ALBERT

Description: Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3′-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway.

Description: Key Points Germline activating STAT3 mutations were detected in 3 patients with autoimmunity, hypogammaglobulinemia, and mycobacterial disease. T-cell lymphoproliferation, deficiency of regulatory and helper 17 T cells, natural killer cells, dendritic cells, and eosinophils were common.

Description: The pathogenesis of common variable immunodeficiency (CVID) and many other immunodeficiencies is elusive, and no cure for these diseases exists. The characteristic features of CVID are immunoglobulin deficiency and recurrent infections, but autoimmunity co-manifests in 30% and lymphoproliferation in 50% of CVID cases. The failure to produce sufficient quantities of immunoglobulins is attributed to B cells, but as T cells are critical players in adaptive immune response and autoimmune disease, they have also suggested to play a role in CVID. As monogenic germline mutations account only for 2-10% of CVID cases, the etiology of CVID remains unknown in most cases. To study if somatic mutations in T cells contribute to immunodeficiencies, we recruited patients with broad immune dysregulation: 8 patients with late-onset CVID, and 9 patients with other type of immunodeficiency and/or severe autoimmunity. All patients signed informed consent and the declaration of Helsinki principles were followed. Study design and key patient characteristics are depicted in the Figure. To discover somatic mutations in T cells, we sequenced both CD4+ and CD8+ cells with a custom gene panel comprising of 2500 immune-related genes with an average coverage of 500x. Somatic variants were identified using the GATK MuTect2 toolset by using a panel of 21 healthy controls' CD4+ and CD8+ cells as a background. Variants were called using paired samples (CD4+ vs CD8+ and vice versa). This approach identifies variants that have occurred in mature T cells. To complement this approach, we performed variant calling also in single-sample mode to identify variants originating from hematopoietic progenitors. Paired-sample analyses revealed 44 somatic mutations in mature T cells in 10/17 (59%) patients: 30 (68%) in CD8+ and 14 (32%) in CD4+ cells. The mutations included 2 frameshift-, 37 missense-, and 5 nonsense variants. In silico tools (both Polyphen-2 and SIFT) predicted 19 (51%) of the missense mutations to be damaging. The Catalogue Of Somatic mutations In Cancer (COSMIC) database included 9/44 (20%) of all mutations. Also, pathway analysis annotated 20% of the mutated genes to be involved in inflammatory response regulation, 27.5% in protein phosphorylation regulation, and 22.5% in positive regulation of cell proliferation. Interesting mutation findings included two patients with STAT5B mutations (N418K and T628S) with a low variant-allele (VAF) frequencies (3.2-3.6% in CD4+ cells), one patient with a KRAS T58I mutation (VAF 7.9% in CD8+), and two patients with distinct C5AR1 mutations (R197W and T62M). Complementing the paired-sample variant calling strategy, single-sample analyses revealed mutations associated with clonal hematopoiesis with low VAFs (2.2-5.5%) in T cells in 4 (23.5%) patients. Three patients had DNMT3A mutations and one patient 4 distinct TET2 mutations (2 nonsense-, 1 frameshift-, and 1 missense mutations). CD4+ and CD8+ T-cell receptor (TCR) repertoires were profiled with deep TCR beta chain (TCRB) sequencing. Healthy controls' CD4+ and CD8+ cells (n=27) were used as comparators. Although some CVID patients harbor major (17-20% of CD8+) T-cell clones, overall CD4+ or CD8+ clonality did not significantly differ from age-matched healthy controls. As a marker of selective pressure, CVID patients' CD4+ and CD8+ cells harbored more clones that shared a CDR3 amino-acid but had distinct nucleotide CDR3 sequences than healthy controls (7.0% vs 3.3% of all amino-acid rearrangements, p=0.021 for CD8+; 4.3% vs 3.5% p=0.013 for CD4+). These TCRs were not enriched with pathogen-specific (such as cytomegalovirus or Epstein-Barr virus) TCRs. Moreover, high CD4+ clonality was associated with a lower frequency of switched-memory-B cells. In conclusion, somatic mutations in T cells occurred in 59% of patients with immunodeficiency in this study, and some of the mutated genes (such as STAT5B) have previously been implicated for the pathogenesis of autoimmunity and lymphoproliferation. Also, clonal hematopoiesis in T cells was discovered in 23.5% of patients. The overall T-cell clonality was not increased, but CVID patients showed slight selective pressure in the TCR repertoire. Our results demonstrate that further research on somatic mutations in immunodeficiencies is needed, as they may contribute to disease pathogenesis in a subset of immunodeficiency patients. Disclosures Martelius: Gilead: Other: lecture fee; Octapharma: Other: travel grant; CSL Behring: Other: lecture fee and travel grant; MSD: Other: lecture fee and travel grant; Sanguin: Other: travel grant and grants. Kankainen:Medix Biochemica: Consultancy. Seppänen:CSL Behring: Other: Chairing and speaker fees. Mustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding.

Description: BACKGROUND A complex interaction between blasts and surrounding cells in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment sustains blast proliferation and confers chemoresistance. T- and NK-cells have been shown to be dysfunctional in AML, which might be associated with immune evasion and poor prognosis. Here, we present a comprehensive analysis of the immune contexture of the AML BM at diagnosis and study its interaction with clinicopathological variables. METHODS Diagnostic BM biopsies (n=69) were collected from AML patients treated in the Helsinki University Hospital during 2005-2017 and age and gender-matched controls (n=12) to construct tissue microarrays (TMA). Using 8-plex immunohistochemistry (mIHC) and computerized image analysis, we determined cell abundance and immunophenotypic states of millions of immune cells. Immunoprofiles were integrated with a total of 120 clinicopathological variables including cytogenetics and molecular genetics, ELN (European Leukemia Net) risk classification, disease burden parameters, and patient demographics. RESULTS Unsupervised hierarchical clustering of the immunologic contexture defined by mIHC analysis grouped AML patients distinctly from control subjects (Fig 1a). By extracting significant differences (Mann-Whitney U test, q

Description: There is a Blood Commentary on this article in this issue.

Description: Background In most solid tumors, CD8+ cytotoxic T-cells and type 1 T-helper cells are associated with a positive prognosis, but a strong immunosuppressive microenvironment may hamper their effectiveness. This notion has contributed to the development of new immune-activating therapies, such as immune checkpoint inhibitors. Although having demonstrated long-term remissions in many different solid tumor types, immune checkpoint inhibitors have not been evaluated comprehensively in hematological malignancies. In this study, we aimed to characterize the cellular and molecular immunological profiles of chronic myeloid leukemia (CML) patients' bone marrow (BM) samples. Methods BM biopsies were taken at the time of diagnosis from chronic phase CML patients (n=57) treated in the Helsinki University Hospital during years 2005-2015. We used non-leukemic (NL) BM biopsies (n=10) as controls. Using hematopathologic expertise, we constructed tissue microarray (TMA) blocks from duplicate BM spots characterized with high leukemic cell infiltration. We stained TMA slides using multiplexed immunohistochemistry (IHC) combining fluorescent and chromogenic staining allowing detection of up to six markers and nuclei simultaneously. Marker panels included T and B-lymphoid (CD3, CD4, CD8, CD20), myeloid dendritic (CD11c, BDCA-1, BDCA-3), macrophage (CD68, pSTAT1, c-MAF), natural killer cell (CD3 and CD56) and leukemia cell (CD34) markers. In addition, we examined immune checkpoint molecules (PD1, CTLA4, OX40, LAG3, TIM3) and their ligands in leukemic cells (HLA-G, PD-L1, PD-L2, HLA-ABC), as well as activation markers (CD25, CD27, CD57, Granzyme B and CD45RO). We analyzed leukemia patients' immune checkpoint expression profiles quantitatively using the image analysis software Cell Profiler and cell analysis software FlowJo and compared results with NL BMs' immune cell profiles. Results The proportion of CD3+ T cells of all cells was significantly higher in CML BM vs. NL BM (median 6.0% [interquartile range (IQR) 3.6-10.7] vs. 2.1% [IQR 1.5-4.5], p=0.001). There was no significant difference in CD8+ cytotoxic T cell levels, but CD4+ helper T cells were 8-fold more abundant in CML as compared to non-leukemic BM (p

Description: Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Description: Background: Dasatinib is a 2nd generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML). Its kinase inhibition profile is broad and includes several kinases important in the immune cell function such as SRC kinases. Furthermore, it is known that dasatinib has immunomodulatory effects in vivo. Recently, we observed that dasatinib induces a rapid and marked mobilization of lymphocytes, which closely follows the drug plasma concentration. The phenomenon is accompanied by an increase of NK-cell cytotoxicity. In addition, we have shown that dasatinib alters T-cell responses long-term favoring Th1 type of responses. Interestingly, the dasatinib induced immune effects have been associated with better treatment responses. We now aimed to characterize the dasatinib-induced antitumor immune responses in a syngeneic murine melanoma model to address whether dasatinib-induced immunoactivation affects tumor growth. Methods: Direct cytotoxic effect of dasatinib on B16.OVA melanoma cells in vitro was assessed with an MTS cell viability assay. T-cell cytotoxicity was assessed by preincubating splenocytes isolated from naïve and OT-I mouse spleen with 100 nM dasatinib and measured their cytotoxic capacity against B16.OVA cells. To further evaluate the dasatinib induced antitumor immune effects in vivo, B16.OVA cells were implanted subcutaneously in C57BL/6J mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 11. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was performed by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b, NK1.1, CTLA4, PD-1 and CD107. Immunohistochemical staining of CD8+ T-cells was performed from the paraffin embedded tumor samples. Results: In vitro incubation of B16.OVA cells with dasatinib showed only a moderate unspecific cytotoxicity with the two highest concentrations of dasatinib (1- and 10 µM), whereas in K562 cells (a CML blast crisis cell line) almost complete killing was observed already with the 100nM concentration. The cell viability of B16.OVA cells was 90% with at 100 nM of dasatinib concentration (as compared to 21% of K562 cells) suggesting that there was no direct dasatinib sensitive target oncokinase in this cell line. In contrast, a significant enhancement in the cytotoxic capacity of splenocytes was observed when they were pretreated with 100nM dasatinib (60% of target cells were alive when incubated with dasatinib pretreated naïve splenocytes compared to 100% with control treated splenocytes, p=0.004). The in vivo tumor experiments demonstrated that the tumor volumes were smaller in dasatinib group, and there was a significant decrease in the specific tumor growth rate (0.06 vs. 0.18, p=0.01) on the 11th day of treatment. Interestingly, dasatinib treated mice had increased proportion of CD8+cells in the circulation (17.9% vs. 14.4%, p=0.005) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells in PB increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). When the tumor content was analyzed, dasatinib treated mice had significantly more tumor infiltrated CD8+ T-cells (median 17 vs. 4/counted fields, p=0.03). In dasatinib group 80% of the tumor infiltrating CD8+ cells expressed PD-1 antigen compared to

Description: Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein are an effective therapy for Philadelphia chromosome positive (Ph+) leukemia. Dasatinib, a 2nd generation pan-TKI, also inhibits wild-type kinases, which may result in unexpected clinical responses. Recent data suggest that dasatinib has a potent immunosuppressive effect on T- and NK-cells in vitro. In contrast, we have noticed a marked expansion of lymphocytes in blood in a subset of dasatinib patients, but its clinical significance and molecular mechanisms are unclear. In this multicenter study, 19 Ph+ leukemia patients with marked lymphoproliferation in blood while on dasatinib (9 CML CP, 2 CML AP, 2 CML BC, 6 Ph+ALL) were identified. Clonality, immunophenotype, and intracellular signaling was analyzed and related clinical information was collected. In addition, prevalence and prognostic significance of the phenomenon was retrospectively assessed in a Phase II clinical study on 46 Ph+ ALL patients. An abrupt lymphocytosis (peak count range 4–20×109/L) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy (range 1–8 months). In most patients, LGL lymphocytosis was long-lasting and continued throughout dasatinib therapy, albeit with marked fluctuations in absolute lymphocyte count (Fig. 1a). In all evaluable patients (n=4), lymphocyte counts normalized after drug discontinuation. All patients were previously exposed to imatinib without similar changes in lymphocyte count or morphology. By immunophenotyping, 14 patients had a cytotoxic T-cell and 5 patients a NK-cell phenotype. After initiation of dasatinib therapy, the CD4/CD8 ratio shifted, expression of activation antigens HLA-DR and CD57 increased, expression of homing antigen CD62L decreased and relative numbers of regulatory T-cells were significantly decreased (p