA+ GZMH+ GZMM+ PRF1+ and GNLY+) than mutated cells. Finally, we also found that in addition to the leukaemic cells, CD4+ T effector memory cells, monocytes and non-leukaemic CD8+ T cells are actively involved in maintaining pathogenesis. In Study III, we studied patients with different forms of immune-mediated arthritis (IMA). We characterised tissue-homing, inflammatory and pathogenic T cells in three patients with seronegative (SN) RA, seropositive (SP) RA and seronegative juvenile (SNJ) RA using single-cell RNA and TCRαβ sequencing from paired synovial tissue (ST) and peripheral blood (PB). We found that in these different types of arthritis, CD4+ T cells represent a uniformly inflammatory cellular milieu, whereas the CD8+ T cell pool represents a more heterogeneous and patient-specific signature. Our findings show that not only CD4+ T cells but also CD8+ T cells home to synovial joints in patients with IMA. Both CD4+ and CD8+ synovial tissue (ST) cells are highly cytotoxic (GZMB), highly activated (LAG3 and PDCD1) and proliferative (NME1, FABP5, G0S2 and MKI67). Overall, these studies highlight the role of cytotoxic T cells in T-LGLL and IMA.Suurten granulaaristen lymfosyyttien T-soluinen leukemia (T-LGLL) on harvinainen leukemian, verisyövän, muoto, joka johtaa sytotoksisten T-solujen lisääntymiseen. Tauti jaetaan kahteen alatyyppiin (CD4+ ja CD8+) leukemiasolujen ilmentämien pintamolekyylien perusteella. Aktivoivia somaattisia STAT3-geenin mutaatioita havaitaan 40 %:lla CD8+ T-LGLL-potilaista, joilla tautiin usein liittyvä nivelreuma on yleisempi. Vastaavasti CD4+ T-LGLL:ssä somaattisia mutaatioita STAT5B-geenissä on aiemmin havaittu jopa 55 %:lla potilaista. Aiemmat tutkimukset viittaavat siihen, että T-LGLL-solut jakautuvat immuuniaktivaation seurauksena, mutta leukemiasolujen kohteena olevaa antigeeniä ei ole vielä löydetty. Nivelreuma on yleisin immuunivälitteisen niveltulehduksen muoto, joka koskettaa jopa 1%:a maailman väestöstä. Useissa aiemmissa tutkimuksissa on kuvattu CD4+ T-muistisolujen kertymistä nivelreumapotilaiden nivelkudokseen, mutta tarkempaa mekanismia näiden solujen kertymisen taustalla ei tunneta. Tässä väitöskirjassa tavoitteena oli kuvata poikkeavia T-soluja sekä T-LGLL:ssä että muissa immuunivälitteisissä niveltulehduksissa. Ensimmäisessä osatyössä löysimme kuusi uutta somaattista STAT5B-mutaatiota suurimmassa tähän asti tutkitussa CD4+ T-LGLL-potilasjoukossa. Mutaatioista kaksi osoittautuivat jatkotutkimuksissa aktivoiviksi. Lisäksi osoitimme T-solureseptorin (TCR) beetaketjun sekvensoinnin avulla, että TCR-geeniperhe Vβ13.1 on CD4+ T-LGL-leukemiasoluissa yliedustettuna. Toisessa osatyössä kuvasimme immuunisoluja CD8+ T-LGLL:ssä käyttäen yksittäissolu-RNA- ja -TCRαβ-sekvensointia (scRNA+TCRαβ-seq). Havaitsimme, että leukemiasolut ovat normaaleja T-soluja sytotoksisempia ja uupuneempia. Lisäksi osoitimme, että STAT3-mutaatio vaikuttaa leukemiasolujen sytotoksisuuteen ja kypsymiseen. Näytimme myös, että leukemiasolujen lisäksi CD4+ T-solut, monosyytit ja ei-leukeemiset CD8+ T-solut osallistuvat immuuniaktivaation ylläpitoon CD8+ T-LGLL:ssä. Kolmannessa osatyössä tutkimme T-soluja kolmessa erilaista immuunivälitteistä niveltulehdusta sairastavassa potilaassa scRNA+TCRαβ-seq-tekniikalla. Vertaamalla nivelkudos- ja verinäytteitä ja eri potilaita toisiinsa havaitsimme, että näissä niveltulehduksen eri muodoissa CD4+ T-solut muistuttavat toisiaan, kun taas CD8+ T-solujen ilmiasu oli erilainen jokaisella potilaalla. Osoitimme, että CD4+ T-solujen lisäksi myös CD8+ T-solut hakeutuvat tulehtuneeseen nivelkudokseen, ja että molemmat T-solutyypit ovat näissä taudeissa sytotoksisia, jakautuvia ja aktivoituneita. Tämä väitöskirjatyö korostaa sytotoksisten T-solujen merkitystä T-LGLL:ssä että immuunivälitteisessä niveltulehduksessa

The spectrum of somatic mutations in large granular lymphocyte leukemia, rheumatoid arthritis, and Felty's syndrome

Funding Information: PS was supported by Blood Research Foundation and HUS Diagnostic Center. DB was supported by the Doctoral Programme in Clinical Research. JH was supported by Finnish Hematology Association, Blood Disease Research Foundation, Helsinki Institute for Life Science, Biomedicum Helsinki Foundation, Finnish Medical Foundation, K. Albin Johansson Foundation, Kaute Foundation, Suomalais-Norjalainen Lääketieteen Säätiö and Emil Aaltonen Foundation. Publisher Copyright: © 2022 Elsevier Inc.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.Peer reviewe

Increased Physiological GDNF Levels Have No Effect on Dopamine Neuron Protection and Restoration in a Proteasome Inhibition Mouse Model of Parkinson’s Disease

Publisher Copyright: © 2023 Olfat et al.Parkinson’s disease (PD) is a progressive neurodegenerative disease that comprises a range of motor and nonmotor symptoms. Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopamine neurons in vitro and in vivo, and intracranial delivery of GDNF has been tested in six clinical trials for treating PD. However, clinical trials with ectopic GDNF have yielded variable results, which could in part result from abnormal expression site and levels caused by ectopic overexpression. Therefore, an important open question is whether an increase in endogenous GDNF expression could be potent in reversing PD progression. Here, we tested the therapeutic potential of endogenous GDNF using mice in which endogenous GDNF can be conditionally upregulated specifically in cells that express GDNF naturally (conditional GDNF hypermorphic mice; GdnfcHyper). We analyzed the impact of endogenous GDNF upregulation in both neuroprotection and neurorestoration procedures, and for both motor and nonmotor symptoms in the proteasome inhibitor lactacystin (LC) model of PD. Our results showed that upregulation of endogenous GDNF in the adult striatum is not protective in LC-induced PD model in mice. Since age is the largest risk factor for PD, we also analyzed the effect of deletion of endogenous GDNF in aged Gdnf conditional knock-out mice. We found that GDNF deletion does not increase susceptibility to LC-induced damage. We conclude that endogenous GDNF does not impact the outcome in the LC-induced proteasome inhibition mouse model of Parkinson’s disease.Peer reviewe

Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+T Lymphocyte Signature

Correction: Volume12, Article Number710831 DOI10.3389/fimmu.2021.710831 PublishedMAY 31 2021Rheumatoid 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 (TCR beta) 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 TCR beta 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.Peer reviewe

Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

The successful use of expanded tumor-infiltrating lymphocytes (TIL) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n = 58) from treatment-naive patients with renal cell carcinoma (RCC), "pre-rapidly expanded" TILs (pre-REP TIL, n= 15) and "rapidly expanded" TILs (REP TIL, n = 25) according to a clinical-grade TIL production protocol, with single-cell RNA (scRNA)+TCR alpha beta-seq (TCR alpha beta sequencing), TCR beta-sequencing (TCR beta-seq), and flow cytometry. REP TILs encompassed a greater abundance of CD4(+) than CD8(+) T cells, with increased LAG-3 and low PD-1 expressions in both CD4(+) and CD8(+) T cell compartments compared with the pre-REP TIL and tumor T cells. The REP protocol preferentially expanded small clones of the CD4(+) phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T cell clones in the tumor do not expand during the expansion protocol. In addition, by generating a catalog of RCC-associated TCR motifs from >1,000 scRNA+TCR alpha beta-seq and TCR beta-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs.Significance: TILs are a heterogenous group of immune cells that recognize and attack the tumor, thus are utilized in various clinical trials. In our study, we explored the TILs in patients with kidney cancer by expanding the TILs using a clinical-grade protocol, as well as observed their characteristics and ability to recognize the tumor using in-depth experimental and computational tools.Peer reviewe

Table S1 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Supplementary Table S1 shows the RCC patient data including clinical parameters and status</p