Episodic alcohol exposure attenuates mesenchymal stem cell chondrogenic differentiation during bone fracture callus formation.

BACKGROUND: During bone fracture repair, mesenchymal stem cells (MSC) differentiate into chondrocytes and osteoblasts to form a fracture callus. Our laboratory previously reported that alcohol-exposed rodents with a surgically created tibia fracture display deficient fracture callus formation and diminished signs of endochondral ossification characterized by the absence of chondrocytes and mature hypertrophic chondrocytes, suggesting that alcohol may inhibit MSC differentiation. These findings led to our hypothesis that alcohol exposure inhibits mesenchymal stem cell chondrogenic differentiation within the developing fracture callus. METHODS: In the present study, we utilized a lineage-tracing approach to determine which stage(s) of chondrogenic differentiation are affected by alcohol exposure. We utilized lineage-specific reporter mice to determine the effects of alcohol on MSC and early and late chondrogenic cell frequencies within the fracture callus. In addition, serially sectioned slides were stained immunofluorescently and immunohistochemically and quantified to determine the effect of alcohol on cell proliferation and apoptosis, respectively, within the fracture callus of alcohol-administered rodents. RESULTS: Alcohol-administered rodents had a reduced fracture callus area at 4, 6, and 9 days postfracture. Alcohol had no effect on apoptosis in the fracture callus at any of the examined timepoints. Alcohol-administered rodents had significantly fewer proliferative cells in the fracture callus at 9 days postfracture, but no effect on cell proliferation was observed at earlier fracture callus timepoints. Alcohol-administered rodents had reduced Collagen2a1- and Collagen10a1-expressing cells in the developing fracture callus, suggesting that alcohol inhibits both early chondrogenic differentiation and later chondrocyte maturation during fracture callus development. CONCLUSION: The data suggest that alcohol could affect normal fracture healing through the mitigation of MSC chondrogenic differentiation at the callus site.

Ethanol Inhibits Mesenchymal Stem Cell Osteochondral Lineage Differentiation Due in Part to an Activation of Forkhead Box Protein O-Specific Signaling.

BACKGROUND: During bone fracture repair, resident mesenchymal stem cells (MSCs) differentiate into chondrocytes, to form a cartilaginous fracture callus, and osteoblasts, to ossify the collagen matrix. Our laboratory previously reported that alcohol administration led to decreased cartilage formation within the fracture callus of rodents and this effect was mitigated by postfracture antioxidant treatment. Forkhead box protein O (FoxO) transcription factors are activated in response to intracellular reactive oxygen species (ROS), and alcohol has been shown to increase ROS. Activation of FoxOs has also been shown to inhibit canonical Wnt signaling, a necessary pathway for MSC differentiation. These findings have led to our hypothesis that alcohol exposure decreases osteochondrogenic differentiation of MSCs through the activation of FoxOs. METHODS: Primary rat MSCs were treated with ethanol (EtOH) and assayed for FoxO expression, FoxO activation, and downstream target expression. Next, MSCs were differentiated toward osteogenic or chondrogenic lineages in the presence of 50 mM EtOH and alterations in osteochondral lineage marker expression were determined. Lastly, osteochondral differentiation experiments were repeated with FoxO1/3 knockdown or with FoxO1/3 inhibitor AS1842856 and osteochondral lineage marker expression was determined. RESULTS: EtOH increased the expression of FoxO3a at mRNA and protein levels in primary cultured MSCs. This was accompanied by an increase in FoxO1 nuclear localization, FoxO1 activation, and downstream catalase expression. Moreover, EtOH exposure decreased expression of osteogenic and chondrogenic lineage markers. FoxO1/3 knockdown restored proosteogenic and prochondrogenic lineage marker expression in the presence of 50 mM EtOH. However, FoxO1/3 inhibitor only restored proosteogenic lineage marker expression. CONCLUSIONS: These data show that EtOH has the ability to inhibit MSC differentiation, and this ability may rely, at least partially, on the activation of FoxO transcription factors.

Correction to: Clinical and immunologic evaluation of three metastatic melanoma patients treated with autologous melanoma-reactive TCR-transduced T cells.

The authors would like to make the following corrections to the published article.

Clinical and immunologic evaluation of three metastatic melanoma patients treated with autologous melanoma-reactive TCR-transduced T cells.

Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.

Alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 can support immune responses toward tumors overexpressing ganglioside D3 in mice.

An immunotherapeutic strategy is discussed supporting anti-tumor activity toward malignancies overexpressing ganglioside D3. GD3 can be targeted by NKT cells when derived moieties are presented in the context of CD1d. NKT cells can support anti-tumor responses by secreting inflammatory cytokines and through cytotoxicity toward CD1d+GD3+ tumors. To overexpress GD3, we generated expression vector DNA and an adenoviral vector encoding the enzyme responsible for generating GD3 from its ubiquitous precursor GM3. We show that DNA encoding α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (SIAT8) introduced by gene gun vaccination in vivo leads to overexpression of GD3 and delays tumor growth. Delayed tumor growth is dependent on CD1d expression by host immune cells, as shown in experiments engaging CD1d knockout mice. A trend toward greater NKT cell populations among tumor-infiltrating lymphocytes is associated with SIAT8 vaccination. A single adenoviral vaccination introduces anti-tumor activity similarly to repeated vaccination with naked DNA. Here, greater NKT tumor infiltrates were accompanied by marked overexpression of IL-17 in the tumor, later switching to IL-4. Our results suggest that a single intramuscular adenoviral vaccination introduces overexpression of GD3 by antigen-presenting cells at the injection site, recruiting NKT cells that provide an inflammatory anti-tumor environment. We propose adenoviral SIAT8 (AdV-SIAT8) can slow the growth of GD3 expressing tumors in patients.

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists.

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In ß-arrestin recruitment assays, ubiquitin did not sufficiently recruit ß-arrestin2 to CXCR4 (EC50 > 10 µM), whereas the EC50 for CXCL12 was 4.6 nM (95% confidence interval 3.1-6.1 nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.

Positioning ganglioside D3 as an immunotherapeutic target in lymphangioleiomyomatosis.

Tumors that develop in lymphangioleiomyomatosis (LAM) as a consequence of biallelic loss of TSC1 or TSC2 gene function express melanoma differentiation antigens. However, the percentage of LAM cells expressing these melanosomal antigens is limited. Here, we report the overexpression of ganglioside D3 (GD3) in LAM. GD3 is a tumor-associated antigen otherwise found in melanoma and neuroendocrine tumors; normal expression is largely restricted to neuronal cells in the brain. We also observed markedly reduced serum antibody titers to GD3, which may allow for a population of GD3-expressing LAM cells to expand within patients. This is supported by the demonstrated sensitivity of cultured LAM cells to complement mediated cytotoxicity via GD3 antibodies. GD3 can serve as a natural killer T (NKT) cell antigen when presented on CD1d molecules expressed on professional antigen-presenting cells. Although CD1d-expressing monocyte derivatives were present in situ, enhanced NKT-cell recruitment to LAM lung was not observed. Cultured LAM cells retained surface expression of GD3 over several passages and also expressed CD1d, implying that infiltrating NKT cells can be directly cytotoxic toward LAM lung lesions. Immunization with antibodies to GD3 may thus be therapeutic in LAM, and enhancement of existing NKT-cell infiltration may be effective to further improve antitumor responses. Overall, we hereby establish GD3 as a suitable target for immunotherapy of LAM.

Enhanced bleaching treatment: opportunities for immune-assisted melanocyte suicide in vitiligo.

Depigmentation in vitiligo occurs by progressive loss of melanocytes from the basal layer of the skin, and can be psychologically devastating to patients. T cell-mediated autoimmunity explains the progressive nature of this disease. Rather than being confronted with periods of rapid depigmentation and bouts of repigmentation, patients with long-standing, treatment-resistant vitiligo can undergo depigmentation treatment. The objective is to remove residual pigmentation to achieve a cosmetically acceptable result--that of skin with a uniform appearance. In the United States, only the use of mono-benzyl ether of hydroquinone (MBEH) is approved for this purpose. However, satisfactory results can take time to appear, and there is a risk of repigmentation. MBEH induces necrotic melanocyte death followed by a cytotoxic T-cell response to remaining, distant melanocytes. As cytotoxic T-cell responses are instrumental to depigmentation, we propose that combining MBEH with immune adjuvant therapies will accelerate immune-mediated melanocyte destruction to achieve faster, more definitive depigmentation than with MBEH alone. As Toll-like Receptor (TLR) agonists--imiquimod, CpG, and Heat Shock Protein 70 (HSP 70)--all support powerful Th1 responses, we propose that using MBEH in combination with these agents can achieve superior depigmentation results for vitiligo patients.

Targeting melanocyte and melanoma stem cells by 8-hydroxy-2-dipropylaminotetralin.

Monobenzyl ether of hydroquinone (MBEH) is cytotoxic towards melanocytes. Its treatment efficacy is limited by an inability to eradicate stem cells. By contrast, 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-DPAT) affects melanocyte stem cell survival. MBEH and 8-DPAT were added to melanocytes and melanoma cells to compare cytotoxicity. Stem cell content among viable cells was determined by fluorocytometry using markers CD34, Pax3, and CD271. Immunostaining was used to identify stem cells in skin explants treated with MBEH or 8-DPAT ex vivo. Mice were exposed to MBEH or 8-DPAT and scanned for depigmentation before harvesting skin. MBEH exposure prompted a relative increase in stem cells among cultured melanocytes and melanoma cells, as treatment preferentially eliminated differentiated cells and spared the stem cells. Viability of this remaining, enriched stem cell population was however rapidly reduced by exposure to 8-DPAT within melanocyte and melanoma cell cultures. In human skin explants, the abundance of melanocyte stem cells was also visibly reduced after 8-DPAT treatment, in contrast to tissue exposed to MBEH. Meanwhile, significant depigmentation of the mouse pelage and loss of differentiated melanocytes was observed in vivo in response to topical application of MBEH, but not 8-DPAT. Prolonged application of the latter agent instead appeared to effectively reduce the abundance of melanocyte stem cells in the dermis. This furthers the idea that MBEH and 8-DPAT target complementary cell populations. Results indicate that combination treatment may demonstrate superior therapeutic activity by eliminating both differentiated and tumor initiating populations.

A coreceptor-independent transgenic human TCR mediates anti-tumor and anti-self immunity in mice.

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4(+) T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2-restricted TCR was positively selected on both CD4(+) and CD8(+) single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3(+)CD4(-)CD8(-) double-negative T cells. TIL 1383I TCR transgenic CD4(+), CD8(+), and CD4(-)CD8(-) T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2(+)/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3(+) T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.

A central role for inducible heat-shock protein 70 in autoimmune vitiligo.

Inducible heat-shock protein 70 (HSP70i) is a protein regulated by stress that protects cells from undergoing apoptosis. Such proteins are marvellously well conserved throughout evolution, which has placed them in the spotlight for helping to understand the intriguing relationship between infection and immunity. In the presence of stress proteins, dendritic cells (DCs) will sense this alarm signal and respond by recruiting immune cells of different plumage to fit the occasion. In times of stress, melanocytes will secrete antigen-bound HSP70i to act as an alarm signal in activating DCs that comes equipped with an address of origin to drive the autoimmune response in vitiligo. Here we pose that if the autoimmune response is funnelled through HSP70i, then blocking the stress protein from activating DCs can lend new treatment opportunities for vitiligo.

Dietary Alcohol Consumption Elicits Corneal Toxicity Through the Generation of Cellular Oxidative Stress.

Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.

A current viewpoint of lymphangioleiomyomatosis supporting immunotherapeutic treatment options.

Lymphangioleiomyomatosis (LAM) leads to hyperproliferation of abnormal smooth muscle cells in the lungs, associated with diffuse pulmonary parenchymal cyst formation and progressive dyspnea on exertion. The disease targets women of child-bearing age. Complications include pneumothoraces and chylous pleural effusions. Ten-year survival is estimated at 70%, and lung transplantation remains the only validated treatment. It has been observed that LAM cells express markers associated with melanocytic differentiation, including gp100 and MART-1. Other melanocytic markers have also been observed. The same proteins are targeted by T cells infiltrating melanoma tumors as well as by T cells infiltrating autoimmune vitiligo skin, and these antigens are regarded as relatively immunogenic. Consequently, vaccines have been developed for melanoma targeting these and other immunogenic melanocyte differentiation proteins. Preliminary data showing susceptibility of LAM cells to melanoma derived T cells suggest that vaccines targeting melanosomal antigens can be successful in treating LAM.

Impact of Alcohol on Bone Health, Homeostasis and Fracture repair.

PURPOSE OF REVIEW: Alcohol use continues to rise globally. We review the current literature on the effect of alcohol on bone health, homeostasis and fracture repair to highlight what has been learned in people and animal models of alcohol consumption. RECENT FINDINGS: Recently, forkhead box O (FoxO) has been found to be upregulated and activated in mesenchymal stem cells (MSC) exposed to alcohol. FoxO has also been found to modulate Wnt/ß-catenin signaling, which is necessary for MSC differentiation. Recent evidence suggests alcohol activates FoxO signaling, which may be dysregulating Wnt/ß-catenin signaling in MSCs cultured in alcohol. SUMMARY: This review highlights the negative health effects learned from people and chronic and episodic binge alcohol consumption animal models. Studies using chronic alcohol exposure or alcohol exposure then bone fracture repair model have explored several different cellular and molecular signaling pathways important for bone homeostasis and fracture repair, and offer potential for future experiments to explore additional signaling pathways that may be dysregulated by alcohol exposure.

Alcohol-induced Wnt signaling inhibition during bone fracture healing is normalized by intermittent parathyroid hormone treatment.

Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol-inhibited bone fracture repair. Male C57BL/6 mice received two-3 day alcohol binges separated by 4 days before receiving a mid-shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/ß-catenin signaling was analyzed at 9 days post-fracture. As previously observed, acute alcohol exposure resulted in a >2-fold decrease in total and the active form of ß-catenin and a 2-fold increase in inactive ß-catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on ß-catenin within the fracture callus. Upstream of ß-catenin, alcohol-treated animals had a 2-fold decrease in total LRP6, the Wnt co-receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK-3ß. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol-treated mice.

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo.

Vitiligo is an autoimmune skin disease characterized by melanocyte destruction. Regulatory T cells (Tregs) are greatly reduced in vitiligo skin, and replenishing peripheral skin Tregs can provide protection against depigmentation. Ganglioside D3 (GD3) is overexpressed by perilesional epidermal cells, including melanocytes, which prompted us to generate GD3-reactive chimeric antigen receptor (CAR) Tregs to treat vitiligo. Mice received either untransduced Tregs or GD3-specific Tregs to test the hypothesis that antigen specificity contributes to reduced autoimmune reactivity in vitro and in vivo. CAR Tregs displayed increased IL-10 secretion in response to antigen, provided superior control of cytotoxicity towards melanocytes, and supported a significant delay in depigmentation compared to untransduced Tregs and vehicle control recipients in a TCR transgenic mouse model of spontaneous vitiligo. The latter findings were associated with a greater abundance of Tregs and melanocytes in treated mice versus both control groups. Our data support the concept that antigen-specific Tregs can be prepared, used, and stored for long-term control of progressive depigmentation.

Effects of ethanol and ethanol metabolites on intrinsic function of mesenteric resistance arteries.

Evidence suggests that ethanol-induced hypertension is associated with increased cardiovascular responsiveness to vasopressors in vivo and enhanced reactivity of isolated arteries to vasopressors ex vivo. The underlying mechanisms are not well understood and the contribution of ethanol metabolites to vascular effects induced by ethanol consumption are unclear. Mesenteric resistance arteries were harvested from Sprague-Dawley rats. Pressure myography was utilized to test effects of ethanol, acetaldehyde and phosphatidylethanol on myogenic tone and on vasoconstriction induced by phenylephrine, arginine vasopressin (aVP), endothelin-1 and KCl. Ethanol, acetaldehyde and phosphatidylethanol concentrations were monitored during the experiments. Ethanol concentrations in the vessel bath decreased with a half-life of 25min; acetaldehyde and phosphatidylethanol concentrations remained constant. Pretreatment with ethanol dose-dependently increased the potency of phenylephrine to induce vasoconstriction 4-fold (p<0.01). These effects were comparable when arteries were pre-treated with a single dose of ethanol for 30min and when ethanol concentrations were kept constant during 30min and 60min of pretreatment. While ethanol also dose-dependently increased the potency of aVP to induce vasoconstriction 1.7-fold (p<0.05), it did not affect vasoconstriction induced by endothelin-1 or KCl. Acetaldehyde pre-treatment (30 min) dose-dependently increased the potency of phenylephrine to induce vasoconstriction 2.7-fold (p<0.01) but did not affect other vasoconstrictor responses. Phosphatidylethanol did not affect any vasoconstrictor responses. Ethanol and its metabolites did not affect myogenic tone. These data suggest that ethanol and acetaldehyde selectively sensitize intrinsic constrictor responses upon activation of vascular α1-adrenergic and/or vasopressin receptors at clinically relevant concentrations. Our findings support the concept that enhanced vasoreactivity to vasoactive hormones contributes to the development of hypertension induced by ethanol consumption. Ex vivo exposure of resistance arteries to ethanol and acetaldehyde resembles effects of chronic ethanol consumption on intrinsic vascular function, and thus could serve as test platform to evaluate interventions aimed to mitigate vascular effects associated with ethanol consumption.

Molecular properties of gp100-reactive T-cell receptors drive the cytokine profile and antitumor efficacy of transgenic host T cells.

To study the contribution of T-cell receptors (TCR) to resulting T-cell responses, we studied three different human αß TCRs, reactive to the same gp100-derived peptide presented in the context of HLA-A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen-induced IFN-γ responses, which correlated with TCR affinity for peptide-MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL-17A release. Importantly, in vivo, SILv44-transgenic T cells mediated superior antitumor responses to 888-A2 + human melanoma tumor cells upon adoptive transfer into tumor-challenged mice while maintaining IL-17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T-cell receptor in defining host T-cell physiology than traditionally assumed, while parameters beyond IFN-γ secretion and TCR affinity ultimately determine the reactivity of tumor-reactive T cells.

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle.

Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in ß-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit ß-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced ß-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

Biased antagonism of CXCR4 avoids antagonist tolerance.

Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and ß-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not ß-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit ß-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.