PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

TRPV1 enhances cholecystokinin signaling in primary vagal afferent neurons and mediates the central effects on spontaneous glutamate release in the NTS.

The gut peptide cholecystokinin (CCK) is released during feeding and promotes satiation by increasing excitation of vagal afferent neurons that innervate the upper gastrointestinal tract. Vagal afferent neurons express CCK1 receptors (CCK1Rs) in the periphery and at central terminals in the nucleus of the solitary tract (NTS). While the effects of CCK have been studied for decades, CCK receptor signaling and coupling to membrane ion channels are not entirely understood. Previous findings have implicated L-type voltage-gated calcium channels as well as transient receptor potential (TRP) channels in mediating the effects of CCK, but the lack of selective pharmacology has made determining the contributions of these putative mediators difficult. The nonselective ion channel transient receptor potential vanilloid subtype 1 (TRPV1) is expressed throughout vagal afferent neurons and controls many forms of signaling, including spontaneous glutamate release onto NTS neurons. Here we tested the hypothesis that CCK1Rs couple directly to TRPV1 to mediate vagal signaling using fluorescent calcium imaging and brainstem electrophysiology. We found that CCK signaling at high concentrations (low-affinity binding) was potentiated in TRPV1-containing afferents and that TRPV1 itself mediated the enhanced CCK1R signaling. While competitive antagonism of TRPV1 failed to alter CCK1R signaling, TRPV1 pore blockade or genetic deletion (TRPV1 KO) significantly reduced the CCK response in cultured vagal afferents and eliminated its ability to increase spontaneous glutamate release in the NTS. Together, these results establish that TRPV1 mediates the low-affinity effects of CCK on vagal afferent activation and control of synaptic transmission in the brainstem.NEW & NOTEWORTHY Cholecystokinin (CCK) signaling via the vagus nerve reduces food intake and produces satiation, yet the signaling cascades mediating these effects remain unknown. Here we report that the capsaicin receptor transient receptor potential vanilloid subtype 1 (TRPV1) potentiates CCK signaling in the vagus and mediates the ability of CCK to control excitatory synaptic transmission in the nucleus of the solitary tract. These results may prove useful in the future development of CCK/TRPV1-based therapeutic interventions.

Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps.

Interleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2Rß and γc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 "superkine" with enhanced affinity for IL-2Rß. Here, we describe next-generation IL-2 variants that function as "receptor signaling clamps." They retained high affinity for IL-2Rß, inhibiting binding of endogenous IL-2, but their interaction with γc was weakened, attenuating IL-2Rß-γc heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2Rα or IL-2Rß. Furthermore, this mutein prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.

Secular trends in the utility of SGLT-2 inhibitors in heart failure patients with type 2 diabetes mellitus across Metro South Health hospitals in South-East Queensland.

BACKGROUND: The 2018 Australian Heart Failure (HF) guidelines strongly recommended commencing sodium-glucose co-transporter-2 inhibitors (SGLT-2is) in HF patients with type 2 diabetes mellitus (T2DM). The uptake of SGLT-2is for HF patients with T2DM in our health service is unknown. AIMS: To determine the adoption of the 2018 HF guidelines by assessing the temporal trends of SGLT-2is' usage in HF patients with T2DM at Metro South Health (MSH) hospitals, in South-East Queensland. METHODS: Retrospective analysis of all HF patients (ejection fraction (EF) < 50%) with T2DM who were managed within MSH hospitals between June 2018 and June 2021. RESULTS: A total of 666 patients met the inclusion criteria with 918 HF encounters. Mean age was 72 years and 71% were male (473/666). Mean EF was 30% (SD ± 11%), and mean estimated glomerular filtration rate was 48 mL/min/1.73 m2 (SD ± 25). Fifty-four per cent (362/666) had contraindications to SGLT-2is. Among those without contraindications, there was a five-fold increase in the utility of SGLT-2is, 7% (2/29) before versus 38% (103/275) after implementation of the HF guidelines (P < 0.001). Patients on SGLT-2is were younger (64 years vs 69 years, P = 0.002) and had a lower number of HF hospitalisations (1.1 vs 2.1, P = 0.01). CONCLUSIONS: During the study period, 54% of our HF patients with T2DM were not on SGLT-2is due to prescribing guidelines/limitations in the Australian context. We observed a five-fold significant increase in the uptake of SGLT-2is before and after implementation of HF guidelines among patients without contraindications to SGLT-2is. There were significantly fewer HF hospitalisations among patients on SGLT-2is compared to those without.

Long-term follow-up after lymphodepleting autologous haematopoietic cell transplantation for treatment-resistant systemic lupus erythematosus.

OBJECTIVE: Autologous haematopoietic cell transplantation (AHSCT) improves immunologic dysfunction in patients with SLE. However, the curative potential of this therapy remains uncertain. This study reports outcomes in SLE patients receiving a lymphodepleting, reduced intensity regimen for AHSCT in SLE. METHODS: Eight patients with SLE refractory to treatment, including i.v. cyclophosphamide (CYC), were enrolled. Five had LN and three CNS involvement as primary indications for transplant. Haematopoietic cell mobilization with CYC, G-CSF and rituximab was followed by collection of CD34+ positively selected cells. The conditioning regimen consisted of concurrent administration of CYC, fludarabine and rituximab. All immunosuppressive medications were discontinued at the start of mobilization and CS were rapidly tapered after the transplant. RESULTS: Five of eight patients achieved a complete response, including a decline in the SLEDAI to zero, which was sustained in four patients for a median of 165 months (range 138-191). One patient achieved a partial response, which was followed by relapse at month 18. Two patients with nephritis and underlying comorbidities in most organs had early deaths from infection and multiorgan failure. AHSCT resulted in profound lymphodepletion, followed by expansion of Treg cells and repopulation of naive T and B cells. Patients with a complete response showed a sustained suppression of the SLE-associated IFN-induced gene signature, marked depletion of memory and plasmablast B cells and resultant sustained elimination of anti-dsDNA antibody. CONCLUSION: Durable clinical and serologic remissions with suppression in the IFN gene signature can be achieved in refractory SLE following lymphodepleting AHSCT. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT00076752.

STAT3 transcription factor promotes instability of nTreg cells and limits generation of iTreg cells during acute murine graft-versus-host disease.

Acute graft-versus-host disease (GvHD) is a major cause of mortality in allogeneic bone marrow transplantation (BMT), for which administration of FoxP3(+) regulatory T (Treg) cells has been proposed as a therapy. However, the phenotypic stability of Treg cells is controversial, and STAT3-dependent cytokines can inhibit FoxP3 expression. We assessed whether the elimination of STAT3 in T cells could limit the severity of GvHD. We found STAT3 limited FoxP3(+) Treg cell numbers following allogeneic BMT by two pathways: instability of natural Treg (nTreg) cells and inhibition of induced Treg (iTreg) cell polarization from naive CD4(+) T cells. Deletion of STAT3 within only the nTreg cell population was not sufficient to protect against lethal GvHD. In contrast, transfer of STAT3-deficient naive CD4(+) T cells increased FoxP3(+) Treg cells post-BMT and prevented lethality, suggesting that the consequence of STAT3 signaling may be greater for iTreg rather than nTreg cells during GvHD.

Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons.

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for Vδ2+ γδ T cells in the modulation of immune response to tumour?

Release of granulysin by γδ T cells contributes to tumour cell killing. A cytolytic 9000 MW isoform of granulysin kills tumour cells directly, whereas a 15 000 MW precursor has been hypothesized to cause both the maturation and migration of dendritic cell (DC) populations. Recruiting DC to a tumour is beneficial as these cells initiate adaptive immune responses, which contribute to the eradication of malignancies. In this study, Vδ2+ γδ T cells were activated by stimulation of peripheral blood mononuclear cells with zoledronic acid or Bacillus Calmette-Guérin (BCG), or were isolated and cultured with tumour targets. Although a large proportion of resting Vδ2+ γδ T cells expressed 15 000 MW granulysin, 9000 MW granulysin expression was induced only after stimulation with BCG. Increased levels of activation and granulysin secretion were also observed when Vδ2+ γδ T cells were cultured with the human B-cell lymphoma line Daudi. High concentrations of recombinant 15 000 MW granulysin caused migration and maturation of immature DC, and also initiated fugetaxis in mature DC. Conversely, low concentrations of recombinant 15 000 MW granulysin resulted in migration of mature DC, but not immature DC. Our data therefore support the hypothesis that Vδ2+ γδ T cells can release granulysin, which may modulate recruitment of DC, initiating adaptive immune responses.

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.

Cytomegalovirus Infection Incidence and Risk Factors Across Diverse Hematopoietic Cell Transplantation Platforms Using a Standardized Monitoring and Treatment Approach: A Comprehensive Evaluation from a Single Institution.

Human cytomegalovirus (CMV) infection and disease remains a significant cause of morbidity and mortality for hematopoietic cell transplantation (HCT) recipients. Disruption of or weak reconstitution of virus-specific cellular immune function, such as with certain HCT approaches, poses significant risk for CMV-related complications. The incidence of and risk factors for CMV infection and the nature of CMV disease were evaluated retrospectively among 356 consecutive HCT recipients transplanted at the National Institutes of Health using all graft sources, including bone marrow, peripheral blood stem cell (PBSC), and umbilical cord blood (UCB), and a range of in vivo and ex vivo approaches for graft-versus-host disease (GVHD) prophylaxis. The cumulative incidence of CMV infection was higher for CMV-seropositive recipients at 33%, regardless of donor CMV serostatus. Patients transplanted with CMV-seropositive donors had a significantly shorter duration of antiviral therapy. Among graft sources UCB was associated with the highest cumulative incidence of CMV infection at 65% and significantly longer treatment duration at a median of 36days, whereas PBSC HCT was associated with the lowest incidence at 26% and the shortest CMV treatment duration at a median of 21days. There were significant differences in the cumulative incidence of CMV infection by T cell manipulation strategy when systemic steroids were included as a risk-modifying event. Over one-third of CMV infections occurred in the setting of systemic steroid administration. CMV disease occurred in 5% of HCT recipients, with 70% of cases in the setting of treatment for GVHD. Although factors related to serostatus, graft source, and GVHD prophylaxis were associated with varied CMV infection incidence, unplanned post-HCT corticosteroid therapy contributed greatly to the incidence of both CMV infection and disease across HCT approaches, highlighting this post-HCT intervention as a key time to potentially tailor the approach to monitoring, preemptive therapy, and even prophylaxis.

Transfusion support for matched sibling allogeneic hematopoietic stem cell transplantation (1993-2010): factors that predict intensity and time to transfusion independence.

BACKGROUND: Patients undergoing allogeneic hematopoietic stem cell transplant require variable, often extensive transfusion support. Identification of factors that predict urgent, intensive, or special needs should improve management of these patients. STUDY DESIGN AND METHODS: This is a retrospective study of red blood cell (RBC) and platelet transfusion support provided for sequential matched sibling donor allogeneic transplants conducted at the Clinical Center, National Institutes of Health, from 1993 through 2010. Factors potentially important for predicting quantity of RBC and platelet transfusions, and time to transfusion independence through Day 200 following hematopoietic stem cell transplantation were evaluated. RESULTS: Subjects (n = 800) received 10,591 RBC and 10,199 platelet transfusions. Multivariable analysis demonstrated that the need for RBC pretransplant, CD34+ dose, transplant year, diagnostic category, and ABO match were significantly independently associated with quantity of RBC transfusions during Days 0 through 30. Only pretransplant need for RBCs, CD34+ dose, and transplant year had significance during Days 0 through 100. Similar analyses for quantity of platelet transfusions demonstrated that for both Days 0 through 30 and 0 through 100 significant factors were need for platelet support before transplant, CD34+ dose, transplant year, and transplant regimen. Of note, long term, during Days 101 through 200, only CD34+ dose remained significant for quantity of RBC and platelet transfusions. Analysis of time to transfusion independence demonstrated that patients with ABO major mismatches required longer to achieve freedom from RBC transfusion support compared to identical matches or those with minor mismatches. CONCLUSION: Patient-specific factors including CD34+ dose and ABO match of the graft should be given particular consideration by transfusion services when planning support of patients receiving allogeneic hematopoietic stem cell transplant.

Upregulation of IFN-Inducible and Damage-Response Pathways in Chronic Graft-versus-Host Disease.

Although chronic graft-versus-host disease (CGVHD) is the primary nonrelapse complication of allogeneic transplantation, understanding of its pathogenesis is limited. To identify the main operant pathways across the spectrum of CGVHD, we analyzed gene expression in circulating monocytes, chosen as in situ systemic reporter cells. Microarrays identified two interrelated pathways: 1) IFN-inducible genes, and 2) innate receptors for cellular damage. Corroborating these with multiplex RNA quantitation, we found that multiple IFN-inducible genes (affecting lymphocyte trafficking, differentiation, and Ag presentation) were concurrently upregulated in CGVHD monocytes compared with normal subjects and non-CGVHD control patients. IFN-inducible chemokines were elevated in both lichenoid and sclerotic CGHVD plasma and were linked to CXCR3+ lymphocyte trafficking. Furthermore, the levels of the IFN-inducible genes CXCL10 and TNFSF13B (BAFF) were correlated at both the gene and the plasma levels, implicating IFN induction as a factor in elevated BAFF levels in CGVHD. In the second pathway, damage-/pathogen-associated molecular pattern receptor genes capable of inducing type I IFN were upregulated. Type I IFN-inducible MxA was expressed in proportion to CGVHD activity in skin, mucosa, and glands, and expression of TLR7 and DDX58 receptor genes correlated with upregulation of type I IFN-inducible genes in monocytes. Finally, in serial analyses after transplant, IFN-inducible and damage-response genes were upregulated in monocytes at CGVHD onset and declined upon therapy and resolution in both lichenoid and sclerotic CGVHD patients. This interlocking analysis of IFN-inducible genes, plasma analytes, and tissue immunohistochemistry strongly supports a unifying hypothesis of induction of IFN by innate response to cellular damage as a mechanism for initiation and persistence of CGVHD.

Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.

Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.

Rapamycin-resistant effector T-cell therapy.

Pharmacologic inhibition of the mechanistic target of rapamycin (mTOR) represents a stress test for tumor cells and T cells. Mechanisms exist that allow cells to survive this stress, including suboptimal target block, alternative signaling pathways, and autophagy. Rapamycin-resistant effector T (T-Rapa) cells have an altered phenotype that associates with increased function. Ex vivo rapamycin, when used in combination with polarizing cytokines and antigen-presenting-cell free costimulation, is a flexible therapeutic approach as polarization to T-helper 1 (Th1)- or Th2-type effectors is possible. Murine T-Rapa cells skewed toward a Th2-type prevented graft rejection and graft-versus-host disease (GVHD) more potently than control Th2 cells and effectively balanced GVHD and graft-versus-tumor (GVT) effects. A phase II clinical trial using low-intensity allogeneic hematopoietic cell transplantation demonstrated that interleukin-4 polarized human T-Rapa cells had a mixed Th2/Th1 phenotype; T-Rapa cell recipients had a balanced Th2/Th1 cytokine profile, conversion of mixed chimerism toward full donor chimerism, and a potentially favorable balance between GVHD and GVT effects. In addition, a phase I clinical trial evaluating autologous T-Rapa cells skewed toward a Th1- and Tc1-type is underway. Use of ex vivo rapamycin to modulate effector T-cell function represents a promising new approach to transplantation therapy.

Predictors for Permanent Discontinuation of Systemic Immunosuppression in Severely Affected Chronic Graft-Versus-Host Disease Patients.

Predicting the duration of systemic therapy in patients with chronic graft-versus-host disease (cGVHD) is of critical clinical importance when counseling patients and for treatment planning. cGVHD characteristics associated with this outcome have not been studied in severely affected patients. The National Institutes of Health (NIH) cGVHD scoring provides a standardized set of organ severity measures that could represent clinically useful and reproducible predictive characteristics. We analyzed 227 previously treated patients most with moderate (n = 54) or severe (n = 170) cGVHD defined by NIH criteria who were prospectively enrolled in a natural history protocol (NCT00092235). Patients received a median of 4 prior systemic therapy regimens and were seen at the NIH for a single time-point visit and were then monitored for survival and ability to discontinue cGVHD systemic therapy. With a median follow-up of 71.1 months, the cumulative incidence of systemic therapy discontinuation was 9.5% (95% confidence interval, 6.0% to 13.9%) at 2 years and 27.7% (95% confidence interval, 20.9% to 34.8%) by 5 years after the initial visit. Factors associated with a higher incidence of immunosuppression discontinuation included lower NIH global severity (P = .019) and lung (P = .030) scores and less extensive deep sclerosis (<37% body surface area, P = .024). Lower patient- and clinician-reported 0 to 10 severity NIH scores and noncyclosporine prophylaxis regimens were also associated with higher incidence of immunosuppression discontinuation (P <.05). In conclusion, we found low success rates for immune suppression discontinuation in previously treated patients who were severely affected with cGVHD. NIH scoring and clinical measures provide new standardized disease-specific tools to predict discontinuation of systemic therapy.

The Biology of Chronic Graft-versus-Host Disease: A Task Force Report from the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease.

Chronic graft-versus-host disease (GVHD) is the leading cause of late, nonrelapse mortality and disability in allogeneic hematopoietic cell transplantation recipients and a major obstacle to improving outcomes. The biology of chronic GVHD remains enigmatic, but understanding the underpinnings of the immunologic mechanisms responsible for the initiation and progression of disease is fundamental to developing effective prevention and treatment strategies. The goals of this task force review are as follows: This document is intended as a review of our understanding of chronic GVHD biology and therapies resulting from preclinical studies, and as a platform for developing innovative clinical strategies to prevent and treat chronic GVHD.

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2+ γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2+ T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2+ T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2+ T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2+ T cell cytotoxicity. Vδ2+ T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2+ T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy.

Ethyl Vanillin Activates TRPA1.

The nonselective cation channel transient receptor potential ankryn subtype family 1 (TRPA1) is expressed in neurons of dorsal root ganglia and trigeminal ganglia and also in vagal afferent neurons that innervate the lungs and gastrointestinal tract. Many TRPA1 agonists are reactive electrophilic compounds that form covalent adducts with TRPA1. Allyl isothiocyanate (AITC), the common agonist used to identify TRPA1, contains an electrophilic group that covalently binds with cysteine residues of TRPA1 and confers a structural change on the channel. There is scientific motivation to identify additional compounds that can activate TRPA1 with different mechanisms of channel gating. We provide evidence that ethyl vanillin (EVA) is a TRPA1 agonist. Using fluorescent calcium imaging and whole-cell patch-clamp electrophysiology on dissociated rat vagal afferent neurons and TRPA1-transfected COS-7 cells, we discovered that EVA activates cells also activated by AITC. Both agonists display similar current profiles and conductances. Pretreatment with A967079, a selective TRPA1 antagonist, blocks the EVA response as well as the AITC response. Furthermore, EVA does not activate vagal afferent neurons from TRPA1 knockout mice, showing selectivity for TRPA1 in this tissue. Interestingly, EVA appears to be pharmacologically different from AITC as a TRPA1 agonist. When AITC is applied before EVA, the EVA response is occluded. However, they both require intracellular oxidation to activate TRPA1. These findings suggest that EVA activates TRPA1 but via a distinct mechanism that may provide greater ease for study in native systems compared with AITC and may shed light on differential modes of TRPA1 gating by ligand types.

Syk and tired of current chronic GVHD therapies.

In this issue of Blood, Flynn et al1 provide key data that lend further support to the development of clinical trials of spleen tyrosine kinase (Syk) inhibition for more effective chronic graft-versus-host disease (cGVHD) treatment.

Haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia.

We studied adults with acute myeloid leukemia (AML) after haploidentical (n = 192) and 8/8 HLA-matched unrelated donor (n = 1982) transplantation. Haploidentical recipients received calcineurin inhibitor (CNI), mycophenolate, and posttransplant cyclophosphamide for graft-versus-host disease (GVHD) prophylaxis; 104 patients received myeloablative and 88 received reduced intensity conditioning regimens. Matched unrelated donor transplant recipients received CNI with mycophenolate or methotrexate for GVHD prophylaxis; 1245 patients received myeloablative and 737 received reduced intensity conditioning regimens. In the myeloablative setting, day 30 neutrophil recovery was lower after haploidentical compared with matched unrelated donor transplants (90% vs 97%, P = .02). Corresponding rates after reduced intensity conditioning transplants were 93% and 96% (P = .25). In the myeloablative setting, 3-month acute grade 2-4 (16% vs 33%, P < .0001) and 3-year chronic GVHD (30% vs 53%, P < .0001) were lower after haploidentical compared with matched unrelated donor transplants. Similar differences were observed after reduced intensity conditioning transplants, 19% vs 28% (P = .05) and 34% vs 52% (P = .002). Among patients receiving myeloablative regimens, 3-year probabilities of overall survival were 45% (95% CI, 36-54) and 50% (95% CI, 47-53) after haploidentical and matched unrelated donor transplants (P = .38). Corresponding rates after reduced intensity conditioning transplants were 46% (95% CI, 35-56) and 44% (95% CI, 0.40-47) (P = .71). Although statistical power is limited, these data suggests that survival for patients with AML after haploidentical transplantation with posttransplant cyclophosphamide is comparable with matched unrelated donor transplantation.