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

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

Co-stimulatory versus cell death aspects of agonistic cd40 monoclonal antibody selicrelumab in chronic lymphocytic leukemia

Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab

Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia

Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab

Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL

Chronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Because ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment, combined with dissecting functional interactions of Bcl-2 family members, in an in vitro model of venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than did cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed; significantly, the pretreatment profile reappeared in patients who relapsed on ibrutinib. However, in response to venetoclax, Bcl-2 members displayed an early increase, underlining the different modes of action of these 2 drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1, and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that proapoptotic Bim interacts with antiapoptotic Bcl-2 members in a sequential manner: Bcl-2 &gt; Bcl-XL &gt; Mcl-1 &gt; Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than is Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.</p

Three-dimensional histochemistry and imaging of human gingiva

In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 +/- 7% as surface area and 24 +/- 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and diseas