B-cell targeting with anti-CD38 daratumumab:implications for differentiation and memory responses

B cell–targeted therapies, such as CD20-targeting mAbs, deplete B cells but do not target the autoantibody-producing plasma cells (PCs). PC-targeting therapies such as daratumumab (anti-CD38) form an attractive approach to treat PC-mediated diseases. CD38 possesses enzymatic and receptor capabilities, which may impact a range of cellular processes including proliferation and differentiation. However, very little is known whether and how CD38 targeting affects B-cell differentiation, in particular for humans beyond cancer settings. Using in-depth in vitro B-cell differentiation assays and signaling pathway analysis, we show that CD38 targeting with daratumumab demonstrated a significant decrease in proliferation, differentiation, and IgG production upon T cell–dependent B-cell stimulation. We found no effect on T-cell activation or proliferation. Furthermore, we demonstrate that daratumumab attenuated the activation of NF-?B in B cells and the transcription of NF-?B–targeted genes. When culturing sorted B-cell subsets with daratumumab, the switched memory B-cell subset was primarily affected. Overall, these in vitro data elucidate novel non-depleting mechanisms by which daratumumab can disturb humoral immune responses. Affecting memory B cells, daratumumab may be used as a therapeutic approach in B cell–mediated diseases other than the currently targeted malignancies

B-cell targeting with anti-CD38 daratumumab:implications for differentiation and memory responses

B cell–targeted therapies, such as CD20-targeting mAbs, deplete B cells but do not target the autoantibody-producing plasma cells (PCs). PC-targeting therapies such as daratumumab (anti-CD38) form an attractive approach to treat PC-mediated diseases. CD38 possesses enzymatic and receptor capabilities, which may impact a range of cellular processes including proliferation and differentiation. However, very little is known whether and how CD38 targeting affects B-cell differentiation, in particular for humans beyond cancer settings. Using in-depth in vitro B-cell differentiation assays and signaling pathway analysis, we show that CD38 targeting with daratumumab demonstrated a significant decrease in proliferation, differentiation, and IgG production upon T cell–dependent B-cell stimulation. We found no effect on T-cell activation or proliferation. Furthermore, we demonstrate that daratumumab attenuated the activation of NF-κB in B cells and the transcription of NF-κB–targeted genes. When culturing sorted B-cell subsets with daratumumab, the switched memory B-cell subset was primarily affected. Overall, these in vitro data elucidate novel non-depleting mechanisms by which daratumumab can disturb humoral immune responses. Affecting memory B cells, daratumumab may be used as a therapeutic approach in B cell–mediated diseases other than the currently targeted malignancies.</p

Liposomal drug delivery in an in vitro 3D bone marrow model for multiple myeloma

Purpose: Liposomal drug delivery can improve the therapeutic index of treatments for multiple myeloma. However, an appropriate 3D model for the in vitro evaluation of liposomal drug delivery is lacking. In this study, we applied a previously developed 3D bone marrow (BM) myeloma model to examine liposomal drug therapy. Material and methods: Liposomes of different sizes (~75-200 nm) were tested in a 3D BM myeloma model, based on multipotent mesenchymal stromal cells, endothelial progenitor cells, and myeloma cells cocultured in hydrogel. The behavior and efficacy of liposomal drug therapy was investigated, evaluating the feasibility of testing liposomal drug delivery in 3D in vitro. Intracellular uptake of untargeted and integrin α4β1 (very late antigen-4) targeted liposomes was compared in myeloma and supporting cells, as well as the effectivity of free and liposome-encapsulated chemotherapy (bortezomib, doxorubicin). Either cocultured myeloma cell lines or primary CD138+ myeloma cells received the treatments. Results: Liposomes (~75-110 nm) passively diffused throughout the heterogeneously porous (~80-850 nm) 3D hydrogel model after insertion. Cellular uptake of liposomes was observed and was increased by targeting very late antigen-4. Liposomal bortezomib and doxorubicin showed increased cytotoxic effects toward myeloma cells compared with the free drugs, using either a cell line or primary myeloma cells. Cytotoxicity toward supporting BM cells was reduced using liposomes. Conclusion: The 3D model allows the study of liposome-encapsulated molecules on multiple myeloma and supporting BM cells, looking at cellular targeting, and general efficacy of the given therapy. The advantages of liposomal drug delivery were demonstrated in a primary myeloma model, enabling the study of patient-to-patient responses to potential drugs and treatment regimes

Additional file 3: Figure S2. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Biological characterization of isolated hMSCs from acoustically stimulated BM at 300 Hz for 5 min at different volumes, 11.5, 10, 8, 6 and 5 ml. The results are presented as the fold change over the non-stimulated bone marrow (baseline). (A) Graphic representation of the bone marrow volumes, donor dependent. (B) Proliferation of hMSCs calculated as PD/day from P1 to P2, donor and volume dependent. (C) CFU potential of hMSCs, donor and volume dependent. (D) ECM production, quantification of nodule size area in mm2, donor and volume dependent. (E) Osteogenic potential calculated as percentage of ALP positive colonies within the CFUs, donor and volume dependent. (F) Adipogenic potential, quantification of Oil red O staining relative to 100% Oil red O staining solution, donor and volume dependent. Values are represented as mean ± standard deviation of at least three independent experiments (n ≥ 3). Statistically significant differences were found with ***p < 0.001, **p < 0.01 and *p < 0.05. (PDF 694 kb

Additional file 4: Figure S3. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Surface marker expression (in percentage) of the acoustic stimulated cells represented as a bar plot. Each bar represents the average expression obtained from three independent donors. Represented are only the surface markers that were expressed in the obtained populations. Negative markers are not shown. No statistically significant differences were found between the two conditions. (PDF 184 kb

Additional file 2: Figure S1. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Macroscopic appearance of bone marrow aspirated from different locations: ilium, proximal femur, distal femur and proximal tibia. (PDF 311 kb

Additional file 5: Figure S4. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Alizarin red staining of calcium nodules after osteogenic induction of hMSC isolated under varying culture condition from different donors. No differences were observed between the culture conditions, though differences between the donors were identified. Donor 2 and 11 showed less calcium nodules formation than the rest of the donors. All the controls stained negative for calcium nodules formation. Values are represented as mean ± standard deviation of at least three independent experiments (n = 3). (PDF 2096 kb

Means of enhancing bone fracture healing: Optimal cell source, isolation methods and acoustic stimulation

Background: The human body has an extensive capacity to regenerate bone tissue after trauma. However large defects such as long bone fractures of the lower limbs cannot be restored without intervention and often lead to nonunion. Therefore, the aim of the present study was to assess the pool and biological functions of human mesenchymal stromal cells (hMSCs) isolated from different bone marrow locations of the lower limbs and to identify novel strategies to prime the cells prior to their use in bone fracture healing. Following, bone marrow from the ilium, proximal femur, distal femur and proximal tibia was aspirated and the hMSCs isolated. Bone marrow type, volume, number of mononuclear cells/hMSCs and their self-renewal, multilineage potential, extracellular matrix (ECM) production and surface marker profiling were analyzed. Additionally, the cells were primed to accelerate bone fracture healing either by using acoustic stimulation or varying the initial hMSCs isolation conditions. Results: We found that the more proximal the bone marrow aspiration location, the larger the bone marrow volume was, the higher the content in mononuclear cells/hMSCs and the higher the self-renewal and osteogenic differentiation potential of the isolated hMSCs were. Acoustic stimulation of bone marrow, as well as the isolation of hMSCs in the absence of fetal bovine serum, increased the osteogenic and ECM production potential of the cells, respectively. Conclusion: We showed that bone marrow properties change with the aspiration location, potentially explaining the differences in bone fracture healing between the tibia and the femur. Furthermore, we showed two new priming methods capable of enhancing bone fracture healing