PD-1 checkpoint inhibition enhances the antilymphoma activity of CD19-CAR-iNKT cells that retain their ability to prevent alloreactivity.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. METHODS: iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. RESULTS: In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. CONCLUSION: In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.

Human invariant natural killer T cells promote tolerance by preferential apoptosis induction of conventional dendritic cells.

Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We recently showed in murine studies and in vitro human models that adoptively transferred invariant natural killer T (iNKT) cells protect from GvHD and promote graft-versus-leukemia effects. The cellular mechanisms underlying GvHD prevention by iNKT cells in humans, however, remain unknown. In order to study relevant cellular interactions, dendritic cells (DC) were either generated from monocytes or isolated directly from blood of healthy donors or GvHD patients and co-cultured in a mixed lymphocyte reaction (MLR) with T cells obtained from healthy donors or transplantation bags. Addition of culture-expanded iNKT cells to the MLR-induced DC apoptosis in a cell contact-dependent manner, thereby preventing T-cell activation and proliferation. Annexin V/propidium iodide staining and image stream assays showed that CD4+CD8-, CD4-CD8+ and double negative iNKT cells are similarly able to induce DC apoptosis. Further MLR assays revealed that conventional DC (cDC) but not plasmacytoid DC (pDC) could induce alloreactive T-cell activation and proliferation. Interestingly, cDC were also more susceptible to apoptosis induced by iNKT cells, which correlates with their higher CD1d expression, leading to a bias in favor of pDC. Remarkably, these results could also be observed in GvHD patients. We propose a new mechanism how ex vivo expanded human iNKT cells prevent alloreactivity of T cells. iNKT cells modulate T-cell responses by selective apoptosis of DC subsets, resulting in suppression of T-cell activation and proliferation while enabling beneficial immune responses through pDC.

Only Hematopoietic Stem and Progenitor Cells from Cord Blood Are Susceptible to Malignant Transformation by MLL-AF4 Translocations.

Mixed lineage leukemia (MLL) (KMT2A) rearrangements (KMT2Ar) play a crucial role in leukemogenesis. Dependent on age, major differences exist regarding disease frequency, main fusion partners and prognosis. In infants, up to 80% of acute lymphoid leukemia (ALL) bear a MLL translocation and half of them are t(4;11), resulting in a poor prognosis. In contrast, in adults only 10% of acute myeloid leukemia (AML) bear t(9;11) with an intermediate prognosis. The reasons for these differences are poorly understood. Recently, we established an efficient CRISPR/Cas9-based KMT2Ar model in hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood (huCB) and faithfully mimicked the underlying biology of the disease. Here, we applied this model to HSPCs from adult bone marrow (huBM) to investigate the impact of the cell of origin and fusion partner on disease development. Both genome-edited infant and adult KMT2Ar cells showed monoclonal outgrowth with an immature morphology, myelomonocytic phenotype and elevated KMT2Ar target gene expression comparable to patient cells. Strikingly, all KMT2Ar cells presented with indefinite growth potential except for MLL-AF4 huBM cells ceasing proliferation after 80 days. We uncovered FFAR2, an epigenetic tumor suppressor, as potentially responsible for the inability of MLL-AF4 to immortalize adult cells under myeloid conditions.

MAT2A as Key Regulator and Therapeutic Target in MLLr Leukemogenesis.

Epigenetic dysregulation plays a pivotal role in mixed-lineage leukemia (MLL) pathogenesis, therefore serving as a suitable therapeutic target. S-adenosylmethionine (SAM) is the universal methyl donor in human cells and is synthesized by methionine adenosyltransferase 2A (MAT2A), which is deregulated in different cancer types. Here, we used our human CRISPR/Cas9-MLL-rearranged (CRISPR/Cas9-MLLr) leukemia model, faithfully mimicking MLLr patients' pathology with indefinite growth potential in vitro, to evaluate the unknown role of MAT2A. Comparable to publicly available patient data, we detected MAT2A to be significantly overexpressed in our CRISPR/Cas9-MLLr model compared to healthy controls. By using non-MLLr and MLLr cell lines and our model, we detected an MLLr-specific enhanced response to PF-9366, a new MAT2A inhibitor, and small interfering (si) RNA-mediated knockdown of MAT2A, by alteration of the proliferation, viability, differentiation, apoptosis, cell cycling, and histone methylation. Moreover, the combinational treatment of PF-9366 with chemotherapy or targeted therapies against the SAM-dependent methyltransferases, disruptor of telomeric silencing 1 like (DOT1L) and protein arginine methyltransferase 5 (PRMT5), revealed even more pronounced effects. In summary, we uncovered MAT2A as a key regulator in MLL leukemogenesis and its inhibition led to significant anti-leukemic effects. Therefore, our study paves the avenue for clinical application of PF-9366 to improve the treatment of poor prognosis MLLr leukemia.

Inhibition of effector B cells by ibrutinib in systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease with a significant morbidity and reduced survival of patients. Effective treatment and clinical control of the disease remain challenging. In particular, the development of pulmonary and cardiac fibrosis and pulmonary hypertension are severe complications responsible for excessive mortality. Currently available treatment strategies only alleviate symptoms and slow disease progression. Here, we investigated the therapeutic potential of ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor used in B cell malignancies, to alter B cell pathology in SSc in an in vitro model of autoimmunity. METHODS: PBMCs and sorted B cells of 24 patients with SSc were used for functional testing after stimulation with hypomethylated DNA fragments (CpG) to induce an innate immune response. The effects of ibrutinib on cytokine production, autoantibody release, and activation of the transcription factor NFκB were evaluated. RESULTS: Ibrutinib was able to reduce the production of the profibrotic hallmark cytokines IL-6 and TNF-α mainly from the effector B cell population in patients with SSc. Importantly, small doses of ibrutinib (0.1 µM) preserved the production of immunoregulatory IL-10 while effectively inhibiting hyperactivated, profibrotic effector B cells. In a flow cytometry analysis of phosphorylated NFκB, an important transcription factor in the induction of innate immune responses in B cells, significantly less activation was observed with ibrutinib treatment. CONCLUSION: Our data could pave the avenue for a clinical application of ibrutinib for patients with SSc as a novel treatment option for the underlying pathogenetic immune imbalance contributing to disease onset and progression.

Invariant natural killer T cells are functionally impaired in patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a potentially fatal autoimmune disease that leads to extensive fibrosis of the skin and internal organs. Invariant natural killer T (iNKT) cells are potent immunoregulatory T lymphocytes being able to orchestrate dysregulated immune responses. The purpose of this study was to evaluate numbers and function of iNKT cells in patients with SSc and to analyze their correlation with disease parameters. METHODS: Human iNKT cells from 88 patients with SSc and 33 healthy controls were analyzed by flow cytometry. Their proliferative capacity and cytokine production were investigated following activation with CD1d ligand α-galactosylceramide (α-GalCer). RESULTS: We observed an absolute and relative decrease of iNKT cells in patients with SSc compared with healthy controls. Interestingly, the subtype of SSc, disease severity, or treatment with immunosuppressive drugs did not affect iNKT cell numbers. However, T helper (Th) cell immune polarization was biased towards a Th17 immunophenotype in SSc patients. Moreover, iNKT cells from patients with SSc showed a significantly decreased expansion capacity upon stimulation with α-GalCer. CONCLUSION: iNKT cells are deficient and functionally impaired in patients with SSc. Therefore, adoptive transfer strategies using culture-expanded iNKT cells could be a novel approach to treat SSc patients.

Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9.

MLL rearrangements play a crucial role in leukemogenesis and comprise a poor prognosis. Therefore, new treatment strategies are urgently needed. We used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4 or -AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient cells faithfully mimicking the nature of the disease. Thus, it serves as a fundamental basis for pharmacological studies: inhibition of histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is one specific therapeutic approach currently tested in clinical trials. However, success was limited by restricted response warranting further investigation of drug combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines and in MLL mouse models. Here, we used DOT1L and PRMT5 inhibitors in our human MLL-rearranged model demonstrating dose-dependent reduced proliferation, impairment of cell cycle, increasing differentiation, apoptosis, downregulation of target genes and sensitization to chemotherapy. Strikingly, the combination of both compounds led to synergistic anti-tumoral effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias.

Invariant NKT Cells From Donor Lymphocyte Infusions (DLI-iNKTs) Promote ex vivo Lysis of Leukemic Blasts in a CD1d-Dependent Manner.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.

Chromobodies to Quantify Changes of Endogenous Protein Concentration in Living Cells.

Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.

Culture-Expanded Human Invariant Natural Killer T Cells Suppress T-Cell Alloreactivity and Eradicate Leukemia.

Graft-versus-host disease (GVHD) is a major cause of significant morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses, protect from lethal GVHD, and promote graft-versus-leukemia effects in murine studies. Since iNKT cells constitute less than 0.5% of human peripheral blood mononuclear cells (PBMCs), in vitro expansion with their glycolipid ligands is required before they can be used for cytotherapy and experimental purposes. Three weeks of cell culture and autologous restimulation with either KRN7000, PBS44, or PBS57 resulted in a robust proliferation of iNKT cells from human PBMCs. Next, iNKT cells were sorted to a purity higher than 90% being crucial for further experimental and clinical applications. These iNKT cells significantly decreased activation and proliferation of allogeneic CD3+ T lymphocytes. In addition, leukemia cell lines and primary leukemia cells were efficiently lysed by culture-expanded iNKT cells. Importantly, culture-expanded donor iNKT cells promoted robust antileukemia activity against HLA-matched allogeneic patient leukemia cells. Our data indicate that the adoptive transfer of culture-expanded iNKT cells could be a powerful cytotherapeutic approach to induce immune tolerance and prevent leukemia relapse after allogeneic HCT in humans.