Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Deletion of CD38 and supplementation of NAD+ attenuate axon degeneration in a mouse facial nerve axotomy model.

Following facial nerve axotomy, nerve function is not fully restored even after reconstruction. This may be attributed to axon degeneration/neuronal death and sustained neuroinflammation. CD38 is an enzyme that catalyses the hydrolysis of nicotinamide adenine dinucleotide (NAD+) and is a candidate molecule for regulating neurodegeneration and neuroinflammation. In this study, we analyzed the effect of CD38 deletion and NAD+ supplementation on neuronal death and glial activation in the facial nucleus in the brain stem, and on axon degeneration and immune cell infiltration in the distal portion of the facial nerve after axotomy in mice. Compared with wild-type mice, CD38 knockout (KO) mice showed reduced microglial activation in the facial nucleus, whereas the levels of neuronal death were not significantly different. In contrast, the axon degeneration and demyelination were delayed, and macrophage accumulation was reduced in the facial nerve of CD38 KO mice after axotomy. Supplementation of NAD+ with nicotinamide riboside slowed the axon degeneration and demyelination, although it did not alter the level of macrophage infiltration after axotomy. These results suggest that CD38 deletion and supplementation of NAD+ may protect transected axon cell-autonomously after facial nerve axotomy.

Oxytocin and CD38 in the paraventricular nucleus play a critical role in paternal aggression in mice.

In mammals, the development of healthy offspring requires maternal care. Behavior by lactating mothers toward other individuals is an important component of maternal aggression. However, it is unclear whether fathers display aggression primed by pups (an external factor), and the protection mechanism is poorly understood. To address this question, we examined paternal aggression in the ICR mouse strain. We found that sires exposed to cues from pups and lactating dams showed stronger aggression toward intruders than did sires that were deprived of family cues or exposed to nonlactating mates. c-Fos immunohistochemistry showed that cells in both the paraventricular and supraoptic nuclei (PVN and SON, respectively) in the hypothalamus of sires exposed to any cues were highly activated. However, c-Fos activation in oxytocinergic neurons was increased only in sires exposed to pup cues and solely in the PVN. In Cd38-knockout sires, the presence of pups induced no or reduced parental aggression; however, this phenotype was recovered, that is, aggression increased to the wild-type level, after intraperitoneal administration of oxytocin (OT). Specific c-Fos activation patterns induced by pup cues were not found in the PVN of knockout sires. These results demonstrate that the PVN is one of the primary hypothalamic areas involved in paternal aggression and suggest that a CD38-dependent OT mechanism in oxytocinergic neurons is critical for part of the behavior associated with the protection of offspring by nurturing male mice.

Daratumumab in multiple myeloma.

The development of effective monoclonal antibodies for the treatment of myeloma has been a long journey of clinical and drug development. Identification of the right target antigen was a critical part of the process. CD38 as a target has been considered for some time, but clinically, daratumumab, a CD38 monoclonal antibody, was the first to be tested, and it has delivered the best clinical responses as a single agent to date. Its proven safety and efficacy in combination with other antimyeloma agents have led to several US Food and Drug Administration approvals for treating myeloma. Furthermore, the results of early trials in the induction therapy setting have demonstrated a beneficial role when it is added to the existing induction regimens. This review summarizes the importance of CD38 as a target and examines the clinical development of the CD38 monoclonal antibody daratumumab and its clinical significance in combination regimens in both patients with relapsed/refractory myeloma and patients with newly diagnosed myeloma.

Effect of Withania somnifer on CD38 expression on CD8+ T lymphocytes among patients of HIV infection.

CD38 on CD8 + T cells is considered a reliable marker of HIV disease progression. Withania somnifer, a traditional ayurvedic medicine, has Th1 immunomodulatory properties. PBMCs from 38 HIV patients were exposed to Withania somnifer root extract at standardized concentration. An overall decline in the percentage of CD38 expressing CD8 + T lymphocytes was observed, though the statistical significance was varied with different categories of HIV patients. Withania somnifer could have promising impact on HIV disease and therefore warrants a further study on larger parameters.

Evaluation of cationic channel TRPV2 as a novel biomarker and therapeutic target in Leukemia-Implications concerning the resolution of pulmonary inflammation.

Patients treated during leukemia face the risk of complications including pulmonary dysfunction that may result from infiltration of leukemic blast cells (LBCs) into lung parenchyma and interstitium. In LBCs, we demonstrated that transient receptor potential vanilloid type 2 channel (TRPV2), reputed for its role in inflammatory processes, exhibited oncogenic activity associated with alteration of its molecular expression profile. TRPV2 was overexpressed in LBCs compared to normal human peripheral blood mononuclear cells (PBMCs). Additionally, functional full length isoform and nonfunctional short form pore-less variant of TRPV2 protein were up-regulated and down-regulated respectively in LBCs. However, the opposite was found in PBMCs. TRPV2 silencing or pharmacological targeting by Tranilast (TL) or SKF96365 (SKF) triggered caspace-mediated apoptosis and cell cycle arrest. TL and SKF inhibited chemotactic peptide fMLP-induced response linked to TRPV2 Ca2+ activity, and down-regulated expression of surface marker CD38 involved in leukemia and lung airway inflammation. Challenging lung airway epithelial cells (AECs) with LBCs decreased (by more than 50%) transepithelial resistance (TER) denoting barrier function alteration. Importantly, TL prevented such loss in TER. Therefore, TRPV2 merits further exploration as a pharmacodynamic biomarker for leukemia patients (with pulmonary inflammation) who might be suitable for a novel [adjuvant] therapeutic strategy based on TL.

Oxytocin pathway gene networks in the human brain.

Oxytocin is a neuropeptide involved in animal and human reproductive and social behavior. Three oxytocin signaling genes have been frequently implicated in human social behavior: OXT (structural gene for oxytocin), OXTR (oxytocin receptor), and CD38 (oxytocin secretion). Here, we characterized the distribution of OXT, OXTR, and CD38 mRNA across the human brain by creating voxel-by-voxel volumetric expression maps, and identified putative gene pathway interactions by comparing gene expression patterns across 20,737 genes. Expression of the three selected oxytocin pathway genes was enriched in subcortical and olfactory regions and there was high co-expression with several dopaminergic and muscarinic acetylcholine genes, reflecting an anatomical basis for critical gene pathway interactions. fMRI meta-analysis revealed that the oxytocin pathway gene maps correspond with the processing of anticipatory, appetitive, and aversive cognitive states. The oxytocin signaling system may interact with dopaminergic and muscarinic acetylcholine signaling to modulate cognitive state processes involved in complex human behaviors.

Beta-carotene as a novel therapy for the treatment of "Autistic like behavior" in animal models of Autism.

Autism-affected individuals are characterized by lower plasma oxytocin and its ectoenzyme regulator CD38. Oxytocin, a hypothalamic hormone secreted upon the release of CD38, plays a role in social behavior and bonding. All-trans retinoic acid is a potent inducer of CD38 and can be used as a novel therapeutic strategy in autism. We investigated the role of beta-carotene in rescuing autistic-like behavior in BALB/c and BTBR mice. Beta-carotene derivatives are preferred as they are neither toxic nor teratogenic. Beta-carotene at 0.1-5.0 mg/kg was administered orally to BALB/c and BTBR newborn mice on days 1-7. They were tested at age 2-3 months for five behavioral tests for "autism"; in addition, brain CD38, oxytocin, oxytocin receptor, Brain Derived Neurotrophic Factor (BDNF) and retinoic acid receptor gene expression, serum oxytocin levels, and neurological score were evaluated. Beta-carotene administered at birth significantly increased T-maze alternations and led to longer time spent with an unfamiliar mouse in the "three-chamber test" and less time spent in the empty chamber. Furthermore, enhanced activity in the open field test; increased time spent in the reciprocal social interaction test; decreased grooming and bedding behaviors; and enhanced brain CD38, oxytocin, oxytocin receptor, BDNF, retinoic acid gene expression, and serum oxytocin levels. No changes in neurological score were observed. Beta-carotene oral supplementation to BALB/c and BTBR mice at birth significantly reduced restricted and stereotyped behaviors and interests, increased social interactions and communication, CD38, and oxytocin, probably by enhancing brain neuroplasticity without toxicity. Thus, beta-carotene administered after birth to newborns of families predisposed to "autism" has the potential to prevent/ameliorate" autistic like behavior". These results support further clinical studies.

CD38 produces nicotinic acid adenosine dinucleotide phosphate in the lysosome.

Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca2+-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellular NAADP production. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface-localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produce NAADP in human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production.

Lenalidomide enhances MOR202-dependent macrophage-mediated effector functions via the vitamin D pathway.

Macrophages are key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD38 antibody MOR202, currently introduced in multiple myeloma (MM) therapy. Therefore, it is important to understand how antibody-mediated effector functions of myeloma-associated macrophages (MAMs) are regulated. Here, we focused on the effects of vitamin D, a known regulator of macrophage effector functions. Consequently, it was the aim of this study to assess whether modulation of the vitamin D pathway alters the tumoricidal activity of MAMs. Here, we demonstrate that MAMs display a defective vitamin D pathway with reduced expression level of CYP27B1 and limited tumoricidal activity which can be restored by the IMiD lenalidomide in vitro. Furthermore, our data indicate that the vitamin D pathway of MAMs from MM patients does recover during an IMiD-containing therapy shown by an improved MOR202-mediated cytotoxic activity of these MAMs against primary MM cells ex vivo. Here, the ex vivo cytotoxic activity could be further enhanced by vitamin D supplementation. These data suggest that vitamin D holds a key role for the effector functions of MAMs and that vitamin D supplementation in IMiD combination trials could further increase the therapeutic efficacy of anti-CD38 antibodies such as MOR202, which remains to be investigated in clinical studies.

ROS-Mediated 15-Hydroxyprostaglandin Dehydrogenase Degradation via Cysteine Oxidation Promotes NAD+-Mediated Epithelial-Mesenchymal Transition.

Nicotinamide adenine dinucleotide (NAD) levels decrease with aging as a result of aging-associated CD38 upregulation. Here, we established a cell model with decreased cellular NAD levels by overexpressing CD38 or treating cells with FK866, an inhibitor of nicotinamide phosphoribosyltransferase. We revealed that decreased NAD triggered reactive oxygen species (ROS)-mediated degradation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which drove cells to undergo epithelial-mesenchymal transition (EMT). Moreover, we showed that oxidation of the Cys44 residue to sulfonic acid in 15-PGDH led to its degradation via non-canonical ubiquitination-proteasome and autophagy pathways. Mutation of Cys44 to alanine abolished ROS-induced 15-PGDH degradation. We demonstrated that 15-PGDH silencing promoted EMT, whereas supplementation with NAD precursors increased NAD and 15-PGDH stability, and reversed the EMT process. Taken together, these results suggest that declining NAD levels contribute to age-dependent increases in cancer incidence, and repletion of NAD precursors is beneficial for increasing 15-PGDH expression.

Production of IL-17 by MAIT Cells Is Increased in Multiple Sclerosis and Is Associated with IL-7 Receptor Expression.

Multiple sclerosis (MS) is a T cell-driven inflammatory disease of the CNS. Research on T cell subsets involved in MS pathogenesis has mainly focused on classical CD4+ T cells, especially Th17 cells, as they produce the proinflammatory, MS-associated cytokine IL-17. However, the abundant unconventional mucosal-associated invariant T (MAIT) cells are also able to produce IL-17. MAIT cells are characterized by high CD161 expression and a semi-invariant Vα7.2 TCR, with which they recognize bacterial and yeast Ags derived from the riboflavin (vitamin B2) metabolism. In this study, we characterized MAIT cells from the peripheral blood of MS patients in comparison with healthy individuals with respect to their type-17 differentiation. We found a specific increase of IL-17+ MAIT cells as well as an increased expression of retinoic acid-related orphan receptor (ROR)γt and CCR6 in MAIT cells from MS patients, whereas the expression of T cell activation markers HLA-DR and CD38 was not different. IL-17 production by MAIT cells furthermore correlated with the surface expression level of the IL-7 receptor α-chain (CD127), which was significantly increased on MAIT cells from MS patients in comparison with healthy individuals. In summary, our findings indicate an augmented type-17 differentiation of MAIT cells in MS patients associated with their IL-7 receptor surface expression, implicating a proinflammatory role of these unconventional T cells in MS immunopathology.

Nanobodies effectively modulate the enzymatic activity of CD38 and allow specific imaging of CD38+ tumors in mouse models in vivo.

The cell surface ecto-enzyme CD38 is a promising target antigen for the treatment of hematological malignancies, as illustrated by the recent approval of daratumumab for the treatment of multiple myeloma. Our aim was to evaluate the potential of CD38-specific nanobodies as novel diagnostics for hematological malignancies. We successfully identified 22 CD38-specific nanobody families using phage display technology from immunized llamas. Crossblockade analyses and in-tandem epitope binning revealed that the nanobodies recognize three different non-overlapping epitopes, with four nanobody families binding complementary to daratumumab. Three nanobody families inhibit the enzymatic activity of CD38 in vitro, while two others were found to act as enhancers. In vivo, fluorochrome-conjugated CD38 nanobodies efficiently reach CD38 expressing tumors in a rodent model within 2 hours after intravenous injection, thereby allowing for convenient same day in vivo tumor imaging. These nanobodies represent highly specific tools for modulating the enzymatic activity of CD38 and for diagnostic monitoring CD38-expressing tumors.

Erythropoietin modulation is associated with improved homing and engraftment after umbilical cord blood transplantation.

Umbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34+ cells. A significant population of UCB CD34+ HSPC expresses cell surface EPOR. Exposure of UCB CD34+ HSPC to EPO inhibits their migration and enhances erythroid differentiation. This migratory inhibitory effect was reversed by depleting EPOR expression on HSPC. Moreover, systemic reduction in EPO levels by hyperbaric oxygen (HBO) used in a preclinical mouse model and in a pilot clinical trial promoted homing of transplanted UCB CD34+ HSPC to BM. Such a systemic reduction of EPO in the host enhanced myeloid differentiation and improved BM homing of UCB CD34+ cells, an effect that was overcome with exogenous EPO administration. Of clinical relevance, HBO therapy before human UCB transplantation was well-tolerated and resulted in transient reduction in EPO with encouraging engraftment rates and kinetics. Our studies indicate that systemic reduction of EPO levels in the host or blocking EPO-EPOR signaling may be an effective strategy to improve BM homing and engraftment after allogeneic UCB transplantation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT02099266).

Transient receptor potential melastatin-2 and temperature participate in the process of CD38-regulated oxytocin secretion.

In recent studies, oxytocin showed potential for the treatment of mental diseases. CD38 is essential for oxytocin release, and hence plays a critical role in social behavior. CD38 catalyzes ß-NAD into cyclic ADP ribose (cADPR), which could elevate the intracellular Ca by Ca-permeable channels for oxytocin secretion. The temperature-sensitive cation channel, transient receptor potential melastatin-2 (TRPM2), is a cation-nonselective cation and has been shown to affect oxytocin indirectly. The aim of the present study was to verify the participation of temperature and TRPM2 in CD38-regulated oxytocin release. The crude membranes were prepared to isolate the nerve terminals from the posterior pituitary. At 34°C, 37°C, and 39°C, agonists (ß-NAD, ADPR, cADPR) and antagonists (8-Br-cADPR, 2-APB) were used to stimulate the nerve terminals. Oxytocin releases were investigated by enzyme-linked immunosorbent assay. In addition, the expression of TRPM2 and CD38 in the hypothalamus and pituitary was detected by western blotting and quantitative PCR. CD38 agonists (ß-NAD, cADPR) and antagonist (8-Br-cADPR) could increase or reduce the oxytocin release, respectively. TRPM2 agonist (ADPR) and antagonist (2-APB) alone could also regulate oxytocin release. Furthermore, temperature could increase agonist stimulation and attenuate the antagonist inhibition on oxytocin release. In addition, CD38 and TRPM2 were expressed in the hypothalamus and pituitary at both the mRNA and the protein level. TRPM2 in pituitary nerve terminals plays a role in oxytocin release. Temperature- enhanced oxytocin release by CD38 and TRPM2. TRPM2 might be involved in the process of CD38-regulated oxytocin release.

Blocking NAD(+)/CD38/cADPR/Ca(2+) pathway in sepsis prevents organ damage.

BACKGROUND: Although the nicotinamide adenine dinucleotide (NAD(+))/CD38/cyclic ADP ribose (cADPR)/Ca(2+) signaling pathway has been shown to regulate intracellular calcium homeostasis and functions in multiple inflammatory processes, its role in sepsis remains unknown. The aim of this study was to determine whether the NAD(+)/CD38/cADPR/Ca(2+) signaling pathway is activated during sepsis and whether an inhibitor of this pathway, 8-Br-cADPR, protects the organs from sepsis-induced damage. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham laparotomies. NAD(+), cADPR, CD38, and intracellular Ca(2+) levels were measured in the hearts, livers, and kidneys of septic rats at 0, 6, 12, 24, and 48 h after CLP surgery. Rats were also divided into sham, CLP, and CLP+8-Br-cADPR groups, and the hearts, livers, and kidneys were hematoxylin-eosin-stained and assayed for malondialdehyde and superoxide dismutase activities. RESULTS: NAD(+), cADPR, CD38, and intracellular Ca(2+) levels increased in the hearts, livers, and kidneys of septic rats as early as 6-24 h after CLP surgery. Treatment with 8-Br-cADPR inhibited sepsis-induced intracellular Ca(2+) mobilization, attenuated tissue injury, reduced malondialdehyde levels, and increased superoxide dismutase activity in septic rats. CONCLUSIONS: The NAD(+)/CD38/cADPR/Ca(2+) signaling pathway was activated during sepsis in the CLP rat model. Blocking this pathway with 8-Br-cADPR protected hearts, livers, and kidneys from sepsis-induced damage.

Monoclonal antibodies targeting CD38 in hematological malignancies and beyond.

CD38 is a multifunctional cell surface protein that has receptor as well as enzyme functions. The protein is generally expressed at low levels on various hematological and solid tissues, while plasma cells express particularly high levels of CD38. The protein is also expressed in a subset of hematological tumors, and shows especially broad and high expression levels in plasma cell tumors such as multiple myeloma (MM). Together, this triggered the development of various therapeutic CD38 antibodies, including daratumumab, isatuximab, and MOR202. Daratumumab binds a unique CD38 epitope and showed strong anti-tumor activity in preclinical models. The antibody engages diverse mechanisms of action, including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, modulation of enzymatic activity, and immunomodulatory activity. CD38-targeting antibodies have a favorable toxicity profile in patients, and early clinical data show a marked activity in MM, while studies in other hematological malignancies are ongoing. Daratumumab has single agent activity and a limited toxicity profile, allowing favorable combination therapies with existing as well as emerging therapies, which are currently evaluated in the clinic. Finally, CD38 antibodies may have a role in the treatment of diseases beyond hematological malignancies, including solid tumors and antibody-mediated autoimmune diseases.

CD38 Monoclonal Antibody Therapies for Multiple Myeloma.

The goal of this review is to provide historical, recent preclinical, and current clinical summaries of efforts to understand the CD38 molecule and to develop monoclonal antibodies that target it. We focus particularly on efforts involving multiple myeloma, a malignancy of terminally differentiated B cells that remains incurable despite many advances. An era of anti-CD38 monoclonal antibody therapy for myeloma is approaching, one that, we hope, will enable patients to live longer and better lives.

Preclinical Evidence for the Therapeutic Potential of CD38-Targeted Immuno-Chemotherapy in Multiple Myeloma Patients Refractory to Lenalidomide and Bortezomib.

PURPOSE: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. EXPERIMENTAL DESIGN: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. RESULTS: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. CONCLUSIONS: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients.

Vitamin D-binding protein controls T cell responses to vitamin D.

BACKGROUND: In vitro studies have shown that the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), can regulate differentiation of CD4+ T cells by inhibiting Th1 and Th17 cell differentiation and promoting Th2 and Treg cell differentiation. However, the serum concentration of 1,25(OH)2D3 is far below the effective concentration of 1,25(OH)2D3 found in in vitro studies, and it has been suggested that 1,25(OH)2D3 must be produced locally from the inactive precursor 25-hydroxyvitamin D3 (25(OH)D3) to affect ongoing immune responses in vivo. Although it has been reported that activated T cells express the 25(OH)D-1α-hydroxylase CYP27B1 that converts 25(OH)D3 to 1,25(OH)2D3, it is still controversial whether activated T cells have the capacity to produce sufficient amounts of 1,25(OH)2D3 to affect vitamin D-responsive genes. Furthermore, it is not known how the vitamin D-binding protein (DBP) found in high concentrations in serum affects T cell responses to 25(OH)D3. RESULTS: We found that activated T cells express CYP27B1 and have the capacity to produce sufficient 1,25(OH)2D3 to affect vitamin D-responsive genes when cultured with physiological concentrations of 25(OH)D3 in serum-free medium. However, if the medium was supplemented with serum or purified DBP, DBP strictly inhibited the production of 1,25(OH)2D3 and 25(OH)D3-induced T cell responses. In contrast, DBP did not inhibit the effect of exogenous 1,25(OH)2D3. Actin, arachidonic acid and albumin did not affect the sequestration of 25(OH)D3 by DBP, whereas carbonylation of DBP did. CONCLUSIONS: Activated T cells express CYP27B1 and can convert 25(OH)D3 to 1,25(OH)2D3 in sufficiently high concentrations to affect vitamin D-responsive genes when cultured in serum-free medium. However, DBP sequesters 25(OH)D3 and inhibits the production of 1,25(OH)2D3 in T cells. To fully exploit the immune-regulatory potential of vitamin D, future studies of the mechanisms that enable the immune system to exploit 25(OH)D3 and convert it to 1,25(OH)2D3 in vivo are required.