Novel Ex Vivo DOAC Removal Methods Reduce Interference in Lupus Anticoagulant Testing.

Direct oral anticoagulants (DOAC) interfere in laboratory coagulation testing. The aim here was to study how commercial DOAC removal methods, DOAC Filter® and DOAC-Stop™, perform to eliminate DOAC concentrations and false positive results in lupus anticoagulant (LAC) testing. We acquired 50 patient samples with high concentrations of DOACs: apixaban (n = 18, range 68-572 ng/mL), dabigatran (n = 8, range 47-154 ng/mL), edoxaban (n = 8, range 35-580 ng/mL) and rivaroxaban (n = 16, range 69-285 ng/mL). DOACs were removed ex vivo with either DOAC Filter® (n = 28) or DOAC-Stop™ (n = 22). Additionally, commercial control and calibrator samples were studied (n = 13 for DOAC Filter®, n = 14 for DOAC-Stop™). LAC screening was performed before and after DOAC removal. Both DOAC Filter® and DOAC-Stop™ were effective in removing DOAC concentrations in samples: DOAC concentrations decreased to median of 0 ng/mL (range 0-48 ng/mL). Only one sample had more than residual 25 ng/mL of DOAC (apixaban). Before DOAC removal, 96% (48/50) of patient samples and over 90% (12/13 DOAC Filter®, 13/14 DOAC-Stop™) of control/calibrator samples were positive in the LAC screening. In patient samples, LAC screening turned negative in 61% (17/28) after DOAC Filter® and 45% (10/22) after DOAC-Stop™ treatment. All control samples became negative after DOAC removal. In conclusion, DOAC removal ex vivo reduces false positives in LAC screening. DOAC removal halved the need for confirmation or mixing tests- Although a subset of patients would require further testing, DOAC removal reduces unnecessary repeated LAC testing.

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

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.

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

[This corrects the article DOI: 10.3389/fimmu.2020.578848.].

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 (TCRß) 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 TCRß 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.

Somatic mutations and T-cell clonality in patients with immunodeficiency.

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.

A robust pipeline with high replication rate for detection of somatic variants in the adaptive immune system as a source of common genetic variation in autoimmune disease.

The role of somatic variants in diseases beyond cancer is increasingly being recognized, with potential roles in autoinflammatory and autoimmune diseases. However, as mutation rates and allele fractions are lower, studies in these diseases are substantially less tolerant of false positives, and bio-informatics algorithms require high replication rates. We developed a pipeline combining two variant callers, MuTect2 and VarScan2, with technical filtering and prioritization. Our pipeline detects somatic variants with allele fractions as low as 0.5% and achieves a replication rate of >55%. Validation in an independent data set demonstrates excellent performance (sensitivity > 57%, specificity > 98%, replication rate > 80%). We applied this pipeline to the autoimmune disease multiple sclerosis (MS) as a proof-of-principle. We demonstrate that 60% of MS patients carry 2-10 exonic somatic variants in their peripheral blood T and B cells, with the vast majority (80%) occurring in T cells and variants persisting over time. Synonymous variants significantly co-occur with non-synonymous variants. Systematic characterization indicates somatic variants are enriched for being novel or very rare in public databases of germline variants and trend towards being more damaging and conserved, as reflected by higher phred-scaled combined annotation-dependent depletion (CADD) and genomic evolutionary rate profiling (GERP) scores. Our pipeline and proof-of-principle now warrant further investigation of common somatic genetic variation on top of inherited genetic variation in the context of autoimmune disease, where it may offer subtle survival advantages to immune cells and contribute to the capacity of these cells to participate in the autoimmune reaction.

Sex bias in MHC I-associated shaping of the adaptive immune system.

HLA associations, T cell receptor (TCR) repertoire bias, and sex bias have independently been shown for many diseases. While some immunological differences between the sexes have been described, they do not fully explain bias in men toward many infections/cancers, and toward women in autoimmunity. Next-generation TCR variable beta chain (TCRBV) immunosequencing of 824 individuals was evaluated in a multiparametric analysis including HLA-A -B/MHC class I background, TCRBV usage, sex, age, ethnicity, and TCRBV selection/expansion dynamics. We found that HLA-associated shaping of TCRBV usage differed between the sexes. Furthermore, certain TCRBVs were selected and expanded in unison. Correlations between these TCRBV relationships and biochemical similarities in HLA-binding positions were different in CD8 T cells of patients with autoimmune diseases (multiple sclerosis and rheumatoid arthritis) compared with healthy controls. Within patients, men showed higher TCRBV relationship Spearman's rhos in relation to HLA-binding position similarities compared with women. In line with this, CD8 T cells of men with autoimmune diseases also showed higher degrees of TCRBV perturbation compared with women. Concerted selection and expansion of CD8 T cells in patients with autoimmune diseases, but especially in men, appears to be less dependent on high HLA-binding similarity than in CD4 T cells. These findings are consistent with studies attributing autoimmunity to processes of epitope spreading and expansion of low-avidity T cell clones and may have further implications for the interpretation of pathogenic mechanisms of infectious and autoimmune diseases with known HLA associations. Reanalysis of some HLA association studies, separating the data by sex, could be informative.

Somatic STAT3 mutations in Felty syndrome: an implication for a common pathogenesis with large granular lymphocyte leukemia.

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.

Dasatinib Changes Immune Cell Profiles Concomitant with Reduced Tumor Growth in Several Murine Solid Tumor Models.

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8+ T cells in PB, together with a higher amount of tumor-infiltrating CD8+ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4+ and CD8+ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials. Cancer Immunol Res; 5(2); 157-69. ©2017 AACR.

Oncolytic Adenovirus Expressing Monoclonal Antibody Trastuzumab for Treatment of HER2-Positive Cancer.

Monoclonal anti-HER2 antibody trastuzumab has significantly improved the survival of patients with HER2-overexpressing tumors. Nevertheless, systemic antibody therapy is expensive, limited in efficacy due to physical tumor barriers, and carries the risk of severe side effects such as cardiomyopathy. Oncolytic viruses mediate cancer-selective transgene expression, kill infected cancer cells while mounting antitumor immune responses, and have recently demonstrated promising efficacy in combination treatments. Here, we armed an oncolytic adenovirus with full-length trastuzumab to achieve effective in situ antibody production coupled with progressive oncolytic cancer cell killing. We constructed an infectivity-enhanced serotype 5 oncolytic adenovirus, Ad5/3-Δ24-tras, coding for human trastuzumab antibody heavy- and light-chain genes, connected by an internal ribosome entry site. Infected cancer cells were able to assemble full-length functional antibody, as confirmed by Western blot, ELISA, and antibody-dependent cell-mediated cytotoxicity assay. Importantly, oncolysis was required for release of the antibody into tumors, providing additional spatial selectivity. Ad5/3-Δ24-tras showed potent in vitro cytotoxicity and enhanced antitumor efficacy over oncolytic control virus Ad5/3-Δ24 or commercial trastuzumab in HER2-positive cancer models in vivo (both P < 0.05). Furthermore, Ad5/3-Δ24-tras resulted in significantly higher tumor-to-systemic antibody concentrations (P < 0.001) over conventional delivery. Immunological analyses revealed dendritic cell activation and natural killer cell accumulation in tumor-draining lymph nodes. Thus, Ad5/3-Δ24-tras is an attractive anticancer approach combining oncolytic immunotherapy with local trastuzumab production, resulting in improved in vivo efficacy and immune cell activation in HER2-positive cancer. Moreover, the finding that tumor cells can produce functional antibody as directed by oncolytic virus could lead to many valuable antitumor approaches. Mol Cancer Ther; 15(9); 2259-69. ©2016 AACR.

LGL leukemia and autoimmunity - the borderline between autoimmune disease and cancer is becoming blurred.

Large granular lymphocyte (LGL) leukemia is a chronic hematological disease, in which the diseased cells consist of clonal large, mature T or NK cells. Major symptoms and findings of the disease include anemia, neutropenia and rheumatoid arthritis. Immunosuppressive treatments, such as methotrexate, usually relieve the symptoms in patients. In LGL leukemia, next-generation sequencing has recently revealed mutations in the STAT3 and STAT5B genes that lead to the activation of these proteins. Similar mutations have been detected in hereditary autoimmune diseases, disorders of bone marrow and malignancies of lymphocyte origin.