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The advent of tyrosine kinase inhibitors (TKIs) as treatment of chronic myeloid leukemia (CML) is a paradigm in molecularly targeted cancer therapy. Nonetheless, TKI-insensitive leukemia stem cells (LSCs) persist in most patients even after years of treatment and are imperative for disease progression as well as recurrence during treatment-free remission (TFR). Here, we have generated high-resolution single-cell multiomics maps from CML patients at diagnosis, retrospectively stratified by BCR::ABL1IS (%) following 12 months of TKI therapy. Simultaneous measurement of global gene expression profiles together with >40 surface markers from the same cells revealed that each patient harbored a unique composition of stem and progenitor cells at diagnosis. The patients with treatment failure after 12 months of therapy had a markedly higher abundance of molecularly defined primitive cells at diagnosis compared to the optimal responders. The multiomic feature landscape enabled visualization of the primitive fraction as a mixture of molecularly distinct BCR::ABL1+ LSCs and BCR::ABL1-hematopoietic stem cells (HSCs) in variable ratio across patients, and guided their prospective isolation by a combination of CD26 and CD35 cell surface markers. We for the first time show that BCR::ABL1+ LSCs and BCR::ABL1- HSCs can be distinctly separated as CD26+CD35- and CD26-CD35+, respectively. In addition, we found the ratio of LSC/HSC to be higher in patients with prospective treatment failure compared to optimal responders, at diagnosis as well as following 3 months of TKI therapy. Collectively, this data builds a framework for understanding therapy response and adapting treatment by devising strategies to extinguish or suppress TKI-insensitive LSCs.

Abstract Background Immune-mediated arthritis is a group of autoinflammatory diseases, where the patient’s own immune system attacks and destroys synovial joints. Sustained remission is not always achieved with available immunosuppressive treatments, warranting more detailed studies of T cell responses that perpetuate synovial inflammation in treatment-refractory patients. Methods In this study, we investigated CD4 + and CD8 + T lymphocytes from the synovial tissue and peripheral blood of patients with treatment-resistant immune-mediated arthritis using paired single-cell RNA and TCR-sequencing. To gain insights into the trafficking of clonal families, we compared the phenotypes of clones with the exact same TCRß amino acid sequence between the two tissues. Results Our results show that both CD4 + and CD8 + T cells display a more activated and inflamed phenotype in the synovial tissue compared to peripheral blood both at the population level and within individual T cell families. Furthermore, we found that both cell subtypes exhibited clonal expansion in the synovial tissue. Conclusions Our findings suggest that the local environment in the synovium drives the proliferation of activated cytotoxic T cells, and both CD4 + and CD8 + T cells may contribute to tissue destruction and disease pathogenesis.

Abstract Exploring non-genetic evolution of cell states during cancer treatments has become attainable by recent advances in lineage-tracing methods. However, transcriptional changes that drive cells into resistant fates may be subtle, necessitating high resolution analysis. Here, we present ReSisTrace that uses shared transcriptomic features of sister cells to predict the states priming treatment resistance. Applying ReSisTrace in ovarian cancer cells perturbed with olaparib, carboplatin or natural killer (NK) cells reveals pre-resistant phenotypes defined by proteostatic and mRNA surveillance features, reflecting traits enriched in the upcoming subclonal selection. Furthermore, we show that DNA repair deficiency renders cells susceptible to both DNA damaging agents and NK killing in a context-dependent manner. Finally, we leverage the obtained pre-resistance profiles to predict and validate small molecules driving cells to sensitive states prior to treatment. In summary, ReSisTrace resolves pre-existing transcriptional features of treatment vulnerability, facilitating both molecular patient stratification and discovery of synergistic pre-sensitizing therapies.

Background Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level.Methods Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity.Results We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity.Conclusions Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Previous studies have characterized the diversity and dynamics of the T cell receptor (TCR) repertoire in patients with solid cancer. Here, by analyzing TCR repertoire data from multiple datasets, the authors report that melanoma-associated antigen-specific TCRs can be used to separate metastatic melanoma patients from healthy controls and to follow anti-tumor responses in patients treated with immunotherapy.

Abstract Renal cell carcinoma (RCC) accounts for 90% of all renal cancers and is considered highly immunogenic. Although many studies have reported the circulating peripheral cytokine profiles, the signatures between the tumor tissue and matching healthy adjacent renal tissue counterparts have not been explored. We aimed to comprehensively investigate the cytokine landscape of RCC tumors and its correlation between the amount and phenotype of the tumor infiltrating lymphocytes (TILs). We analyzed the secretion of 42 cytokines from the tumor (n = 46), adjacent healthy kidney tissues (n = 23) and matching plasma samples (n = 33) with a Luminex-based assay. We further explored the differences between the tissue types, as well as correlated the findings with clinical data and detailed immunophenotyping of the TILs. Using an unsupervised clustering approach, we observed distinct differences in the cytokine profiles between the tumor and adjacent renal tissue samples. The tumor samples clustered into three distinct profiles based on the cytokine expressions: high (52.2% of the tumors), intermediate (26.1%), and low (21.7%). Most of the tumor cytokines positively correlated with each other, except for IL-8 that showed no correlation with any of the measured cytokine expressions. Furthermore, the quantity of lymphocytes in the tumor samples analyzed with flow cytometry positively correlated with the chemokine-family of cytokines, CXCL10 (IP-10) and CXCL9 (MIG). No significant correlations were found between the tumor and matching plasma cytokines, suggesting that circulating cytokines poorly mirror the tumor cytokine environment. Our study highlights distinct cytokine profiles in the RCC tumor microenvironment and provides insights to potential biomarkers for the treatment of RCC.

BCL-XL and BCL-2 are key anti-apoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/BCL-2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the von Hippel-Lindau (VHL) E3 ligase, leading to BCLX L/BCL-2 ubiquitination and degradation selectively in cells expressing VHL. Because platelets lack VHL expression, 753B spares on-target platelet toxicity caused by the first-generation dual BCL-XL/BCL-2 inhibitor navitoclax (ABT-263). Here, we report pre-clinical single-agent activity of 753B against different leukemia subsets. 753B effectively reduced cell viability and induced dose-dependent degradation of BCL-XL and BCL-2 in a subset of hematopoietic cell lines, acute myeloid leukemia (AML) primary samples, and in vivo patient-derived xenograft AML models. We further demonstrated the senolytic activity of 753B, which enhanced the efficacy of chemotherapy by targeting chemotherapy-induced cellular senescence. These results provide a pre-clinical rationale for the utility of 753B in AML therapy, and suggest that 753B could produce an added therapeutic benefit by overcoming cellular senescence-induced chemoresistance when combined with chemotherapy.

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.

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.

Abstract 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.

Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3high, 25/52) and NK cells (NKhigh, 27/52). CD3high tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NKhigh tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-γ, TNF-α via NF-κB, and T cell receptor signaling) and kidney metabolism pathways in the CD3high subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1, and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3high subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies.

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.

In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome–negative (Ph–) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph– samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL.

Abstract T-cell prolymphocytic leukemia (T-PLL) is a poor prognostic disease with very limited options of efficient therapies. Most patients are refractory to chemotherapies and despite high response rates after alemtuzumab, virtually all patients relapse. Therefore, there is an unmet medical need for novel therapies in T-PLL. As the chemokine receptor CCR7 is a molecule expressed in a wide range of malignancies and relevant in many tumor processes, the present study addressed the biologic role of this receptor in T-PLL. Furthermore, we elucidated the mechanisms of action mediated by an anti-CCR7 monoclonal antibody (mAb) and evaluated whether its anti-tumor activity would warrant development towards clinical applications in T-PLL. Our results demonstrate that CCR7 is a prognostic biomarker for overall survival in T-PLL patients and a functional receptor involved in the migration, invasion, and survival of leukemic cells. Targeting CCR7 with a mAb inhibited ligand-mediated signaling pathways and induced tumor cell killing in primary samples. In addition, directing antibodies against CCR7 was highly effective in T-cell leukemia xenograft models. Together, these findings make CCR7 an attractive molecule for novel mAb-based therapeutic applications in T-PLL, a disease where recent drug screen efforts and studies addressing new compounds have focused on chemotherapy or small molecules.

Chronic graft versus host disease (cGvHD) is a major cause of morbidity and mortality in allogeneic bone marrow transplantation. Here the authors identify a recurrent activating mTOR mutation in expanded donor T-cell clones of 3 cGvHD patients, which suggests somatic mutations may contribute to GvHD pathogenesis and opens avenues to targeted therapies.

Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007-1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

Background Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms.Methods The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids.Results Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations.Conclusion Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.

Elevated MYC levels can sensitize tumor cells to apoptosis. In this study, the authors demonstrate that AMPK activation and BCL-2/BCL-XL inhibition have a synergistic effect on apoptosis, and that together with anti PD-1 therapy they can suppress Myc-driven mammary tumor growth.

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.

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.

Innate drug sensitivity in healthy cells aids identification of lineage specific anti-cancer therapies and reveals off-target effects. To characterize the diversity in drug responses in the major hematopoietic cell types, we simultaneously assessed their sensitivity to 71 small molecules utilizing a multi-parametric flow cytometry assay and mapped their proteomic and basal signaling profiles. Unsupervised hierarchical clustering identified distinct drug responses in healthy cell subsets based on their cellular lineage. Compared to other cell types, CD19+/B and CD56+/NK cells were more sensitive to dexamethasone, venetoclax and midostaurin, while monocytes were more sensitive to trametinib. Venetoclax exhibited dose-dependent cell selectivity that inversely correlated to STAT3 phosphorylation. Lineage specific effect of midostaurin was similarly detected in CD19+/B cells from healthy, acute myeloid leukemia and chronic lymphocytic leukemia samples. Comparison of drug responses in healthy and neoplastic cells showed that healthy cell responses are predictive of the corresponding malignant cell response. Taken together, understanding drug sensitivity in the healthy cell-of-origin provides opportunities to obtain a new level of therapy precision and avoid off-target toxicity.

Tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here, we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL), and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T-cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes - the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T-cells and macrophages, together entitled as a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T-cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy.

Abstract Background Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete. Results We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17 years, and healthy age-matched controls. The proportion of B cells (CD19+) and naïve B cells (CD27−, IgD+) were high while memory B cells (CD27+) and switched memory B cells (CD27+IgM−IgD−), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21low, CD38low) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4+ T cells was reduced significantly and the proportion of CD8+ T central memory significantly elevated. An increased proportion of CD57+ CD8+ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16+ and CD27− NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity. The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L+ T cells, naïve CD4+ and CD27+ CD8+ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8+ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied. Conclusions This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

Common variable immunodeficiency 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 common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta 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 common variable immunodeficiency 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 (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency 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.

Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in TMEM173 and IFIH1 showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways in vitro, and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in TMEM173 and IFIH1 as likely modifiers of the phenotype.

Abstract: Telomere length (TL) in peripheral blood (PB) cells of patients with chronic myeloid leukemia (CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL−, nonleukemic CD34+CD38− hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL+ leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clone's size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment.

Aggressive natural killer-cell leukemia (ANKL) has few targeted therapies. Here ANKL patients are reported to harbor STAT3, RAS-MAPK pathway, DDX3X and epigenetic modifier mutations; and drug sensitivity profiling uncovers the importance of the JAK-STAT pathway, revealing potential ANKL therapeutic targets.

A Correction to this paper has been published: https://doi.org/10.1038/s41408-021-00427-1

Changes in the immune system induced by tyrosine kinase inhibitors (TKI) have been shown to positively correlate with therapy responses in chronic myeloid leukemia (CML). However, only a few longitudinal studies exist and no randomized comparisons between two TKIs have been reported. Therefore, we prospectively analyzed the immune system of newly diagnosed CML patients treated with imatinib (n = 20) or bosutinib (n = 13), that participated in the randomized BFORE trial (NCT02130557). Comprehensive immunophenotyping, plasma protein profiling, and functional assays to determine activation levels of T and NK cells were performed at diagnosis, 3, and 12 months after therapy start. All results were correlated with clinical parameters such as Sokal risk and BCR-ABL load measured according to IS%. At diagnosis, low Sokal risk CML patients had a higher frequency of cytotoxic cells (CD8 + T and NK cells), increased cytotoxic potential of NK cells and lower frequency of naïve and central memory CD4 + T cells. Further, soluble plasma protein profile divided patients into two distinct clusters with different disease burden at diagnosis. During treatment, BCR-ABL IS% correlated with immunological parameters such as plasma proteins, together with different memory subsets of CD4+ and CD8 + T cells. Interestingly, the proportion and cytotoxic potential of NK cells together with several soluble proteins increased during imatinib treatment. In contrast, no major immunological changes were observed during bosutinib treatment. In conclusion, imatinib and bosutinib were shown to have differential effects on the immune system in this randomized clinical trial. Increased number and function of NK cells were especially observed during imatinib therapy.

Primary testicular lymphoma is a rare lymphoid malignancy, most often, histologically, representing diffuse large B-cell lymphoma. The tumor microenvironment and limited immune surveillance have a major impact on diffuse large B-cell lymphoma pathogenesis and survival, but the impact on primary testicular lymphoma is unknown. Here, the purpose of the study was to characterize the tumor microenvironment in primary testicular lymphoma, and associate the findings with outcome. We profiled the expression of 730 immune response genes in 60 primary testicular lymphomas utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize the immune cell phenotypes in the tumor tissue. We identified a gene signature enriched for T-lymphocyte markers differentially expressed between the patients. Low expression of the signature predicted poor outcome independently of the International Prognostic Index (progression-free survival: HR=2.810, 95%CI: 1.228-6.431, P=0.014; overall survival: HR=3.267, 95%CI: 1.406-7.590, P=0.006). The T-lymphocyte signature was associated with outcome also in an independent diffuse large B-cell lymphoma cohort (n=96). Multiplex immunohistochemistry revealed that poor survival of primary testicular lymphoma patients correlated with low percentage of CD3+CD4+ and CD3+CD8+ tumor-infiltrating lymphocytes (P

Primary testicular lymphoma is a rare and aggressive lymphoid malignancy, most often representing diffuse large B-cell lymphoma histologically. Tumor-associated macrophages and tumor-infiltrating lymphocytes have been associated with survival in diffuse large B-cell lymphoma, but their prognostic impact in primary testicular lymphoma is unknown. Here, we aimed to identify macrophages, their immunophenotypes and association with lymphocytes, and translate the findings into survival of patients with primary testicular lymphoma. We collected clinical data and tumor tissue from 74 primary testicular lymphoma patients, and used multiplex immunohistochemistry and digital image analysis to examine macrophage markers (CD68, CD163, and c-Maf), T-cell markers (CD3, CD4, and CD8), B-cell marker (CD20), and three checkpoint molecules (PD-L1, PD-L2, and PD-1). We demonstrate that a large proportion of macrophages (median 41%, range 0.08–99%) and lymphoma cells (median 34%, range 0.1–100%) express PD-L1. The quantity of PD-L1+ CD68+ macrophages correlates positively with the amount of PD-1+ lymphocytes, and a high proportion of either PD-L1+ CD68+ macrophages or PD-1+ CD4+ and PD-1+ CD8+ T cells translates into favorable survival. In contrast, the number of PD-L1+lymphoma cells or PD-L1− macrophages do not associate with outcome. In multivariate analyses with IPI, PD-L1+ CD68+ macrophage and PD-1+ lymphocyte contents remain as independent prognostic factors for survival. In conclusion, high PD-L1+ CD68+ macrophage and PD-1+ lymphocyte contents predict favorable survival in patients with primary testicular lymphoma. The findings implicate that the tumor microenvironment and PD-1 – PD-L1 pathway have a significant role in regulating treatment outcome. They also bring new insights to the targeted thera py of primary testicular lymphoma.

Tyrosine kinase inhibitors (TKIs) are highly effective for the treatment of chronic myeloid leukemia (CML), but very few patients are cured. The major drawbacks regarding TKIs are their low efficacy in eradicating the leukemic stem cells responsible for disease maintenance and relapse upon drug cessation. Herein, we performed ribonucleic acid sequencing of flow-sorted primitive (CD34+CD38low) and progenitor (CD34+ CD38+) chronic phase CML cells, and identified transcriptional upregulation of 32 cell surface molecules relative to corresponding normal bone marrow cells. Focusing on novel markers with increased expression on primitive CML cells, we confirmed upregulation of the scavenger receptor CD36 and the leptin receptor by flow cytometry. We also delineate a subpopulation of primitive CML cells expressing CD36 that is less sensitive to imatinib treatment. Using CD36 targeting antibodies, we show that the CD36 positive cells can be targeted and killed by antibody-dependent cellular cytotoxicity. In summary, CD36 defines a subpopulation of primitive CML cells with decreased imatinib sensitivity that can be effectively targeted and killed using an anti-CD36 antibody.

Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30–40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8+ T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.

Monitoring of single cell signal transduction in leukemic cellular subsets has been proposed to provide deeper understanding of disease biology and prognosis, but has so far not been tested in a clinical trial of targeted therapy. We developed a complete mass cytometry analysis pipeline for characterization of intracellular signal transduction patterns in the major leukocyte subsets of chronic phase chronic myeloid leukemia. Changes in phosphorylated Bcr-Abl1 and the signaling pathways involved were readily identifiable in peripheral blood single cells already within three hours of the patient receiving oral nilotinib. The signal transduction profiles of healthy donors were clearly distinct from those of the patients at diagnosis. Furthermore, using principal component analysis, we could show that phosphorylated transcription factors STAT3 (Y705) and CREB (S133) within seven days reflected BCR-ABL1IS at three and six months. Analyses of peripheral blood cells longitudinally collected from patients in the ENEST1st clinical trial showed that single cell mass cytometry appears to be highly suitable for future investigations addressing tyrosine kinase inhibitor dosing and effect. (clinicaltrials.gov identifier: 01061177)

In chronic myeloid leukemia (CML), early treatment prediction is important to identify patients with inferior overall outcomes. We examined the feasibility of using reductions in BCR-ABL1 transcript levels after 1 month of tyrosine kinase inhibitor (TKI) treatment to predict therapy response. Fifty-two first-line TKI-treated CML patients were included (imatinib n = 26, dasatinib n = 21, nilotinib n = 5), and BCR-ABL1 transcript levels were measured at diagnosis (dg) and 1, 3, 6, 12, 18, 24, and 36 months. The fold change of the BCR-ABL1 transcripts at 1 month compared to initial BCR-ABL1 transcript levels was used to indicate early therapy response. In our cohort, 21% of patients had no decrease in BCR-ABL1 transcript levels after 1 month and were classified as poor responders. Surprisingly, these patients had lower BCR-ABL1 transcript levels at dg compared to responders (31% vs. 48%, p = 0.0083). Poor responders also significantly more often had enlarged spleen (55% vs. 15%; p

When introducing multiplex platforms to measure protein content in precious clinical material there is an increased risk of cross reactivity, loss of sensitivity as well as accuracy. In this paper, four multiplex platforms and one singleplex platform were compared by running pre- and post-treatment plasma samples from CML patients. We found a variation of absolute protein concentrations between platforms. For some of the analytes and platforms, relative differences between pre- and post-treatment samples correlated. We conclude that absolute concentrations measured by different platforms should be compared with caution and comparing relative differences could be more accurate.

The original version of this Article contained an error in Fig. 7. In panel b, the survival curves were shifted relative to the y axis. This error has been corrected in both the PDF and HTML versions of the Article.

T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.

A small proportion of chronic myeloid leukemia patients treated with interferon-α (IFN-α) monotherapy are able to discontinue the treatment without disease relapse although residual leukemia cells are present. Recently, we showed that these patients have increased amount of NK-cells and a distinct blood cytokine profile. We now aimed to study the function of NK- and T-cells in order to understand the role of the immune system in maintaining the treatment response after IFN-α discontinuation. The study included 13 patients: 5 patients were still treated with IFN-α monotherapy (IFN-ON, median treatment time 163 months) and 8 had stopped the treatment successfully (IFN-OFF, median time without therapy 42 months). Detailed immunophenotype and cytokine production of NK- and T-cells was analyzed with flow cytometry. In addition, the cytotoxicity of NK-cells was studied using K562 as target cells and both the degranulation and direct killing was measured. Compared to healthy controls, IFN-OFF patients had increased proportion of CD4(+) effector memory (CCR7(-)CD45RA(-); median 23% vs. healthy 16%, p = 0.009) and CD8(+) central memory T-cells (CCR7(+)CD45RA(-); median 26% vs. healthy 14%, p = 0.004). Further, upon stimulation the IFN-γ/TNF-α cytokine secretion by CD4(+) T-cells was significantly enhanced in IFN-OFF patients (median 13.7% vs. healthy 7.8%, p = 0.01), and CD4+ effector and central memory cells were the main cytokine producers. No similar increase was observed in IFN-ON group (6.5%). In addition, the proportion of NK-cells was significantly increased in IFN-OFF patients (median IFN-OFF 24%, healthy 13%, p = 0.04), but their direct killing of K562 cells was impaired. The cytotoxicity of NK-cells was also diminished in IFN-ON patients. To conclude, in addition to elevated NK-cell count, IFN-OFF patients have increased amount of memory T-cells, which are able to induce strong cytokine response upon stimulation. This activity may contribute to the maintenance of prolonged remission after successful IFN-α discontinuation.

Immunotherapy (eg interferon α) in combination with tyrosine kinase inhibitors is currently in clinical trials for treatment of chronic myeloid leukemia (CML). Cancer patients commonly have problems with so called immune escape mechanisms that may hamper immunotherapy. Hence, to study the function of the immune system in CML is of interest. In the present paper we have identified immune escape mechanisms in CML with focus on those that directly hamper T cells since these cells are important to control tumor progression. CML patient samples were investigated for the presence of myeloid-derived suppressor cells (MDSCs), expression of programmed death receptor ligand 1/programmed death receptor 1 (PD-L1/PD-1), arginase 1 and soluble CD25. MDSC levels were increased in samples from Sokal high risk patients (p