Assessing Vulnerability to COVID-19 in High-Risk Populations: The Role of SARS-CoV-2 Spike-Targeted Serology.

Individuals at increased risk for severe coronavirus disease-2019 (COVID-19) outcomes, due to compromised immunity or other risk factors, would benefit from objective measures of vulnerability to infection based on vaccination or prior infection. The authors reviewed published data to identify a specific role and interpretation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike-targeted serology testing. Specific recommendations are provided for an evidence-based and clinically-useful interpretation of SARS-CoV-2 spike-targeted serology to identify vulnerability to infection and potential subsequent adverse outcomes. Decreased vaccine effectiveness among immunocompromised individuals is linked to correspondingly high rates of breakthrough infections. Negative results on SARS-CoV-2 antibody tests are associated with increased risk for subsequent infection. "Low-positive" results on semiquantitative SARS-CoV-2 spike-targeted antibody tests may help identify persons at increased risk as well. Standardized SARS-CoV-2 spike-targeted antibody tests may provide objective information on the risk of SARS-CoV-2 infection and associated adverse outcomes. This holds especially for high-risk populations that demonstrate a relatively high rate of seronegativity. The widespread availability of such tests presents an opportunity to refine risk assessment for individuals with suboptimal SARS-CoV-2 antibody levels and to promote effective interventions. Interim federal guidance would support physicians and patients while additional investigations are pursued.

A Combination therapy using an mTOR inhibitor and Honokiol effectively induces autophagy through the modulation of AXL and Rubicon in renal cancer cells and restricts renal tumor growth following organ transplantation.

Development of cancer, including renal cancer, is a major problem in immunosuppressed patients. The mTOR inhibitor Rapamycin (RAPA) is used as an immunosuppressive agent in patients with organ transplants and other immunological disorders; and it also has antitumorigenic potential. However, long-term use of RAPA causes reactivation of Akt, and ultimately leads to enhanced tumor growth. Honokiol (HNK) is a natural compound, which possesses both anti-inflammatory and antitumorigenic properties. In this study, we investigated the effect of a novel combination therapy using RAPA + HNK on allograft survival and post-transplantation renal tumor growth. We observed that it effectively modulated the expression of some key regulatory molecules (like Carabin, an endogenous Ras inhibitor; and Rubicon, a negative regulator of autophagy) that play important roles in tumor cell growth and survival. This combination induced toxic autophagy and apoptosis to promote cancer cell death; and was associated with a reduced expression of the tumor-promoting receptor tyrosine kinase AXL. Finally, we utilized a novel murine model to examine the effect of RAPA + HNK on post-transplantation renal tumor growth. The combination treatment prolonged the allograft survival and significantly inhibited post-transplantation tumor growth. It was associated with reduced tumor expression of Rubicon and the cytoprotective/antioxidant heme oxygenase-1 to overcome therapeutic resistance. It also downregulated the coinhibitory programmed death-1 ligand, which plays major role(s) in the immune escape of tumor cells. Together, this combination treatment has a great potential to restrict renal tumor growth in transplant recipients as well as other immunosuppressed patients.

Inhibition of mevalonate metabolism by statins augments the immunoregulatory phenotype of vascular endothelial cells and inhibits the costimulation of CD4+ T cells.

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.

T Cell-Specific Adaptor Protein Regulates Mitochondrial Function and CD4+ T Regulatory Cell Activity In Vivo following Transplantation.

The T cell-specific adaptor protein (TSAd), encoded by the SH2D2A gene, is an intracellular molecule that binds Lck to elicit signals that result in cytokine production in CD4+ T effector cells (Teff). Nevertheless, using Sh2d2a knockout (KO; also called TSAd-/-) mice, we find that alloimmune CD4+ Teff responses are fully competent in vivo. Furthermore, and contrary to expectations, we find that allograft rejection is accelerated in KO recipients of MHC class II-mismatched B6.C-H-2bm12 heart transplants versus wild-type (WT) recipients. Also, KO recipients of fully MHC-mismatched cardiac allografts are resistant to the graft-prolonging effects of costimulatory blockade. Using adoptive transfer models, we find that KO T regulatory cells (Tregs) are less efficient in suppressing Teff function and they produce IFN-γ following mitogenic activation. In addition, pyrosequencing demonstrated higher levels of methylation of CpG regions within the Treg-specific demethylated region of KO versus WT Tregs, suggesting that TSAd, in part, promotes Treg stability. By Western blot, Lck is absent in the mitochondria of KO Tregs, and reactive oxygen species production by mitochondria is reduced in KO versus WT Tregs. Full transcriptomic analysis demonstrated that the key mechanism of TSAd function in Tregs relates to its effects on cellular activation rather than intrinsic effects on mitochondria/metabolism. Nevertheless, KO Tregs compensate for a lack of activation by increasing the number of mitochondria per cell. Thus, TSAd serves as a critical cell-intrinsic molecule in CD4+Foxp3+ Tregs to regulate the translocation of Lck to mitochondria, cellular activation responses, and the development of immunoregulation following solid organ transplantation.

Calcineurin inhibitors augment endothelial-to-mesenchymal transition by enhancing proliferation in association with cytokine-mediated activation.

Calcineurin Inhibitors (CNIs) are routinely used for immunosuppression following solid organ transplantation. However, the prolonged use of these agents lead to organ fibrosis which limits their efficacy. CNIs induce TGFß expression, which is reported to augment endothelial-to-mesenchymal transition (EndMT), but their role in this process is not known. In these studies, we find that the CNIs FK506 and cyclosporine (CsA) are potent to increase endothelial cell (EC) proliferation using established in vitro assays (P < 0.05). Furthermore, using phosphokinase arrays, we find that each CNI activates the MAPK and Akt/mTOR signaling pathways, and that pharmacological inhibition of each pathway targets CNI-induced proliferative responses (P < 0.001). EndMT was evaluated by FACS for N-cadherin and CD31 expression and by qPCR for the expression of α-smooth muscle actin, N-cadherin and Snail. We find that CNIs do not directly induce dedifferentiation, while TGFß and hypoxia induce EndMT in small numbers of EC. In contrast, the treatment of EC with the inflammatory cytokine TNFα was potent to elicit an EndMT response, and its effects were most notably in EC following proliferation/doubling. Taken together, these observations suggest that CNIs elicit proliferative responses, which enhance EndMT in association with local inflammation. The clinical implications of these findings are that anti-proliferative therapeutics have high potential to target the initiation of this EndMT response.

DEPTOR modulates activation responses in CD4+ T cells and enhances immunoregulation following transplantation.

DEPTOR is an evolutionarily conserved cell-intrinsic binding partner of mTOR that functions as a negative regulator of signaling responses. In this study, we show that DEPTOR is expressed within CD4+ T cells, and we observed that its relative level of expression modulates differentiation as well as glucose utilization within CD4+ T effectors in vitro. Using knock-in mice, we also find that induced expression of DEPTOR within CD4+ T regulatory cells stabilizes Foxp3 expression, shifts metabolism toward oxidative phosphorylation, and increases survival and suppressive function. In vivo, fully MHC mismatched cardiac allograft survival is significantly prolonged in knock-in recipients and sustained recipient expression of DEPTOR in combination with costimulatory blockade induces long-term graft survival. Furthermore, we show that the induced expression of DEPTOR in CD4+ T effectors fails to inhibit acute allograft rejection. Rather, prolonged survival is dominantly mediated via induced expression and function of DEPTOR within recipient CD4+ T regulatory cells. These collective findings identify DEPTOR as a novel protein that functions in CD4+ T cells to augment immunoregulation in vitro and in vivo.

DEPTOR at the Nexus of Cancer, Metabolism, and Immunity.

DEP domain-containing mechanistic target of rapamycin (mTOR)-interacting protein (DEPTOR) is an important modulator of mTOR, a kinase at the center of two important protein complexes named mTORC1 and mTORC2. These highly studied complexes play essential roles in regulating growth, metabolism, and immunity in response to mitogens, nutrients, and cytokines. Defects in mTOR signaling have been associated with the development of many diseases, including cancer and diabetes, and approaches aiming at modulating mTOR activity are envisioned as an attractive strategy to improve human health. DEPTOR interaction with mTOR represses its kinase activity and rewires the mTOR signaling pathway. Over the last years, several studies have revealed key roles for DEPTOR in numerous biological and pathological processes. Here, we provide the current state of the knowledge regarding the cellular and physiological functions of DEPTOR by focusing on its impact on the mTOR pathway and its role in promoting health and disease.

Study rationale, design, and pretransplantation alloantibody status: A first report of Clinical Trials in Organ Transplantation in Children-04 (CTOTC-04) in pediatric heart transplantation.

Anti-HLA donor-specific antibodies are associated with worse outcomes after organ transplantation. Among sensitized pediatric heart candidates, requirement for negative donor-specific cytotoxicity crossmatch increases wait times and mortality. However, transplantation with positive crossmatch may increase posttransplantation morbidity and mortality. We address this clinical challenge in a prospective, multicenter, observational cohort study of children listed for heart transplantation (Clinical Trials in Organ Transplantation in Children-04 [CTOTC-04]). Outcomes were compared among sensitized recipients who underwent transplantation with positive crossmatch, nonsensitized recipients, and sensitized recipients without positive crossmatch. Positive crossmatch recipients received antibody removal and augmented immunosuppression, while other recipients received standard immunosuppression with corticosteroid avoidance. This first CTOTC-04 report summarizes study rationale and design and relates pretransplantation sensitization status using solid-phase technology. Risk factors for sensitization were explored. Of 317 screened patients, 290 were enrolled and 240 underwent transplantation. Core laboratory evaluation demonstrated that more than half of patients were anti-HLA sensitized. Greater than 80% of sensitized patients had class I (with or without class II) HLA antibodies, and one-third of sensitized patients had at least 1 HLA antibody with median fluorescence intensity of ≥8000. Logistic regression models demonstrated male sex, weight, congenital heart disease history, prior allograft, and ventricular assist device are independent risk factors for sensitization.

Convergent and Divergent Migratory Patterns of Human Neutrophils inside Microfluidic Mazes.

Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways.

Vascular endothelial growth factor A is associated with the subsequent development of moderate or severe cardiac allograft vasculopathy in pediatric heart transplant recipients.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is the leading cause of chronic allograft loss after pediatric heart transplantation. We hypothesized that biomarkers of endothelial injury and repair would predict CAV development in pediatric heart transplant recipients. METHODS: Blood was collected from pediatric heart transplant recipients at the time of routine annual coronary angiography, and the concentrations of 13 angiogenesis-related molecules were determined. The primary end point was the presence of moderate or severe CAV by angiography during a 5-year follow-up period. RESULTS: The study enrolled 48 recipients (57% male) with a median age of 15.5 years (range, 2-22 years) and median time post-transplant of 5.8 years (range, 2-15 years). Eight recipients developed moderate/severe CAV at a median follow-up of 4.7 years, of whom 3 died, 3 underwent retransplantation, 1 had a myocardial infarction, and 1 was listed for retransplantation. Clinical characteristics associated with the development of moderate/severe CAV included prednisone use at enrollment (p = 0.03) and positive recipient cytomegalovirus immunoglobulin G at the time of transplant (p = < 0.01). Multivariable Cox proportional hazards regression identified plasma vascular endothelial growth factor (VEGF)-A concentration greater than 90 pg/ml at the time of blood draw as a significant predictor of time to moderate or severe CAV (hazard ratio, 14.3; 95% confidence interval, 1.3-163). Receiver operating characteristic curve analysis demonstrated that VEGF-A shows moderate performance for association with the subsequent development of CAV (area under the curve, 0.77; 95% confidence interval, 0.61-0.92). CONCLUSIONS: VEGF-A levels in pediatric heart transplant recipients are associated with clinically important CAV progression within the subsequent 5 years.

The intragraft microenvironment as a central determinant of chronic rejection or local immunoregulation/tolerance.

PURPOSE OF REVIEW: Chronic rejection is associated with persistent mononuclear cell recruitment, endothelial activation and proliferation, local tissue hypoxia and related biology that enhance effector immune responses. In contrast, the tumor microenvironment elicits signals/factors that inhibit effector T cell responses and rather promote immunoregulation locally within the tissue itself. The identification of immunoregulatory check points and/or secreted factors that are deficient within allografts is of great importance in the understanding and prevention of chronic rejection. RECENT FINDINGS: The relative deficiency of immunomodulatory molecules (cell surface and secreted) on microvascular endothelial cells within the intragraft microenvironment, is of functional importance in shaping the phenotype of rejection. These regulatory molecules include coinhibitory and/or intracellular regulatory signals/factors that enhance local activation of T regulatory cells. For example, semaphorins may interact with endothelial cells and CD4 T cells to promote local tolerance. Additionally, metabolites and electrolytes within the allograft microenvironment may regulate local effector and regulatory cell responses. SUMMARY: Multiple factors within allografts shape the microenvironment either towards local immunoregulation or proinflammation. Promoting the expression of intragraft cell surface or secreted molecules that support immunoregulation will be critical for long-term graft survival and/or alloimmune tolerance.

Cholesterol efflux capacity of high-density lipoprotein correlates with survival and allograft vasculopathy in cardiac transplant recipients.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is a major cause of mortality after cardiac transplantation. High-density lipoprotein (HDL) cholesterol efflux capacity (CEC) is inversely associated with coronary artery disease. In 2 independent studies, we tested the hypothesis that reduced CEC is associated with mortality and disease progression in CAV. METHODS: We tested the relationship between CEC and survival in a cohort of patients with CAV (n = 35). To determine whether reduced CEC is associated with CAV progression, we utilized samples from the Clinical Trials in Organ Transplantation 05 (CTOT05) study to determine the association between CEC and CAV progression and status at 1 year (n = 81), as assessed by average change in maximal intimal thickness (MIT) on intravascular ultrasound. RESULTS: Multivariable Cox proportional hazard models demonstrated that higher levels of CEC were associated with improved survival (hazard ratio 0.26, 95% confidence interval 0.11 to 0.63) per standard deviation CEC, p = 0.002). Patients who developed CAV had reduced CEC at baseline and 1-year post-transplant. We observed a significant association between pre-transplant CEC and the average change in MIT, particularly among patients who developed CAV at 1 year (ß = -0.59, p = 0.02, R2 = 0.35). CONCLUSION: Reduced CEC is associated with disease progression and mortality in CAV patients. These findings suggest the hypothesis that interventions to increase CEC may be useful in cardiac transplant patients for prevention or treatment of CAV.

Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo.

Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4(+) T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4(+) T cells, but it had no demonstrable effects on the migration of purified CD4(+)CD25(+)CD127(dim) T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3(+) T cell infiltrates. Neogenin was also observed on CD3(+) T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4(+) T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.

Translational implications of endothelial cell dysfunction in association with chronic allograft rejection.

Advances in therapeutics have dramatically improved short-term graft survival, but the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection. Key initial events include EC injury, activation and uncontrolled leukocyte-induced angiogenesis. We propose that all of these early changes in the microvasculature lead to abnormal blood flow patterns, local tissue hypoxia, and an associated overexpression of HIF-1α-inducible genes, including vascular endothelial growth factor. We also discuss how cell intrinsic regulators of mTOR-mediated signaling within EC are of critical importance in microvascular stability and may thus have a role in the inhibition of chronic rejection. Finally, we discuss recent findings indicating that miRNAs may regulate EC stability, and we review their potential as novel non-invasive biomarkers of allograft rejection. Overall, this review provides insights into molecular events, genes, and signals that promote chronic rejection and their potential as biomarkers that serve to support the future development of interruption therapeutics.

Microfluidic mazes to characterize T-cell exploration patterns following activation in vitro.

The migration of T-cell subsets within peripheral tissues is characteristic of inflammation and immunoregulation. In general, the lymphocyte migratory response is assumed directional and guided by local gradients of chemoattractants and/or chemorepellents. However, little is known about how cells explore their tissue environment, and whether lymphocyte activation may influence speed and exploratory patterns of migration. To probe migration patterns by T-cells we designed a microfluidic maze device that replicates critical features of a tissue-like microenvironment. We quantified the migration patterns of unstimulated and mitogen-activated human T-cells at single cell resolution and found significant differences in exploration within microfluidic mazes. While unstimulated lymphocytes migrated in a directed manner, activated T-cells migrated through large areas of the mazes in an exploratory pattern in response to the chemoattractants RANTES (CCL5) and IP-10 (CXCL10). The analysis of migration enabled by the microfluidic devices help develop new methods for determining how human circulating T-cells function in vivo to seek out antigens in health and disease states.

Regulation of mTOR Signaling by Semaphorin 3F-Neuropilin 2 Interactions In Vitro and In Vivo.

Semaphorin 3F (SEMA3F) provides neuronal guidance cues via its ability to bind neuropilin 2 (NRP2) and Plexin A family molecules. Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood. Here, we analyze SEMA3F-NRP2 signaling responses in human endothelial, T cell and tumor cells using phosphokinase arrays, immunoprecipitation and Western blot analyses. Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase. We also find that the expression of vascular endothelial growth factor, as well as mTOR-inducible cellular activation responses and cytoskeleton stability are inhibited by SEMA3F-NRP2 interactions in vitro. In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts. Taken together, SEMA3F regulates mTOR signaling in diverse human cell types, suggesting that it has broad therapeutic implications.

Use of [18F]FDG Positron Emission Tomography to Monitor the Development of Cardiac Allograft Rejection.

BACKGROUND: Positron emission tomography (PET) has the potential to be a specific, sensitive and quantitative diagnostic test for transplant rejection. To test this hypothesis, we evaluated F-labeled fluorodeoxyglucose ([F]FDG) and N-labeled ammonia ([N]NH3) small animal PET imaging in a well-established murine cardiac rejection model. METHODS: Heterotopic transplants were performed using minor major histocompatibility complex-mismatched B6.C-H2 donor hearts in C57BL/6(H-2) recipients. C57BL/6 donor hearts into C57BL/6 recipients served as isograft controls. [F]FDG PET imaging was performed weekly between posttransplant days 7 and 42, and the percent injected dose was computed for each graft. [N]NH3 imaging was performed to evaluate myocardial perfusion. RESULTS: There was a significant increase in [F]FDG uptake in allografts from day 14 to day 21 (1.6% to 5.2%; P < 0.001) and uptake in allografts was significantly increased on posttransplant days 21 (5.2% vs 0.9%; P = 0.005) and 28 (4.8% vs 0.9%; P = 0.006) compared to isograft controls. Furthermore, [F]FDG uptake correlated with an increase in rejection grade within allografts between days 14 and 28 after transplantation. Finally, the uptake of [N]NH3 was significantly lower relative to the native heart in allografts with chronic vasculopathy compared to isograft controls on day 28 (P = 0.01). CONCLUSIONS: PET imaging with [F]FDG can be used after transplantation to monitor the evolution of rejection. Decreased uptake of [N]NH3 in rejecting allografts may be reflective of decreased myocardial blood flow. These data suggest that combined [F]FDG and [N]NH3 PET imaging could be used as a noninvasive, quantitative technique for serial monitoring of allograft rejection and has potential application in human transplant recipients.

Chronic allograft rejection: a fresh look.

PURPOSE OF REVIEW: New developments suggest that the graft itself and molecules expressed within the graft microenvironment dictate the phenotype and evolution of chronic rejection. RECENT FINDINGS: Once ischemia-reperfusion injury, cellular and humoral immune responses target the microvasculature, the associated local tissue hypoxia results in hypoxia-inducible factor 1α-dependent expression of pro-inflammatory and proangiogenic growth factors including vascular endothelial growth factor (VEGF) as a physiological response to injury. Local expression of VEGF can promote the recruitment of alloimune T cells into the graft. mTOR/Akt signaling within endothelial cells regulates cytokine- and alloantibody-induced activation and proliferation and their proinflammatory phenotype. Inhibition of mTOR and/or Akt results in an anti-inflammatory phenotype and enables the expression of coinhibitory molecules that limit local T cell reactivation and promotes immunoregulation. Semaphorin family molecules may bind to neuropilin-1 on regulatory T cell subsets to stabilize functional responses. Ligation of neuropilin-1 on Tregs also inhibits Akt-induced responses suggesting common theme for enhancing local immunoregulation and long-term graft survival. SUMMARY: Events within the graft initiated by mTOR/Akt-induced signaling promote the development of chronic rejection. Semaphorin-neuropilin biology represents a novel avenue for targeting this biology and warrants further investigation.

Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.