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The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1– ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.

Group 2 innate lymphoid cells (ILC2s) orchestrate protective type 2 immunity and have been implicated in various immune disorders. In the mouse, circulatory inflammatory ILC2s (iILC2s) were identified as a major source of type 2 cytokines. The human equivalent of the iILC2 subset remains unknown. Here, we identify a human inflammatory ILC2 population that resides in inflamed mucosal tissue and is specifically marked by surface CD45RO expression. CD45RO+ ILC2s are derived from resting CD45RA+ ILC2s upon activation by epithelial alarmins such as IL-33 and TSLP, which is tightly linked to STAT5 activation and up-regulation of the IRF4/BATF transcription factors. Transcriptome analysis reveals marked similarities between human CD45RO+ ILC2s and mouse iILC2s. Frequencies of CD45RO+ inflammatory ILC2 are increased in inflamed mucosal tissue and in the circulation of patients with chronic rhinosinusitis or asthma, correlating with disease severity and resistance to corticosteroid therapy. CD45RA-to-CD45RO ILC2 conversion is suppressed by corticosteroids via induction of differentiation toward an immunomodulatory ILC2 phenotype characterized by low type 2 cytokine and high amphiregulin expression. Once converted, however, CD45RO+ ILC2s are resistant to corticosteroids, which is associated with metabolic reprogramming resulting in the activation of detoxification pathways. Our combined data identify CD45RO+ inflammatory ILC2s as a human analog of mouse iILC2s linked to severe type 2 inflammatory disease and therapy resistance.

Autoreactive B cells mediate autoimmune pathology, but exactly how remains unknown. A hallmark of rheumatoid arthritis (RA), a common autoimmune disease, is the presence of disease-specific anticitrullinated protein antibodies (ACPAs). Here, we showed that ACPA-positive B cells in patients with RA strongly expressed T cell-stimulating ligands, produced abundant proinflammatory cytokines, and were proliferative while escaping inhibitory signals. This activated state was found at different degrees in different stages of disease: highest in patients with recent-onset RA, moderate in patients with established RA, and far less pronounced in ACPA-positive individuals "at risk" for developing disease. The activated autoreactive B cell response persisted in patients who achieved clinical remission with conventional treatment. ACPA-positive B cells in blood and synovial fluid secreted increased amounts of the chemoattractant interleukin-8, which attracted neutrophils, the most abundant immune cell in arthritic joints. Tetanus toxoid-specific B cells from the same patients exhibited properties of memory B cells without the activation and proliferation phenotype, but these cells transiently acquired a similar proliferative phenotype upon booster vaccination. Together, these data indicated that continuous antigenic triggering of autoreactive B cells occurs in human autoimmune disease and support the emerging concept of immunological activity that persists under treatment even in clinical remission, which may revise our current concept of treatment targets for future therapeutic interventions. In addition, our data pointed to a pathogenic role of ACPA-positive B cells in the inflammatory disease process underlying RA and favor approaches that aim at their antigen-specific inactivation or depletion.

Background Colorectal cancer (CRC) associated with Lynch syndrome is characterized by an abundance of infiltrating lymphocytes. To study whether tumor-specific antibodies with therapeutic potential can be isolated from these patients, the B-cell repertoire from a patient with Lynch syndrome who recovered from a stage IV colon carcinoma was screened. Here we describe an antibody, AT1636 that recognizes a previously unidentified O-mannosylated 70kDa form of E-cadherin. The intercellular interactions by E-cadherin on tumor cells have for long been recognized as protective in cancer metastasis, and deregulation of E-cadherin is a hallmark for epithelial-mesenchymal transition (EMT). Methods The study protocol was approved by the Medical Ethical Committee of the Academic Medical Centre, Amsterdam, The Netherlands (NL42718.018.12). AIMM’s BCL6 and Bcl-xL immortalization method1 was used to interrogate the human antibody repertoire. From a carrier of a pathogenic gene variant in the MSH6 gene diagnosed with stage IV CRC and liver metastasis that had been treated with avastin, capecitabine and oxaliplatin, peripheral-blood memory B cells were obtained 9 years after last treatment. Antibodies-containing supernatant of cultured B-cells were screened for binding to 3 different CRC cell lines (DLD1, LS174T and COLO205) and absence of binding to fibroblast by flow cytometry. A high-affinity variant of AT1636 (AT1636IYN) was sorted from the original AT1636, AID-expressing B-cell clone.2 Results Antibodies that demonstrated differential binding to CRC cells were characterized and targets recognized by such antibodies were identified using immunoprecipitation and mass-spectrometry. One of the antibodies, AT1636, recognized a previously unidentified O-mannosylated 70kDa E-cadherin variant (ECV). Although the 70kDa ECV is found in all full-length E-cadherin expressing cells, tumor-specific binding of AT1636 is dependent on the O-mannosylation pattern in the antibody epitope on ECV. Using shRNA knock-down AT1636 binding was shown to depend on the transmembrane O-mannosyltransferase targeting cadherins 3 (TMTC3). 3 In accordance, coexpression of TMTC3 and E-cadherin in tumor cells is predictive for AT1636 binding. In addition, we observed that (over)expression of ECV results in a strong de-adhesive, EMT-like phenotype. Although AT1636 by itself is not able to induce ADCC, the CD3-bispecific antibody (single-chain UCHT1) AT1636 format specifically killed CRC cell lines. Conclusions The AT1636 antibody retrieved from a patient with Lynch syndrome binds a previous unidentified cancer-specific O-mannosylated 70kDa form of E-cadherin. This variant might play a role in tumor-cell invasion and metastasis. More importantly, we provide a rationale to advance AT1636 based therapeutics for treatment of CRC. Ethics Approval The study protocol was approved by the Medical Ethical Committee of the Academic Medical Centre, Amsterdam, The Netherlands (NL42718.018.12) References M.J. Kwakkenbos, et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine 2010;16:123–128. K. Wagner, et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. Proc Natl Acad Sci USA 2014; 111: 16820–16825. I.S.B. Larsen, et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci USA 2017;114:11163–11168.

Background Adaptive immunity to cancer cells forms a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether tumor-targeting antibodies could be isolated from a patient that cured (now 13 years tumor-free) metastatic melanoma following adoptive transfer of ex vivo expanded autologous T cells. Methods Patient‘s peripheral blood B cells were isolated and tested for the presence of tumor-reactive B cells using AIMM’s immmortalisation technology. Antibody AT1412 was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 binds the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities in cancer and induces ADCC and ADCP by effector cells. Results Spontaneous immune rejection of tumors was observed in human immune system (HIS) mouse models implanted with CD9 genetically-disrupted A375 melanoma (A375-CD9KO) tumor cells, while A375wt cells were not cleared. Most notably, no tumor rejection of A375-CD9KO tumors was observed in NSG mice, indicating that blockade of CD9 makes tumor cells susceptible to immune rejection.CD9 has been described to regulate integrin signaling, e.g. LFA-1, VLA-4, VCAM-1 and ICAM-1. AT1412 was shown to modulate CD9 function by enhancing adhesion and transmigration of T cells to endothelial (HUVEC) cells. AT1412 was most potently enhancing transendothelial T-cell migration, in contrast to a high affinity version of AT1412 or other high affinity anti-CD9 reference antibodies (e.g. ALB6). Enhanced immune cell infiltration is also observed in immunodeficient mice harbouring a human immune system (HIS). AT1412 strongly enhanced CD8 T-cell and macrophage infiltration resulting in tumor rejection (A375 melanoma). PD-1 checkpoint blockade is further sustaining this effect. In a second melanoma model carrying a PD-1 resistant and highly aggressive tumor (SK-MEL5) AT1412 together with nivolumab was inducing full tumor rejection, while either one of the antibodies alone did not. Conclusions The safety of AT1412 has been assessed in preclinical development and is well tolerated up to 10 mg/kg (highest dose tested) by non human primates. AT1412 demonstrated a half-life of 8.5 days, supporting 2–3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. First in Human clinical study is planned to start early 2021. Ethics Approval Study protocols were approved by the Medical Ethical Committee of the Leiden University Medical Center (Leiden, Netherlands). Consent Blood was obtained after written informed consent by the patient.

It has come to our attention that there is an error in Fig. 2A and consequently the Graphical Abstract of our manuscript. During the production process, the colors used in the legend to the pie chart were incorrectly assigned, so that those representing AR3 and AR4 were reversed. Please see the corrected Fig. 2 and Graphical Abstract below. We apologize for any inconvenience caused. [Figure presented] [Figure presented]

A novel B cell subset that produces proangiogenic cytokines is increased in pathologies associated with angiogenesis., B cells contribute to immune responses through the production of immunoglobulins, antigen presentation, and cytokine production. Several B cell subsets with distinct functions and polarized cytokine profiles have been reported. In this study, we used transcriptomics analysis of immortalized B cell clones to identify an IgG4+ B cell subset with a unique function. These B cells are characterized by simultaneous expression of proangiogenic cytokines including VEGF, CYR61, ADM, FGF2, PDGFA, and MDK. Consequently, supernatants from these clones efficiently promote endothelial cell tube formation. We identified CD49b and CD73 as surface markers identifying proangiogenic B cells. Circulating CD49b+CD73+ B cells showed significantly increased frequency in patients with melanoma and eosinophilic esophagitis (EoE), two diseases associated with angiogenesis. In addition, tissue-infiltrating IgG4+CD49b+CD73+ B cells expressing proangiogenic cytokines were detected in patients with EoE and melanoma. Our results demonstrate a previously unidentified proangiogenic B cell subset characterized by expression of CD49b, CD73, and proangiogenic cytokines.

Innate lymphoid cells (ILCs) are important for tissue homeostasis and for the initiation of immune responses. Based on their transcriptional regulation and cytokine profiles, ILCs can be categorized into five subsets with defined phenotypes and functional profiles, but they also have the ability to adapt to local environmental cues by changing these profiles. This plasticity raises the question of the extent to which the cytokine production profiles of ILCs are pre-programmed or are a reflection of the tissue microenvironment. Here, we review recent advances in research on ILCs, with a focus on the plasticity of these cells. We highlight the ability of ILCs to communicate with the surrounding microenvironment and discuss the possible consequences of ILC plasticity for our understanding of the biological roles of these cells. Finally, we discuss how we might use this knowledge of ILC plasticity to develop or improve options for the treatment of inflammatory diseases.

ILCs and T helper cells have been shown to exert bi-directional regulation in mice. However, how crosstalk between ILCs and CD4+ T cells influences immune function in humans is unknown. Here we show that human intestinal ILCs co-localize with T cells in healthy and colorectal cancer tissue and display elevated HLA-DR expression in tumor and tumor-adjacent areas. Although mostly lacking co-stimulatory molecules ex vivo, intestinal and peripheral blood (PB) ILCs acquire antigen-presenting characteristics triggered by inflammasome-associated cytokines IL-1β and IL-18. IL-1β drives the expression of HLA-DR and co-stimulatory molecules on PB ILCs in an NF-κB-dependent manner, priming them as efficient inducers of cytomegalovirus-specific memory CD4+ T-cell responses. This effect is strongly inhibited by the anti-inflammatory cytokine TGF-β. Our results suggest that circulating and tissue-resident ILCs have the intrinsic capacity to respond to the immediate cytokine milieu and regulate local CD4+ T-cell responses, with potential implications for anti-tumor immunity and inflammation., Murine ILCs can modulate T cell responses in MHCII-dependent manner. Here the authors show that human ILCs process and present antigens and induce T-cell responses upon exposure to IL-1-family cytokines; along with the article by Lehmann et al, this work elucidates how cytokines set context specificity of ILC-T cell crosstalk by regulating ILC antigen presentation.

SummaryIn this study, we addressed the role of IL-10-producing ILCs in controlling grass pollen allergy. IL-10+ ILCs are comprised in KLRG1+ILC2-lineage cells and absent on CRTH2+ ILC2 which lack KLRG1. We observed that ILC2s in healthy subjects and grass pollen allergic patients show differential gene expression profiles. A cross-sectional study demonstrates that IL-10-producing KLRG1+ ILC2s are strongly reduced in patients with grass pollen and house dust mite allergy when compared to healthy subjects and those who have received grass pollen subcutaneous immunotherapy treatment. In a prospective double-blind, placebo-controlled trial of grass pollen sublingual immunotherapy (SLIT), we observed that the capacity of ILC2 to produce IL-10 was restored in patients who received SLIT, but not patients receiving a placebo. Finally, we demonstrated that these IL-10-producing ILCs are functional and play a crucial role in restoring barrier function and tissue balance in allergic diseases.

Group 2 innate lymphoid cells (ILC2s) are the most well defined group of ILCs. ILC2 development is controlled by the GATA-3 transcription factor and these cells produce archetypal type 2 cytokines, such as IL-5 and IL-13. These cytokines mediate parasite expulsion and tissue repair, but also contribute to type 2 inflammatory diseases, including allergy, asthma and chronic rhinosinusitis with nasal polyps. In response to tightly regulated local environmental cues ILCs can generate characteristics of other subtypes, a process known as plasticity. Recent advances in the ILC2 field has led to the discovery that ILC2s can promptly shift to functional IFN-γ-producing ILC1s or IL-17-producing ILC3s, depending on the cytokines and chemokines produced by antigen presenting cells or epithelial cells. Due to yet unknown triggers, this complex network of signals may become dysregulated. In this review, we will discuss general ILC characteristic, ILC2 development, plasticity, memory function, and implications in disease.

Hepatitis C virus (HCV) infection is a major global public health problem. Early induction of cross-reactive neutralizing antibodies during acute infection correlates with the spontaneous clearance of HCV. Understanding the antibody response in multiple subjects in large-scale studies would greatly benefit vaccine development. To determine the breadth of a polyclonal-serum antibody response, and or, the monoclonal antibodies against the different HCV E1E2 genotypes, we developed a quick and high throughput flow cytometry assay using fluorescent cell barcoding to distinguish cells transfected with different E1E2 sequences in a single measurement. HCV-specific antibodies recognizing conformational epitopes were tested for binding to cells transfected with E1E2 from six genotypes. In this assay, 1500 samples can be analyzed for specific binding to 6 different HCV E1E2 sequences within 8h. Plasma of HCV infected subjects were tested in our assay allowing us to determine the breadth of their antibody response. In summary, we developed a quick and high throughput assay to study the specificity of an antibody response against multiple HCV E1E2 sequences simultaneously. This assay can also be used to facilitate the discovery of novel antibodies, and because other flavi- and picornaviruses have similar intracellular assembly mechanisms, this approach can be used to study the antibody response against such viruses.

Most patients with acute myeloid leukemia (AML) can only be cured when allogeneic hematopoietic stem-cell transplantation induces a graft-versus-leukemia immune response (GVL). Although the role of T cells and natural killer cells in tumor immunology has been established, less is known about the contribution of B cells. From B cells of high-risk patients with AML with potent and lasting GVL responses, we isolated monoclonal antibodies directed against antigens expressed on the cell surface of AML cells but not on normal hematopoietic and nonhematopoietic cells. A number of these donor-derived antibodies recognized the U5 snRNP200 complex, a component of the spliceosome that in normal cells is found in the cell. In AML however, the U5 snRNP200 complex is exposed on the cell membrane of leukemic blasts. U5 snRNP200 complex-specific antibodies induced death of AML cells in an Fc receptor-dependent way in the absence of cytotoxic leukocytes or complement. In an AML mouse model, treatment with U5 snRNP200 complex-specific antibodies led to significant tumor growth inhibition. Thus, donor-derived U5 snRNP200 complex-recognizing AML-specific antibodies may contribute to antitumor responses.

Immunotherapy has proven beneficial in many hematologic and nonhematologic malignancies, but immunotherapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is hampered by the lack of tumor-specific targets. We took advantage of the tumor-immunotherapeutic effect of allogeneic hematopoietic stem cell transplantation and searched the B-cell repertoire of a patient with a lasting and potent graft-versus-AML response for the presence of AML-specific antibodies. We identified an antibody, AT1413, that was of donor origin and that specifically recognizes a novel sialylated epitope on CD43 (CD43s). Strikingly, CD43s is expressed on all World Health Organization 2008 types of AML and MDS. AT1413 induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Of note, AT1413 was highly efficacious against AML cells in a humanized mouse model without affecting nonmalignant human myeloid cells, suggesting AT1413 has potential as a therapeutic antibody.

Objective. The molecular mechanisms steering abnormal B cell responses in autoimmune diseases remain poorly understood. We undertook this study to identify molecular switches controlling pathologic B cell responses in rheumatoid arthritis (RA).Methods. Candidate molecules were identified by gene expression profiling of RA synovitis and validated by quantitative polymerase chain reaction and immunohistochemistry. B cell-specific expression was confirmed by immunofluorescence, immunoblotting, and flow cytometry. The role of Bob1 in pathologic B cell responses was assessed in collagen-induced arthritis (CIA).Results. Transcriptional profiling of RA synovitis revealed a prominent B cell signature, with the transcriptional coactivator Bob1 and its putative target BCMA being among the most up-regulated genes. Further analysis confirmed the microarray data and demonstrated elevated levels of Bob1 in B cells in RA synovium. A functional study showed that Bob1-deficient mice failed to produce pathogenic anti-type II collagen (anti-CII) antibodies and were resistant to CIA. Adoptive transfer of cells from Bob1-deficient and Bob1-sufficient mice to recombination-activating gene 1-null mice demonstrated that Bob1 deficiency exclusively in B cells abrogated germinal center (GC) B cell formation, anti-CII antibody production, and CIA development. Consistent with data from animal studies, immunophenotyping of human B cell subsets revealed increased expression of Bob1, predominantly in centrocytes and centroblasts. Correspondingly, Bob1 expression in RA synovitis was strongly correlated with CD21L, a molecular marker of GCs. In addition, similar Bob1 overexpression and correlation with CD21L expression was evidenced in parotid salivary gland tissue from patients with primary Sjogren's syndrome.Conclusion. These expression and functional data identify the transcriptional coactivator Bob1 as a candidate molecular switch of pathogenic B cell responses in autoimmune diseases in humans.

Genetically encoded tyrosine (Y-tag) can be utilized as a latent anchor for inducible and site-selective conjugation. Upon oxidation of tyrosine with mushroom tyrosinase, strain-promoted cycloaddition (SPOCQ) of the resulting 1,2-quinone with various. bicyclo[6.1.0]nonyne (BCN) derivatives led to efficient conjugation. The method was applied for fluorophore labeling of laminarinase A and for the site-specific preparation of an antibody drug conjugate

Summary Here, we characterize a subset of ILC3s that express Neuropilin1 (NRP1) and are present in lymphoid tissues, but not in the peripheral blood or skin. NRP1+ group 3 innate lymphoid cells (ILC3s) display in vitro lymphoid tissue inducer (LTi) activity. In agreement with this, NRP1+ ILC3s are mainly located in proximity to high endothelial venules (HEVs) and express cell surface molecules involved in lymphocyte migration in secondary lymphoid tissues via HEVs. NRP1 was also expressed on mouse fetal LTi cells, indicating that NRP1 is a conserved marker for LTi cells. Human NRP1+ ILC3s are primed cells because they express CD45RO and produce higher amounts of cytokines than NRP1− cells, which express CD45RA. The NRP1 ligand vascular endothelial growth factor A (VEGF-A) served as a chemotactic factor for NRP1+ ILC3s. NRP1+ ILC3s are present in lung tissues from smokers and patients with chronic obstructive pulmonary disease, suggesting a role in angiogenesis and/or the initiation of ectopic pulmonary lymphoid aggregates., Graphical Abstract, Highlights • NRP1+ ILC3s are present in lymphoid tissues, but not in the peripheral blood or skin • NRP1+ ILC3s express CD45RO and produce higher amounts of cytokines than NRP1− ILC3s • NRP1 is a marker for human ILC3s with LTi phenotype and in vitro LTi activity • NRP1+ ILC3s are present in lung tissues from smokers and COPD patients, Shikhagaie et al. find that NRP1 expressing human ILC3s are LTi-like cells, which are present in fetal tissues and adult lymphoid tissues, but not in peripheral blood or skin. NRP1+ ILC3s cells are primed and migrate in response to VEGF-A. In addition, their presence in the lungs of smokers and COPD patients provides insight into the formation of ectopic lymphoid aggregates.

Innate lymphoid cells (ILCs) are important in the regulation of barrier homeostasis. These cells do not express T cell receptors but share many functional similarities with T helper cells and cytotoxic CD8(+) T lymphocytes. ILCs are divided into three groups, namely group 1 ILCs, group 2 ILCs and group 3 ILCs, based on the transcription factors they depend on for their development and function, and the cytokines they produce. Emerging data indicate that ILCs not only have protective functions but can also have detrimental effects when dysregulated, leading to chronic inflammation and autoimmune diseases, including asthma, inflammatory bowel disease, graft-versus-host disease, psoriasis, rheumatoid arthritis and atopic dermatitis. Elucidation of the cytokine pathways involved in various autoimmune diseases - and the identification of ILCs as potent producers of these cytokines - points towards a potential role for these cellular players in the pathophysiology of these diseases. In this Review we discuss the current knowledge of the role of ILCs in the pathogenesis of rheumatic and other autoimmune diseases

Despite recent advances in treatment of B-acute lymphoblastic leukemia (B-ALL) there is still a need for novel targeted therapies. The tetraspanin CD9 is expressed in 60-80% of B-ALL and correlates with adverse prognosis. Recently, the mouse CD9 antibody ALB6 was shown to induce leukemia rejection in NOD/SCID mice. However, clinical development of ALB6 and other CD9-targeting antibodies was hampered by their CD9 mediated induction of platelet aggregation. It is known that CD9 is still expressed on tumor cells after treatment with chemotherapy or blinatumomab (Leung, 2019; Linder, 2016). AT1412 is a fully human antibody isolated from B cells of a patient that was cured from stage IV metastatic melanoma (Verdegaal, 2011). AT1412 targets CD9, without inducing platelet aggregation in vitro or thrombosis in cynomolgus monkeys after intravenous administration at therapeutic dose levels. By crystallography AT1412 was shown to bind a unique epitope preventing homodimerization of CD9, distinctly different from other CD9 antibodies. AT1412 binds a majority of patient B-ALL samples, but not T-ALL and induces ADCC and ADCP of CD9 positive B-ALL primary cells and the level of cytotoxicity significantly correlated with that of AT1412 binding. In both NSG and immunodeficient mice harboring a human immune system (HIS mice) AT1412 demonstrated a strong, dose-dependent tumor rejection of B-ALL, most pronounced in the extramedullary sites. In HIS mice AT1412 treatment led to an accumulation of T cells and CD14+ myeloid cells at the tumor sites. To support clinical development, pre-clinical safety of AT1412 was evaluated in cynomolgus monkeys. AT1412 demonstrated a half-life of 8.5 days, supporting 2-3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. Taken together, we demonstrate that CD9 on B-ALL cells can be successfully targeted by AT1412. AT1412 targets a unique epitope and does not induce thrombosis. Pre-clinical safety assessment is supporting that AT1412 can be safely administered. A First in Human clinical study is scheduled to start early 2021 in human solid tumors to determine safety and efficacy. AT1412 efficacy will be evaluated in B-ALL in expansion cohorts. Leung, K.T., Zhang, C., Chan, K.Y.Y., Li, K., Cheung, J.T.K., Ng, M.H.L., Zhang, X.-B., Sit, T., Lee, W.Y.W., Kang, W., et al. (2019). CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity. Leukemia. Linder, K., Gandhiraj, D., Hanmantgad, M., Seiter, K., and Liu, D. (2016). Complete remission after single agent blinatumomab in a patient with pre-B acute lymphoid leukemia relapsed and refractory to three prior regimens: HyperCVAD, high dose cytarabine mitoxantrone and CLAG. Exp. Hematol. Oncol. 5, 5-8. Verdegaal, E.M.E., Visser, M., Ramwadhdoebé, T.H., van der Minne, C.E., van Steijn, J. a Q.M.J., Kapiteijn, E., Haanen, J.B. a G., van der Burg, S.H., Nortier, J.W.R., and Osanto, S. (2011). Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha. Cancer Immunol. Immunother. 60, 953-963. Disclosures Schotte: AIMM Therapeutics: Current Employment, Current equity holder in private company. Villaudy:AIMM Therapeutics: Current Employment, Current equity holder in private company. Levie:AIMM Therapeutics: Current Employment, Current equity holder in private company. Go:AIMM Therapeutics: Current Employment, Current equity holder in private company. Yasuda:AIMM Therapeutics: Current Employment, Current equity holder in private company. Frankin:AIMM Therapeutics: Current Employment, Current equity holder in private company. Cercel:AIMM Therapeutics: Current Employment, Current equity holder in private company. van Hal-van Veen:AIMM Therapeutics: Current Employment, Current equity holder in private company. van de Berg:AIMM Therapeutics: Current Employment, Current equity holder in private company. Fatmawati:AIMM Therapeutics: Current Employment, Current equity holder in private company. Kedde:AIMM Therapeutics: Current Employment, Current equity holder in private company. Claassen:AIMM Therapeutics: Current Employment, Current equity holder in private company. Rijneveld:Amgen: Research Funding; Servier: Research Funding. Spits:AIMM Therapeutics: Current equity holder in private company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. van Eenennaam:AIMM Therapeutics: Current Employment, Current equity holder in private company.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often associated with chemotherapy- and radiotherapy-induced host tissue damage, leading to graft-versus-host disease (GVHD). Innate lymphoid cells (ILC) have an essential role in tissue homeostasis and tissue repair via their production of interleukin (IL)-22, which acts on intestinal stem cells. The tissue healing capacities of ILC via IL-22 in the context of allo-HSCT and GVHD has previously been demonstrated in a mouse model for acute GVHD. We investigated potential other ways of ILC-mediated tissue protection against GVHD. Tissue injury leads to the release of danger-associated molecular patterns (DAMPs). DAMPs interact with purinergic receptors and ectoenzymes on immune cells and induce pleiotropic effects, including activation of proinflammatory antigen-presenting cells and immunosuppressive effects via the generation of adenosine. Here, we report a novel subset of human ILC3 that coexpress the ectoenzymes CD39 and CD73 (ecto(+) ILC3). Ecto(+) ILC3 express RORγt and were present in the oral-gastrointestinal tract and bone marrow. ILC3 ectoenzyme expression is modulated by the proinflammatory cytokine IL-1β. Extracellular adenosine triphosphate (eATP) stimulated ecto(+) ILC3 to produce IL-22 and adenosine. Activated ecto(+) ILC3 suppressed autologous T-cell proliferation in coculture experiments via the production of adenosine. In allo-HSCT recipients, intestinal GVHD was associated with reduced proportions of ecto(+) ILC3 and decreased levels of adenosine and its metabolite inosine. Taken together, ecto(+) ILC3 have immunosuppressive properties, but in patients with GVHD, ecto(+) ILC3 are depleted. A lack of ecto(+) ILC3 and subsequent reduced capacity to neutralize DAMPs may contribute to the development of GVHD.

Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell–engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell–targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell–mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell–engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format. Significance: These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.

Innate lymphoid cells (ILCs) are crucial for the immune surveillance at mucosal sites. ILCs coordinate early eradication of pathogens and contribute to tissue healing and remodeling, features that are dysfunctional in patients with cystic fibrosis (CF). The mechanisms by which ILCs contribute to CF-immunopathology are ill-defined. Here, we show that group 2 ILCs (ILC2s) transdifferentiated into IL-17-secreting cells in the presence of the epithelial-derived cytokines IL-1β, IL-23 and TGF-β. This conversion is abrogated by IL-4 or vitamin D3. IL-17 producing ILC2s induce IL-8 secretion by epithelial cells and their presence in nasal polyps of CF patients is associated with neutrophilia. Our data suggest that ILC2s undergo transdifferentiation in CF nasal polyps in response to local cytokines, which are induced by infectious agents., Innate lymphoid cells (ILCs) play critical immunological roles including immune surveillance at mucosal sites. Here the authors show that during nasal inflammation pathogen-induced cytokine production guides the differentiation of ILCs.

Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune responses. As such, ILCs make up interesting therapeutic targets for several diseases. In patients with allergy and asthma, group 2 innate lymphoid cells produce high amounts of IL-5 and IL-13, thereby contributing to type 2-mediated inflammation. Group 3 innate lymphoid cells are implicated in intestinal homeostasis and psoriasis pathology through abundant IL-22 production, whereas group 1 innate lymphoid cells are accumulated in chronic inflammation of the gut (inflammatory bowel disease) and lung (chronic obstructive pulmonary disease), where they contribute to IFN-γ-mediated inflammation. Although the ontogeny of mouse ILCs is slowly unraveling, the development of human ILCs is far from understood. In addition, the growing complexity of the human ILC family in terms of previously unrecognized functional heterogeneity and plasticity has generated confusion within the field. Here we provide an updated view on the function and plasticity of human ILCs in tissue homeostasis and disease.

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

Summary Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen‐presenting cells for the discovery of tumor neoantigens.

AML relapse after rituximab treatment for GvHD: crucial role for B cells in G v L responses

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

INTRODUCTION Colorectal cancer (CRC) associated with Lynch syndrome is characterized by an abundance of infiltrating lymphocytes. To study whether tumor-specific antibodies with therapeutic potential can be isolated from these patients, the B-cell repertoire from a patient with Lynch syndrome who recovered from a stage IV colon carcinoma was screened. Here we describe a novel human antibody, AT1636 that recognizes a previously unidentified O-mannosylated 70kDa form of E-cadherin. The intercellular interactions by E-cadherin on tumor cells have for long been recognized as protective in cancer metastasis, and deregulation of E-cadherin is a hallmark for epithelial-mesenchymal transition (EMT). METHODS AIMM's BCL6 and Bcl-xL immortalization method[1] was used to interrogate the human antibody repertoire against targets on colon cancer cells. From a carrier of a pathogenic gene variant in the MSH6 gene diagnosed with stage IV CRC and liver metastasis that had been treated with avastin, capecitabine and oxaliplatin, peripheral-blood memory B cells were obtained 9 years after last treatment. Antibodies-containing supernatant of cultured B-cells were screened for binding to 3 different CRC cell lines (DLD1, LS174T and COLO205) and absence of binding to fibroblast by flow cytometry. High-affinity variants of AT1636 (AT1636IYN) were sorted from the AID-expressing immortalized B-cells clone[2]. RESULTS Patient derived antibodies that demonstrated differential binding to CRC cells were further characterized. Targets recognized by such antibodies were identified using immunoprecipitation and mass-spectrometry. AT1636 binds to a previously unidentified single O-mannosylated 70kDa E-cadherin variant (ECV). Although the 70 kDa ECV is found in all cells that express full length E-cadherin, tumor-specific binding of AT1636 is dependent on the single O-mannosylation pattern in the antibody epitope on ECV. Using shRNA knock-down AT1636 binding was shown to depend on the transmembrane O-mannosyltransferase targeting cadherins 3 (TMTC3)[3]. In accordance, coexpression of TMTC3 and E-cadherin in tumor cells is predictive for AT1636 binding. In addition, we observed that (over)expression of ECV results in a strong de-adhesive, EMT-like phenotype. Although AT1636 by itself is not able to induce ADCC, the CD3-bispecific antibody (single-chain UCHT1) AT1636 format specifically killed CRC cell lines. CONCLUSION The AT1636 antibody retrieved from a patient with Lynch syndrome binds a previous unidentified cancer-specific O-mannosylated 70kDa form of E-cadherin. This variant might play a role in tumor-cell invasion and metastasis. More importantly, we provide a rationale to advance AT1636 based therapeutics for treatment of CRC. references 1) Kwakkenbos et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine 2010 2) Wagner et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. PNAS 2014 3) Larsen et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. PNAS 2017 Citation Format: Martijn Kedde, Tim Beaumont, Sabrina J. Merat, Mark J. Kwakkenbos, Lina Bartels, Dorien van de Berg, Koen Wagner, Arjen Q. Bakker, Kelly Maijoor, Martino Böhne, Camille Bru, Veronika Kattler, Hans van Eenennaam, Victorine H. Roos, Frank G.J. Kallenberg, Jan Paul Medema, Paul J. Hensbergen, Pauline van Helden, Evelien Dekker, Hergen Spits. A colon cancer survivor-derived antibody recognizes a previously unidentified truncated, O-mannosylated 70kDa variant of E-cadherin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5163.

Background Adaptive immunity to cancer cells has been shown to form a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether anti-tumor antibodies could be isolated from a patient with metastatic melanoma that was tumor-free for 6 years following adoptive transfer of ex vivo expanded autologous T cells (Verdegaal 2011). Methods Peripheral blood memory B cells were immortalized using AIMM's immmortalisation technology (ectopic Bcl-6 and Bcl-xL; Kwakkenbos 2010) expression and analyzed for the presence of tumor-reactive B cells. Results AT1412 antibody was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 was found to bind the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities and disease progression. The crystal structure of the CD9 large extracellular loop in complex with an AT1412 Fab fragment revealed that AT1412 binds the CD9 epitope in an extended and unfolded conformation. In addition to melanoma, AT1412 binds other tumor types including gastric, colon- and pancreatic cancer. AT1412 was shown to induce antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) of cancer cells. In addition, AT1412 demonstrated activation of monocytes, most likely via activation of CD9 on platelets-bound monocytes. In mice carrying a human immune system (HIS-mice; van Lent 2010) AT1412 strongly enhanced tumor rejection of A375 and SKMEL-5 tumor cells. AT1412 treatment was shown to enhance tumor infiltration of CD8 T cells and macrophages. AT1412 efficacy was further synergistically enhanced when combined with an anti-PD1 antibody (nivolumab). Other groups have shown that anti-CD9 antibodies can induce tumor rejection in mice. However, these antibodies could not be advanced due to induction of platelet aggregation. We show that AT1412 does not induce aggregation of both human and cynomolgus platelets. Also affinity matured versions of AT1412 do not induce platelet aggregation, supporting that AT1412 binds a unique epitope on CD9. In addition, we studied the safety of AT1412 during a one month exposure in a weekly administration in cynomolgus monkeys. Besides a transient thrombocytopenia AT1412 was well tolerated up to 10 mg/kg antibody (highest dose tested) and did not lead to other adverse events nor were any coagulation factors affected Conclusions Taken together, applying AIMM's proprietary B-cell immortalization technology we isolated antibody AT1412 that targets a unique epitope on CD9. AT1412 was shown to induce tumor rejection as a single agent and enhances the activity of anti-PD-1 antibodies, while not inducing platelet aggregation. One month exposure of AT1412 in monkeys indicated that AT1412 can be safely administered. Preclinical development of AT1412 is ongoing to initiate clinical evaluation in 2020. Funding Dutch Cancer Society, grant UVA 2010-4822 Citation Format: Remko Schotte, Julien Villaudy, Martijn Kedde, Wouter Pos, Koen Wagner, Viviana Neviani, Daniel Go, Etsuko Yasuda, Christien Fatmawati, Els Verdegaal, Susan van Hal, Yvonne Claasen, Hans van Eenennaam, Pauline van Helden, Piet Gros, Sjoerd van der Burg, Hergen Spits. A patient-derived anti-CD9 antibody induces tumor rejection and synergistically enhances anti-PD1 activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 532.

Introduction Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (BCP-ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19-targeting immunotherapy has significantly improved patient outcomes in (relapsed) BCP-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. An ongoing medical need remains for the identification of alternative immunotherapeutic targets for treatment of ALL. Methods CD9 is expressed in 60-80% of BCP-ALL, is correlated with adverse prognosis and has been proposed as therapeutic target for BCP-ALL. AT1412 is a fully human CD9-targeting antibody, that was identified from a patient cured of metastatic melanoma after adoptive T-cell therapy, using our B-cell immortalization technology (AIMSelect) [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo. Results Binding of AT1412 to BCP-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on CD9 levels that we detected using a commercial CD9 antibody. AT1412-induced antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) correlated with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. These findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=38). AT1412 showed binding to the majority of B-ALL samples but not to T-ALL samples. AT1412 induced ADCC of nearly all B-ALL samples it bound to (10/11) and of none of the non-binding B-ALL or T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were corroborated by the observation that AT1412 induced specific B-ALL cell death when a bone marrow sample from a newly diagnosed BCP-ALL patient was incubated with AT1412. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human BCP-ALL. Conclusion Taken together, the majority of BCP-ALL express CD9 and this is associated with poor prognosis. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Citation Format: Greta de Jong, Sophie E. Levie, Remko Schotte, Wouter Pos, Daniel Go, Etsuko Yasuda, Madalina Cercel, Susan E. van Hal, Esmay Frankin, Aniko Szabo, Martijn Kedde, Els Verdegaal, Julien Villaudy, Sjoerd van der Burg, Pauline M. van Helden, Hans van Eenennaam, Hergen Spits, Anita van Rijneveld, Mette D. Hazenberg. AT1412, a patient-derived antibody in development for the treatment of CD9-positive precursor B-acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 531.

Introduction CD43s, a unique sialylated form of CD43 expressed by myeloid malignancies, is a novel target and AT1413, an antibody targeting CD43s was shown to have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was isolated from a high-risk AML patient who successfully cleared the leukemia after allogeneic stem cell transplantation. Because CD43 is also expressed in non-hematopoietic cells we studied whether CD43s is also present and can also be targeted on non-hematopoietic tumors. Materials and methods AT1413 binding on a panel of tumor cell lines was analyzed by flow cytometry. AT1413 was constructed into a bispecific T-cell engaging format (AT1413 bTCE) by linking the full-length AT1413 IgG to two single chain variable fragments against CD3ϵ with a combination of site-specific enzymatic and chemical coupling. A monovalent T-cell engager was produced as heterotrimer consisting of one AT1413 monovalent chain and one anti-CD3 scFV fused to AT1413 monovalent chain. Point mutations in the IgG heavy chain were introduced to prevent interactions between AT1413 T-cell engagers and Fc-receptors. The cytotoxicity-inducing activities were established using PBMCs as effector and tumor cells as target cells using standard cytotoxic assays in vitro and in vivo in a mouse model carrying human immune cells. Results We observed that AT1413 binds to non-hematopoietic tumor cells, such as melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody dependent cellular cytotoxicity against melanoma cell lines and primary melanoma samples. However, AT1413 was unable to affect growth of melanoma cells in vivo. To increase the efficacy of AT1413 it was formatted as a bispecific T-cell engaging antibody (TCE): one binding bivalently (bTCE) to CD43s and monovalently to CD3ϵ and the other monovalently (KiH) to both CD43s and CD3ϵ. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with different potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth. Conclusion AT1413 recognizes a sialylated epitope on CD43 shared by melanoma, AML and MDS cells. Two different bispecific TCE forms of AT1413 induce strong anti-tumor cytotoxic activities in vitro and in vivo. These data indicate a broad therapeutic potential of AT1413. Citation Format: Greta de Jong, Lina Bartels, Martijn Kedde, Els Verdegaal, Marijn A. Gillissen, Sophie E. Levie, Madalina G. Cercel, Susan E. van Hal-van Veen, Christien Fatmawati, Dorien van de Berg, Etsuko Yasuda, Yvonne Claassen, Arjen Q. Bakker, Remko Schotte, Julien Villaudy, Koen Wagner, Hergen Spits, Mette D. Hazenberg, Pauline M. van Helden. T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 542.

Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8+ T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc.

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse., Ministerio de Economia y Competitividad (MINECO) PLE-2009-0110, SAF2010-15106, SAF2013-44857-R, and SAF2016-75442-R (Agencia Estatal de Investigacion/European Regional Development Fund, European Union), the Fundacion Sandra Ibarra, the Fundacion Asociacion Espanola Contra el Cancer (AECC CI13131229), the Instituto de Salud Carlos III (RTICC RD06/0014/1012), and the European Union Seventh Framework Programme (FP7/2007-20013) ThymiStem project (602587). Institutional grants from the Fundacion Ramon Areces and Banco de Santander

Introduction Recently we identified the transcriptional co-activator BOB.1 as specifically overexpressed in RA synovium, where its levels strongly correlated with the presence of germinal centres (GCs). In accordance with human data, mice lacking functional BOB.1 failed to mount GC response and were resistant to the experimental model of RA. Objectives In this study we investigated whether increased levels of BOB.1 impact the phenotype and function of B cells in a GC-like environment. Methods Expression levels of BOB.1 were assessed by qPCR and immunofluorescence. DNA encoding BOB.1 was introduced into B cells by retroviral-mediated gene transfer. The phenotype, activation and BCR signalling were analysed by FACS. Expression of CCL3 was measured by qPCR. Impact of high levels of BOB.1 in B cells on proliferative responses of autologous TFH cells was assessed by FACS after 5 days of co-culture with SEB-pulsed B cells. Results Immunofluorescence staining of inflamed RA synovium demonstrated the presence of high numbers of BOB.1-positive cells clustered in the lymphoid follicles in a pattern similar to the normal tonsillar tissue. Further analysis revealed that expression of BOB.1 in RA synovial B cells was significantly higher than in tonsillar B cells. To investigate how high levels of BOB.1 impact phenotype and function of B cells in a GC-like environment we overexpressed BOB.1 in human primary B cells cultured with CD40L and IL-21. In these conditions B cells rapidly differentiate into antibody-producing plasma cells, a process that is accompanied by a decrease in expression of CD20 and increase in CD38 and CD27. In contrast to the control, cells transduced with BOB.1 retained CD20 expression while expressed intermediate levels of CD27 and CD38. Accordingly, the percentage of plasmablasts was significantly lower in BOB.1-overexpressing cells, confirming that high levels of BOB.1 suppress plasma cell differentiation. Further analysis revealed that BOB.1-transduced memory B cells expressed high levels of costimulatory receptors CD40, CD80 and PD-L2 involved in B-T cell interactions and showed more rapid Ca2+mobilisation and increased production of T-cell-recruiting chemokine CCL3 following BCR stimulation. Accordingly, TFH cells co-cultured with BOB.1-transduced B cells exhibit a higher rate of proliferation as compared to TFH cells co-cultured with B cells transduced with the control retrovirus. Conclusions These data suggest that increased levels of BOB.1 in B cells during T cell-dependent responses in GCs suppress their terminal differentiation and enhance expression of costimulatory molecules and BCR signalling strength, which is translated into their greater help to TFH cells. Whether this is sufficient to drive/accelerate autoimmune disease will be evaluated in a B cell-specific BOB.1 tg mouse model. Disclosure of interest None declared

Innate lymphoid cells (ILCs) are lymphocytes that do not express the type of diversified antigen receptors expressed on T cells and B cells. ILCs are largely tissue-resident cells and are deeply integrated into the fabric of tissues. The discovery and investigation of ILCs over the past decade has changed our perception of immune regulation and how the immune system contributes to the maintenance of tissue homeostasis. We now know that cytokine-producing ILCs contribute to multiple immune pathways by, for example, sustaining appropriate immune responses to commensals and pathogens at mucosal barriers, potentiating adaptive immunity, and regulating tissue inflammation. Critically, the biology of ILCs also extends beyond classical immunology to metabolic homeostasis, tissue remodeling, and dialog with the nervous system. The last 10 years have also contributed to our greater understanding of the transcriptional networks that regulate lymphocyte commitment and delineation. This, in conjunction with the recent advances in our understanding of the influence of local tissue microenvironments on the plasticity and function of ILCs, has led to a re-evaluation of their existing categorization. In this review, we distill the advances in ILC biology over the past decade to refine the nomenclature of ILCs and highlight the importance of ILCs in tissue homeostasis, morphogenesis, metabolism, repair, and regeneration. Since the discovery of innate lymphoid cells a decade ago, studies in this field have drastically expanded our understanding of the role of the immune system in the maintenance of homeostasis. This Review explores the immune functions of these cells and their connection to metabolism, tissue repair, and the nervous system.

Respiratory pathogens and the human immune response they elicit can now be studied in humanized mice.

The innate immune system is composed of a diverse array of evolutionarily ancient haematopoietic cell types, including dendritic cells, monocytes, macrophages and granulocytes. These cell populations collaborate with each other, with the adaptive immune system and with non-haematopoietic cells to promote immunity, inflammation and tissue repair. Innate lymphoid cells are the most recently identified constituents of the innate immune system and have been the focus of intense investigation over the past five years. We summarize the studies that formally identified innate lymphoid cells and highlight their emerging roles in controlling tissue homeostasis in the context of infection, chronic inflammation, metabolic disease and cancer.

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4+ malignancies. Mol Cancer Ther; 14(11); 2527–40. ©2015 AACR.

Tumor-specific neo-antigens that arise as a consequence of mutations(1,2) are thought to be important for the therapeutic efficacy of cancer immunotherapies(3-5). Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8(+) T cells(3-7), but it is unclear whether neoantigen-specific CD4(+) T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4(+) T-cell responses in tumor control(8-10), we addressed whether neo-antigen-specific CD4(+) T-cell reactivity is a common property in human melanoma

Innate lymphoid cells (ILCs) have emerged as a key cell type involved in surveillance and maintenance of mucosal tissues. Mouse ILCs rely on the transcriptional regulator Inhibitor of DNA-binding protein 2 (Id2) for their development. Here, we show that Id2 also drives development of human ILC because forced expression of Id2 in human thymic progenitors blocked T cell commitment, upregulated CD161 and promyelocytic leukemia zinc finger (PLZF), and maintained CD127 expression, markers that are characteristic for human ILCs. Surprisingly CD5 was also expressed on these in vitro generated ILCs. This was not an in vitro artifact because CD5 was also found on ex vivo isolated ILCs from thymus and from umbilical cord blood. CD5 was also expressed on small proportions of ILC2 and ILC3. CD5+ ILCs were functionally immature, but could further differentiate into mature CD5− cytokine-secreting ILCs. Our data show that Id2 governs human ILC development from thymic progenitor cells toward immature CD5+ ILCs.

International audience; Innate lymphoid cells (ILCs) were first described as playing important roles in the development of lymphoid tissues and more recently in the initiation of inflammation at barrier surfaces in response to infection or tissue damage. It has now become apparent that ILCs play more complex roles throughout the duration of immune responses, participating in the transition from innate to adaptive immunity and contributing to chronic inflammation. The proximity of ILCs to epithelial surfaces and their constitutive strategic positioning in other tissues throughout the body ensures that, in spite of their rarity, ILCs are able to regulate immune homeostasis effectively. Dysregulation of ILC function might result in chronic pathologies such as allergies, autoimmunity, and inflammation. A new role for ILCs in the maintenance of metabolic homeostasis has started to emerge, underlining their importance in fundamental physiological processes beyond infection and immunity.

Immunodeficient mice reconstituted with human hematopoietic stem cells provide a small-animal model for the study of development and function of human hematopoietic cells in vivo. However, in the current models, the immune response, and especially the humoral response by the human immune cells is far from optimal. The B cells found in these mice exhibit an immature and abnormal phenotype correlating with a reduced capacity to produce antigen-specific affinity matured antibodies upon infection or immunization. Herein, we review the current state of knowledge of development, function and antibody production of human B cells and discuss the obstacles for the improvement of these models. (C) 2014 Elsevier B.V. All rights reserved

T cell acute lymphoblastic leukemias (T-ALLs) commonly display constitutively active PI3K/mTOR and Notch signaling. However, controversy surrounds whether these pathways have independent functions and whether Pten loss is sufficient to generate resistance to Notch inhibition. Here we report that Pten−/− T-ALL is sensitive to either PI3K/mTOR or Notch inhibition alone, each pathway controlling distinct downstream signaling events that cannot be rescued by activation of the other pathway, consistent with independent, non-redundant functions. Although many human T-ALLs display constitutively activating Notch1 mutations, primary Pten−/− T-ALLs expressed wild-type Notch1 and depended on the Notch ligand DLL4 in vivo. Pten−/− T-ALLs with or without γc/TCR signaling responded similarly to PI3K/mTOR and Notch inhibition, although extended culture in vitro occasionally induced Notch-independent growth. However, unlike the T-ALLs lacking only Pten, eight of 23 Pten−/− T-ALLs that also lacked γc/TCR signaling accumulated Notch1 mutations, suggesting crosstalk between γc/TCR and Notch signaling. Importantly, we concluded that loss of γc/TCR signaling also inhibited thymic exit of Pten−/− T-ALLs. Our results may be clinically relevant in revealing that Pten loss is not sufficient to engender resistance to Notch inhibition, uncovering a role in T-ALL for ligand-dependent induction of wild-type Notch1, and suggesting that γc/TCR signaling could be targeted for preventing metastasis.

Innate lymphoid cells (ILCs) are lymphoid cells that do not express rearranged receptors and have important effector and regulatory functions in innate immunity and tissue remodeling. ILCs are categorized into 3 groups based on their distinct patterns of cytokine production and the requirement of particular transcription factors for their development and function. Group 1 ILCs (ILC1s) produce interferon γ and depend on Tbet, group 2 ILCs (ILC2s) produce type 2 cytokines like interleukin-5 (IL-5) and IL-13 and require GATA3, and group 3 ILCs (ILC3s) include lymphoid tissue inducer cells, produce IL-17 and/or IL-22, and are dependent on RORγt. Whereas ILCs play essential roles in the innate immune system, uncontrolled activation and proliferation of ILCs can contribute to inflammatory autoimmune diseases. In this review, we provide an overview of the characteristics of ILCs in the context of health and disease. We will focus on human ILCs but refer to mouse studies if needed to clarify aspects of ILC biology.