Background More effective therapies for steroid-refractory acute graft-versus-host disease (SR-aG... more Background More effective therapies for steroid-refractory acute graft-versus-host disease (SR-aGVHD) are urgently needed. Because infections and relapse of the hematological malignancy contribute to the dismal overall survival (OS) therapies that limit the duration of immune suppression after achieving a remission might be preferred. The immunotoxin (IT)-combination (T-GuardTM) consists of two antibody-drug conjugates (i.e. Ricin A) that target CD3 and CD7 on activated T lymphocytes and has shown efficacy as third-line therapy in SR-aGVHD while allowing fast immune reconstitution. Objectives We conducted a prospective phase I/II multicenter trial on the safety and efficacy of T-GuardTM for the treatment of SR-aGVHD (NCT02027805). Methods Adult patients with grade II-IV SR-aGVHD were eligible for inclusion. Exclusion criteria consisted of the presence of uncontrolled infections, signs of chronic GVHD, severe renal impairment and severe hypoalbuminemia. T-Guard was given as 4-hour in...
Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages ... more Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation o...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 9, 2017
Older acute myeloid leukemia (AML) patients have a poor prognosis, therefore novel therapies are ... more Older acute myeloid leukemia (AML) patients have a poor prognosis, therefore novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting an unique scalable NK cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPCs) from partially HLA-matched umbilical cord blood units. Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30x10^6/kg body weight) after lymphodepleting chemotherapy without cytokine boosting. HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1x10^4 T cells/kg and <3x10^5 B cells/kg body weight. HSPC-NK cells were well tolerated and no graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting was given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, ...
After lymphocyte-depleted BMT, CD8+ T cells have been expanded to or above normal levels in 45% o... more After lymphocyte-depleted BMT, CD8+ T cells have been expanded to or above normal levels in 45% of the recipients within 3 months. The mechanisms underlying proliferation of donor-derived CD8+ T cells after BMT are still unclear. We investigated whether these CD8+ T cells proliferate in response to specific antigens by determination of TCR clonality and whether these cells exert specific cytotoxicity. PCR analysis of TCR-gamma gene rearrangements showed a marked clonal predominance in CD8+ T cells of recipients with a high number of these cells. Strong association between expansion of CD8+ T cells and CMV infection suggests involvement of CMV antigens. Therefore, we examined CMV-specific cytotoxicity of freshly isolated CD8+ T cells of two BMT recipients with clonal expansion after the onset of CMV infection. CD8+ T cells exerted HLA-restricted cytotoxicity directed against CMV-infected fibroblasts indicating that CMV stimulates proliferation. The majority of CD8+ T cells in these r...
We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by ... more We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by using class switch variants (CLB-B4-IgG1 and CLB-B4-IgG2a) conjugated to ricin A. Notably, IgG1-IT appeared to be approximately 100-fold more potent than IgG2a-IT toward B-cell lines Daudi and KM3. Binding and internalization studies with 125I-labeled monoclonal antibodies (mAbs) revealed a higher cellular uptake of IgG1 compared to IgG2a, despite similar binding affinities. Following removal of the Fc part, both mAbs internalized at the same rate as IgG2a, indicating that the Fc part of IgG1 is involved in enhanced cellular uptake. The involvement of Fc gamma RII (CD32) in this process was demonstrated by a decreased cytotoxicity of IgG1-IT (and not IgG2a-IT) in the presence of Fc gamma RII-blocking mAbs AT10 or IV.3. To identify the isoform responsible for this phenomenon, internalization of IgG1 and IgG2a in 11 B-cell lines and malignant B-cells of 8 patients was compared with express...
Proceedings of the National Academy of Sciences, 2012
Adaptive features of innate immunity, recently described as “trained immunity,” have been documen... more Adaptive features of innate immunity, recently described as “trained immunity,” have been documented in plants, invertebrate animals, and mice, but not yet in humans. Here we show that bacille Calmette-Guérin (BCG) vaccination in healthy volunteers led not only to a four- to sevenfold increase in the production of IFN-γ, but also to a twofold enhanced release of monocyte-derived cytokines, such as TNF and IL-1β, in response to unrelated bacterial and fungal pathogens. The enhanced function of circulating monocytes persisted for at least 3 mo after vaccination and was accompanied by increased expression of activation markers such as CD11b and Toll-like receptor 4. These training effects were induced through the NOD2 receptor and mediated by increased histone 3 lysine 4 trimethylation. In experimental studies, BCG vaccination induced T- and B-lymphocyte–independent protection of severe combined immunodeficiency SCID mice from disseminated candidiasis (100% survival in BCG-vaccinated m...
A better understanding of human NK cell development in vivo is crucial to exploit NK cells for im... more A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56(bright)CD16⁺/⁻ and CD56(dim)CD16⁺ subset...
Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K, 2012
Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosi... more Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the 2nd and 3rd European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as "normal", "suggestive of" or "diagnostic of" MDS. A FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis if at l...
Background More effective therapies for steroid-refractory acute graft-versus-host disease (SR-aG... more Background More effective therapies for steroid-refractory acute graft-versus-host disease (SR-aGVHD) are urgently needed. Because infections and relapse of the hematological malignancy contribute to the dismal overall survival (OS) therapies that limit the duration of immune suppression after achieving a remission might be preferred. The immunotoxin (IT)-combination (T-GuardTM) consists of two antibody-drug conjugates (i.e. Ricin A) that target CD3 and CD7 on activated T lymphocytes and has shown efficacy as third-line therapy in SR-aGVHD while allowing fast immune reconstitution. Objectives We conducted a prospective phase I/II multicenter trial on the safety and efficacy of T-GuardTM for the treatment of SR-aGVHD (NCT02027805). Methods Adult patients with grade II-IV SR-aGVHD were eligible for inclusion. Exclusion criteria consisted of the presence of uncontrolled infections, signs of chronic GVHD, severe renal impairment and severe hypoalbuminemia. T-Guard was given as 4-hour in...
Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages ... more Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation o...
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 9, 2017
Older acute myeloid leukemia (AML) patients have a poor prognosis, therefore novel therapies are ... more Older acute myeloid leukemia (AML) patients have a poor prognosis, therefore novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting an unique scalable NK cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPCs) from partially HLA-matched umbilical cord blood units. Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30x10^6/kg body weight) after lymphodepleting chemotherapy without cytokine boosting. HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1x10^4 T cells/kg and <3x10^5 B cells/kg body weight. HSPC-NK cells were well tolerated and no graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting was given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, ...
After lymphocyte-depleted BMT, CD8+ T cells have been expanded to or above normal levels in 45% o... more After lymphocyte-depleted BMT, CD8+ T cells have been expanded to or above normal levels in 45% of the recipients within 3 months. The mechanisms underlying proliferation of donor-derived CD8+ T cells after BMT are still unclear. We investigated whether these CD8+ T cells proliferate in response to specific antigens by determination of TCR clonality and whether these cells exert specific cytotoxicity. PCR analysis of TCR-gamma gene rearrangements showed a marked clonal predominance in CD8+ T cells of recipients with a high number of these cells. Strong association between expansion of CD8+ T cells and CMV infection suggests involvement of CMV antigens. Therefore, we examined CMV-specific cytotoxicity of freshly isolated CD8+ T cells of two BMT recipients with clonal expansion after the onset of CMV infection. CD8+ T cells exerted HLA-restricted cytotoxicity directed against CMV-infected fibroblasts indicating that CMV stimulates proliferation. The majority of CD8+ T cells in these r...
We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by ... more We analyzed the effect of isotype variation on effectiveness of B-cell CD19 immunotoxins (IT) by using class switch variants (CLB-B4-IgG1 and CLB-B4-IgG2a) conjugated to ricin A. Notably, IgG1-IT appeared to be approximately 100-fold more potent than IgG2a-IT toward B-cell lines Daudi and KM3. Binding and internalization studies with 125I-labeled monoclonal antibodies (mAbs) revealed a higher cellular uptake of IgG1 compared to IgG2a, despite similar binding affinities. Following removal of the Fc part, both mAbs internalized at the same rate as IgG2a, indicating that the Fc part of IgG1 is involved in enhanced cellular uptake. The involvement of Fc gamma RII (CD32) in this process was demonstrated by a decreased cytotoxicity of IgG1-IT (and not IgG2a-IT) in the presence of Fc gamma RII-blocking mAbs AT10 or IV.3. To identify the isoform responsible for this phenomenon, internalization of IgG1 and IgG2a in 11 B-cell lines and malignant B-cells of 8 patients was compared with express...
Proceedings of the National Academy of Sciences, 2012
Adaptive features of innate immunity, recently described as “trained immunity,” have been documen... more Adaptive features of innate immunity, recently described as “trained immunity,” have been documented in plants, invertebrate animals, and mice, but not yet in humans. Here we show that bacille Calmette-Guérin (BCG) vaccination in healthy volunteers led not only to a four- to sevenfold increase in the production of IFN-γ, but also to a twofold enhanced release of monocyte-derived cytokines, such as TNF and IL-1β, in response to unrelated bacterial and fungal pathogens. The enhanced function of circulating monocytes persisted for at least 3 mo after vaccination and was accompanied by increased expression of activation markers such as CD11b and Toll-like receptor 4. These training effects were induced through the NOD2 receptor and mediated by increased histone 3 lysine 4 trimethylation. In experimental studies, BCG vaccination induced T- and B-lymphocyte–independent protection of severe combined immunodeficiency SCID mice from disseminated candidiasis (100% survival in BCG-vaccinated m...
A better understanding of human NK cell development in vivo is crucial to exploit NK cells for im... more A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56(bright)CD16⁺/⁻ and CD56(dim)CD16⁺ subset...
Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K, 2012
Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosi... more Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the 2nd and 3rd European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as "normal", "suggestive of" or "diagnostic of" MDS. A FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis if at l...
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