Lymphocyte HLA-DR/CD-38 co-expression correlates with Hodgkin lymphoma cell cytotoxicity in vitro independent of PD-1/PD1-L pathway.

The interactions between Hodgkin and Reed Sternberg cells and tumor microenvironment, the changes that occur with therapy and, in particular, checkpoint inhibition are not fully understood. Understanding these is key to optimizing outcomes for patients with Hodgkin lymphoma (HL). We evaluated the immunophenotypic characteristics of cytotoxic, helper T and NK lymphocytes upon in vitro stimulation, cell-mediated cytotoxicity against HL cells, HDLM-2 and KM-H2, and the association with effector cell activation state, as well as changes in cytotoxicity following PD-1 or PDL-1 blockade. Higher HLA-DR/CD38 expression on effector cells was associated with increased cytotoxicity against HL cells. All effector cell types were cytotoxic of HL cells, though achieved maximum activation and cytotoxicity at variable timepoints. HLA-DR/CD38 co-expression correlated with cytotoxicity, but PD-1 expression did not. There was no significant change in cell-mediated cytotoxicity following PD-1/PDL-1 blockade. The mechanism of action of checkpoint inhibitors may not be limited to direct PD-1/PDL-1 blockade.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Molecular dynamics of targeting CD38 in multiple myeloma.

Multiple functions of CD38 need exploring to expand clinical application of anti-CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co-cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody-exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti-CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism-related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody-exposed cells showed increased CD73 and CD39 expression, presence of programmed death-ligand 1 and significant up-/down-modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor-positive (FcR+ ) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR+ cells with significant functional modifications. These observations have relevance for improving anti-CD38 therapeutic antibodies through targeting this mechanism and its sequelae.

Daudi cell stroma: An alternative to dithiothreitol to resolve daratumumab interference in pretransfusion testing.

Treatment of red blood cells with dithiothreitol (DTT) or trypsin effectively denatures CD38; however, this treatment damages other antigens, some of which are of clinical importance. Thus, other avenues to deplete daratumumab (DARA) from plasma samples should be explored. STUDY DESIGN AND METHODS: The Daudi B-cell line was found to express high levels of CD38 and was sonicated in a sonication buffer to achieve complete cell lysis. The resulting stroma preparation was centrifuged at 20 000g for 20 minutes and then mixed with 250 µL of DARA-plasma and incubated for 10 minutes at 37°C. The stroma-DARA-plasma mixture was centrifuged again, and the supernatant was collected and subjected to four additional rounds of adsorption with fresh stroma. DARA-depleted plasma was tested by gel indirect antiglobulin test (IAT). RESULTS: CD38 expression on Daudi cells was confirmed by flow cytometry. Gel IAT analysis showed that the incubation of plasma from DARA-treated patients with Daudi cells stroma resulted in a significant depletion of DARA but allowing detection of other alloantibodies of interest such as anti-K, anti-Yta , and anti-Gya . CONCLUSIONS: Daudi cell stroma is inexpensive, easy to prepare in large batches, and can be used as an off-the-shelf reagent. Incubation of plasma from DARA-treated patients with Daudi cell stroma can efficiently overcome DARA interference in serologic testing without affecting DTT- or trypsin-sensitive antigens.

High CD38 expression in childhood T-cell acute lymphoblastic leukemia is not associated with prognosis.

BACKGROUND: Prognostic factors are not well exploited in childhood T-cell acute lymphoblastic leukemia (T-ALL). OBJECTIVE: The aim of this study was to analyze the prognostic role of CD38 as well as minimal residual disease (MRD) and other biological factors in T-ALL. METHODS: Immunophenotyping of bone marrow (BM) at diagnosis and MRD levels were determined using a standard panel of antibodies by 4-colour flow cytometry. A total of 96 children with T-ALL were enrolled. RESULTS: The results showed that 97.9% of T-ALL patients were positive for CD38 with a median level of 85.3%. CD38-high group had a worse early treatment response than the CD38-low group. However, CD38 levels were not associated with prognosis, albeit CD38-high group had a worse 5-year event free survival rate (55.1% vs. 66.6%, P> 0.05) and a higher 5-year cumulative incidence of relapse (35.6% vs. 19.8%, P> 0.05). Very high MRD levels (> 10%) were related to the worse survival. Neither flow cytometry based minimal residual disease (MRD) levels nor CD38 expression levels showed significant relation to the hazard of relapse (P> 0.05). CONCLUSIONS: We conclude that T-ALL has a high level of CD38 expression which is not associated with prognosis. Very high MRD level (> 10%) is related to the worse survival, however, FCM based MRD detection does not convey a significant prognostic value.

A combination of LMO2 negative and CD38 positive is useful for the diagnosis of Burkitt lymphoma.

BACKGROUND: To evaluate the clinical utility of LIM Domain Only 2 (LMO2) negative and CD38 positive in diagnosis of Burkitt lymphoma (BL). METHODS: LMO2 and CD38 expression determined by immunohistochemistry in 75 BL, 12 High-grade B-cell lymphoma, NOS (HGBL,NOS) and 3 Burkitt-like lymphomas with the 11q aberration. RESULTS: The sensitivity and specificity of LMO2 negative for detecting BL were 98.67 and 100%, respectively; those of CD38 positive were 98.67 and 66.67%, respectively. The sensitivity and specificity of a combination of both for detecting BL were 97.33 and 100%, respectively. In our study, the combined LMO2 negative and CD38 positive results had a higher area under the curve than either LMO2 negative or CD38 positive alone. CONCLUSIONS: A combination of LMO2 negative and CD38 positive is useful for the diagnosis of Burkitt lymphoma.

Quantitative Proteomic Analysis Reveals the Deregulation of Nicotinamide Adenine Dinucleotide Metabolism and CD38 in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) has become a major health challenge worldwide. However, the precise etiological and pathophysiological factors involved in IBD remain unclear. Proteomics can be used for large-scale protein identification analysis. In the current study, using tandem mass tag- (TMT-) based shotgun proteomics, proteomic differences between intestinal tissue from health controls, patients with Crohn's disease (CD), and patients with ulcerative colitis (UC) were compared. Proteins with fold change >2 or <0.5 and P value < 0.05 between groups were considered differentially expressed. ProteinAtlas was used to analyze the tissue specificity of differentially expressed proteins (DEPs). Reactome pathway analysis was applied to cluster functional pathways. A total of 4786 proteins were identified, with 59 proteins showing higher levels and 43 showing lower levels in patients with IBD than in controls. Seventeen proteins, including angiotensin converting enzyme 2 (ACE2) and angiotensin converting enzyme 1 (ACE), showed higher levels in CD than in UC. Several novel proteins such as CD38, chitinase 3-like 1 (CHI3L1), olfactomedin 4 (OLFM4), and intelectin 1 were screened out between patients with IBD and controls. When proteins with fold change >1.2 or <0.84 and P value < 0.05 between groups were considered differentially expressed, the expression of 10 proteins, including CD38, involved in the nicotinamide adenine dinucleotide (NAD) metabolism and signaling pathway showed significant changes in IBD. Using the NCBI GEO database, we confirmed increased CD38 mRNA expression in patients with UC and in mouse colitis models. Protein CD38 expression was higher in CD and UC than in normal controls. CD38 expression was higher in inflamed tissues than in noninflamed tissues, and CD38 was located in F4/80-positive cells. Our study may provide novel insights into the molecular pathogenesis of IBD. Further studies are required on the role of NAD metabolism and CD38 in intestinal inflammation.

CD38 Inhibits Prostate Cancer Metabolism and Proliferation by Reducing Cellular NAD+ Pools.

Tumor cells require increased rates of cell metabolism to generate the macromolecules necessary to sustain proliferation. They rely heavily on NAD+ as a cofactor for multiple metabolic enzymes in anabolic and catabolic reactions. NAD+ also serves as a substrate for PARPs, sirtuins, and cyclic ADP-ribose synthases. Dysregulation of the cyclic ADP-ribose synthase CD38, the main NAD'ase in cells, is reported in multiple cancer types. This study demonstrates a novel connection between CD38, modulation of NAD+, and tumor cell metabolism in prostate cancer. CD38 expression inversely correlates with prostate cancer progression. Expressing CD38 in prostate cancer cells lowered intracellular NAD+, resulting in cell-cycle arrest and expression of p21Cip1 (CDKNA1). In parallel, CD38 diminishes glycolytic and mitochondrial metabolism, activates AMP-activated protein kinase (AMPK), and inhibits fatty acid and lipid synthesis. Pharmacologic inhibition of nicotinamide phosphoribosyltransferase (NAMPT) mimicked the metabolic consequences of CD38 expression, demonstrating similarity between CD38 expression and NAMPT inhibition. Modulation of NAD+ by CD38 also induces significant differential expression of the transcriptome, producing a gene expression signature indicative of a nonproliferative phenotype. Altogether, in the context of prostate cancer, the data establish a novel role for the CD38-NAD+ axis in the regulation of cell metabolism and development.Implications: This research establishes a mechanistic connection between CD38 and metabolic control. It also provides the foundation for the translation of agents that modulate NAD+ levels in cancer cells as therapeutics. Mol Cancer Res; 16(11); 1687-700. ©2018 AACR.

Correlation Between Baseline 18F-FDG PET/CT Findings and CD38- and CD138-Expressing Myeloma Cells in Bone Marrow and Clinical Parameters in Patients with Multiple Myeloma

Objective: The aim of this study was to evaluate the relation between the rate of fluorine-18 (18F) fludeoxyglucose (FDG) uptake and CD38 and CD138 expression in myeloma cells in bone marrow and other clinical parameters in patients with multiple myeloma (MM). Materials and Methods: Patients with the diagnosis of MM who underwent 18F-FDG positron emission tomography/computed tomography (PET/CT) for initial staging were evaluated retrospectively. We analyzed a total of 42 patients (43-83 years old, mean: 64.4±9.9). Hematological and biochemical tests including hemoglobin, hematocrit, C-reactive protein, ß2-microglobulin, creatinine, albumin, calcium, lactate dehydrogenase, and erythrocyte sedimentation rate were recorded. In bone marrow samples, plasma cell ratio and CD38 and CD138 immunohistochemical staining were evaluated. On PET/CT images, mean standardized uptake values (SUVmean) of the right anterior and posterior iliac crest and right proximal femora were calculated. The correlations between the average SUVmean of bone marrow and CD38- and CD138-expressing myeloma cells and other parameters were analyzed by Spearman's correlation test. Values of p<0.05 were considered statistically significant. Results: Types of MM were IgGK (45%), IgGL (21%), IgAK (7%), IgAL (10%), and others (17%). Thirty-two (76%) patients were at stage III according to the Salmon-Durie staging system. There was a statistically significant positive correlation between bone marrow FDG uptake and percentage of plasma cells in bone marrow and CD38 and CD138 expression in plasma cells (r=0.403, r=0.339, and r=0.409) and ß2-microglobulin and C-reactive protein levels (r=0.676, r=0.541). There was a negative correlation between bone marrow FDG uptake and hemoglobin and hematocrit values (r=-0.377 and r=-0.368). Other hematological parameters were not correlated with FDG uptake in bone marrow. Conclusion: Increased FDG uptake is correlated with the percentage of CD38 and CD138 expression in plasma cells in bone marrow. In addition to initial staging, 18F-FDG PET/CT is useful in treatment planning and prognostic evaluation in MM patients.

Human Immunodeficiency Virus Type-1 Elite Controllers Maintain Low Co-Expression of Inhibitory Receptors on CD4+ T Cells.

Human immunodeficiency virus type-1 (HIV-1) elite controllers (ELCs) represent a unique population that control viral replication in the absence of antiretroviral therapy (cART). It is well established that expression of multiple inhibitory receptors on CD8+ T cells is associated with HIV-1 disease progression. However, whether reduced co-expression of inhibitory receptors on CD4+ T cells is linked to natural viral control and slow HIV-1 disease progression remains undefined. Here, we report on the expression pattern of numerous measurable inhibitory receptors, associated with T cell exhaustion (programmed cell death-1, CTLA-4, and TIGIT), on different CD4+ T cell memory populations in ELCs and HIV-infected subjects with or without long-term cART. We found that the co-expression pattern of inhibitory receptors was significantly reduced in ELCs compared with HIV-1 cART-treated and viremic subjects, and similar to healthy controls. Markers associated with T cell exhaustion varied among different memory CD4+ T cell subsets and highest levels were found mainly on transitional memory T cells. CD4+ T cells co-expressing all inhibitory markers were positively correlated to T cell activation (CD38+ HLA-DR+) as well as the transcription factors Helios and FoxP3. Finally, clinical parameters such as CD4 count, HIV-1 viral load, and the CD4/CD8 ratio all showed significant associations with CD4+ T cell exhaustion. We demonstrate that ELCs are able to maintain lower levels of CD4+ T cell exhaustion despite years of ongoing viral replication compared with successfully cART-treated subjects. Our findings suggest that ELCs harbor a "healthy" state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status.

Increased Natural Killer Cell Activation in HIV-Infected Immunologic Non-Responders Correlates with CD4+ T Cell Recovery after Antiretroviral Therapy and Viral Suppression.

The role of natural killer (NK) cell function in HIV disease especially in the setting of long-term antiretroviral therapy (ART) and viral suppression is not fully understood. In the current study, we have investigated NK cell activation in healthy controls and aviremic ART-treated HIV+ subjects with different degrees of immune restoration. We performed a cross sectional study in 12 healthy controls and 24 aviremic ART-treated HIV-infected subjects including 13 HIV+ subjects with CD4+ T cells above 500 cells/µL defined as "immunologic responders" and 11 HIV+ subjects with CD4+ T cells below 350 cells/µL defined as "immunologic non-responders". We analyzed NK cell number, subset, and activation by expression of CD107a and NKG2D and co-expression of CD38 and HLA-DR. NK cell-mediated cytotoxicity against uninfected CD4+ T cells was tested in vitro. We found that NK cell absolute number, percentage of NK cells, and percentage of NK cell subsets were similar in the three study groups. The increased NK cell activation was found predominantly in CD56dimCD16+ subset of immunologic non-responders but not immunologic responders compared to healthy controls. The activation of NK cells was inversely correlated with the peripheral CD4+ T cell count in HIV+ subjects, even after controlling for chronic T cell activation, sex, and age, potential contributors for CD4+ T cell counts in HIV disease. Interestingly, NK cells from immunologic non-responders mediated cytotoxicity against uninfected CD4+ T cells ex vivo. NK cells may play a role in blunted CD4+ T cell recovery in ART-treated HIV disease.

Spleen Tyrosine Kinase Is Involved in the CD38 Signal Transduction Pathway in Chronic Lymphocytic Leukemia.

The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.

CD38 gene-modified dendritic cells inhibit murine asthma development by increasing IL-12 production and promoting Th1 cell differentiation.

Predominant T helper (Th)2 and impaired Th1 cell polarization has a crucial role in the development of asthma. Cluster of differentiation (CD)38 is associated with the increased release of interleukin (IL)­12 from dendritic cells (DCs) and DC­induced Th1 cell polarization. However, whether CD38 expression affects DC function in asthma development remains unknown. In the current study, adenoviruses were constructed containing the murine CD38 gene. Overexpression of CD38 protein level in DCs induced from bone­marrow derived DCs (BMDCs) by recombinant mouse granulocyte macrophage colony­stimulating factor and IL­4 was achieved through 24 h adenovirus infection. The results demonstrated that BMDCs with CD38 overexpression exhibited no phenotypic change; however, following stimulation with lipopolysaccharide (LPS), maturation and IL­12 secretion were increased. In addition, CD38­overexpressing BMDCs stimulated with LPS exhibited more effective Th1 cell differentiation. Mice that were administered CD38­overexpressing BMDCs exhibited milder symptoms of asthma. Furthermore, decreased IL­4, IL­5 and IL­13 levels were detected in bronchoalveolar lavage fluid (BALF), reduced immunoglobulin E levels were measured in the sera, and increased interferon­Î³ was detected in BALF from the recipients of CD38­overexpressing BMDCs. Increased phosphorylated­p38 expression was also detected in LPS-stimulated CD38-overexpressing BMDCs, whereas pretreatment with a p38­specific inhibitor was able to abolish the effects of LPS stimulation and CD38 overexpression on IL­12 release and Th1 cell differentiation in BMDCs. These results suggested that CD38 may be involved in the DC function of alleviating asthma via restoration of the Th1/Th2 balance, thus providing a novel strategy for asthma therapy.

CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer.

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctn(f/f)), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38(high) MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38(high) MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38(low) MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor-derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38(+) MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy.

Concurrent loss of co-stimulatory molecules and functional cytokine secretion attributes leads to proliferative senescence of CD8(+) T cells in HIV/TB co-infection.

The role of T-cell immunosenescence and functional CD8(+) T-cell responses in HIV/TB co-infection is unclear. We examined and correlated surrogate markers of HIV disease progression with immune activation, immunosenescence and differentiation using T-cell pools of HIV/TB co-infected, HIV-infected and healthy controls. Our investigations showed increased plasma viremia and reduced CD4/CD8 T-cell ratio in HIV/TB co-infected subjects relative to HIV-infected, and also a closer association with changes in the expression of CD38, a cyclic ADP ribose hydrolase and CD57, which were consistently expressed on late-senescent CD8(+) T cells. Up-regulation of CD57 and CD38 were directly proportional to lack of co-stimulatory markers on CD8(+) T cells, besides diminished expression of CD127 (IL-7Rα) on CD57(+)CD4(+) T cells. Notably, intracellular IFN-γ, perforin and granzyme B levels in HIV-specific CD8(+) T cells of HIV/TB co-infected subjects were diminished. Intracellular CD57 levels in HIV gag p24-specific CD8(+) T cells were significantly increased in HIV/TB co-infection. We suggest that HIV-TB co-infection contributes to senescence associated with chronic immune activation, which could be due to functional insufficiency of CD8(+) T cells.

CD38 Is Required for Neural Differentiation of Mouse Embryonic Stem Cells by Modulating Reactive Oxygen Species.

CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca(2+) mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production.