Porous Precision-Templated 40 µm Pore Scaffolds Promote Healing through Synergy in Macrophage Receptor with Collagenous Structure and Toll-Like Receptor Signaling.

Porous precision-templated scaffolds (PTS) with uniform, interconnected, 40 µm pores have shown favorable healing outcomes and a reduced foreign body reaction (FBR). Macrophage receptor with collagenous structure (MARCO) and toll-like receptors (TLRs) have been identified as key surface receptors in the initial inflammatory phase of wound healing. However, the role of MARCO and TLRs in modulating monocyte and macrophage phenotypes within PTS remains uncharacterized. In this study, we demonstrate a synergetic relationship between MARCO and TLR signaling in cells inhabiting PTS, where induction with TLR3 or TLR4 agonists to 40 µm scaffold-resident cells upregulates the transcription of MARCO. Upon deletion of MARCO, the prohealing phenotype within 40 µm PTS polarizes to a proinflammatory and profibrotic phenotype. Analysis of downstream TLR signaling shows that MARCO is required to attenuate nuclear factor kappa B (NF-κB) inflammation in 40 µm PTS by regulating the transcription of inhibitory NFKB inhibitor alpha (NFKBIA) and interleukin-1 receptor-associated kinase 3 (IRAK-M), primarily through a MyD88-dependent signaling pathway. Investigation of implant outcome in the absence of MARCO demonstrates an increase in collagen deposition within the scaffold and the development of tissue fibrosis. Overall, these results further our understanding of the molecular mechanisms underlying MARCO and TLR signaling within PTS. Impact statement Monocyte and macrophage phenotypes in the foreign body reaction (FBR) are essential for the development of a proinflammatory, prohealing, or profibrotic response to implanted biomaterials. Identification of key surface receptors and signaling mechanisms that give rise to these phenotypes remain to be elucidated. In this study, we report a synergistic relationship between macrophage receptor with collagenous structure (MARCO) and toll-like receptor (TLR) signaling in scaffold-resident cells inhabiting porous precision-templated 40 µm pore scaffolds through a MyD88-dependent pathway that promotes healing. These findings advance our understanding of the FBR and provide further evidence that suggests MARCO, TLRs, and fibrosis may be interconnected.

Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds.

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO®, an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 µm in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 µm PTS remain unclear. Here, we use HALO® to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 µm PTS compared to 100 µm PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 µm PTS. When subcutaneously implanted in MyD88KO mice, 40 µm PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 µm PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.

Monocytes contribute to a pro-healing response in 40 µm diameter uniform-pore, precision-templated scaffolds.

Porous precision-templated scaffolds (PTS) with uniformly distributed 40 µm spherical pores have shown a remarkable ability in immunomodulating resident cells for tissue regeneration. While the pore size mediated pro-healing response observed only in 40 µm pore PTS has been attributed to selective macrophage polarization, monocyte recruitment and phenotype have largely been uncharacterized in regulating implant outcome. Here, we employ a double transgenic mouse model for myeloid characterization and a multifaceted phenotyping approach to quantify monocyte dynamics within subcutaneously implanted PTS. Within 40 µm PTS, myeloid cells were found to preferentially infiltrate into the scaffold. Additionally, macrophage receptor with collagenous structure (MARCO), an innate activation marker, was significantly upregulated within 40 µm PTS. When 40 µm PTS were implanted in monocyte-depleted mice, the transcription of MARCO was significantly decreased and an increase in pro-inflammatory inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFα) were observed. Typical of a foreign body response (FBR), 100 µm PTS significantly upregulated pro-inflammatory iNOS, secreted higher amounts of TNFα, and displayed a pore size dependent morphology compared to 40 µm PTS. Overall, these results identify a pore size dependent modulation of circulating monocytes and implicates MARCO expression as a defining subset of monocytes that appears to be responsible for regulating a pro-healing host response.

Potential role of exosome-based allorecognition pathways involved in lung transplant rejection.

Innate and adaptive immunity both contribute to allorecognition mechanisms that drive rejection after lung transplantation. Classic allorecognition pathways have been extensively described, but there continues to be several unanswered questions. Exosome research appears to be a novel and potentially significant area of allorecognition research and could be the missing link that answers some existing questions. This article reviews literature that is associated with allorecognition pathways and the role of exosomes in alloreactivity.

Uniform 40-µm-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication.

Implanted porous precision templated scaffolds (PTS) with 40-µm spherical pores reduce inflammation and foreign body reaction (FBR) while increasing vascular density upon implantation. Larger or smaller pores, however, promote chronic inflammation and FBR. While macrophage (MØ) recruitment and polarization participates in perpetuating this pore-size-mediated phenomenon, the driving mechanism of this unique pro-healing response is poorly characterized. We hypothesized that the primarily myeloid PTS resident cells release small extracellular vesicles (sEVs) that induce pore-size-dependent pro-healing effects in surrounding T cells. Upon profiling resident immune cells and their sEVs from explanted 40-µm- (pro-healing) and 100-µm-pore diameter (inflammatory) PTS, we found that PTS pore size did not affect PTS resident immune cell population ratios or the proportion of myeloid sEVs generated from explanted PTS. However, quantitative transcriptomic assessment indicated cell and sEV phenotype were pore size dependent. In vitro experiments demonstrated the ability of PTS cell-derived sEVs to stimulate T cells transcriptionally and proliferatively. Specifically, sEVs isolated from cells inhabiting explanted 100 µm PTS significantly upregulated Th1 inflammatory gene expression in immortalized T cells. sEVs isolated from cell inhabiting both 40- and 100-µm PTS upregulated essential Treg transcriptional markers in both primary and immortalized T cells. Finally, we investigated the effects of Treg depletion on explanted PTS resident cells. FoxP3+ cell depletion suggests Tregs play a unique role in balancing T cell subset ratios, thus driving host response in 40-µm PTS. These results indicate that predominantly 40-µm PTS myeloid cell-derived sEVs affect T cells through a distinct, pore-size-mediated modality.

Prediction Model for Nodal Disease Among Patients With Non-Small Cell Lung Cancer.

BACKGROUND: We characterized the performance characteristics of guideline-recommended invasive mediastinal staging (IMS) for lung cancer and developed a prediction model for nodal disease as a potential alternative approach to staging. METHODS: We conducted a prospective cohort study of adults with suspected/confirmed non-small cell lung cancer without evidence of distant metastatic disease (by computed tomography/positron emission tomography) who underwent nodal evaluation by IMS and/or at the time of resection. The true-positive rate was the proportion of patients with true nodal disease selected to undergo IMS based on guideline recommendations, and the false-positive rate was the proportion of patients without true nodal disease selected to undergo IMS. Logistic regression was used to predict nodal disease using radiographic predictors. RESULTS: Among 123 eligible subjects, 31 (25%) had pathologically confirmed nodal disease. A guideline-recommended invasive staging strategy had a true-positive rate and false-positive rate of 100% and 65%, respectively. The prediction model fit the data well (goodness-of-fit test, p = 0.55) and had excellent discrimination (optimism corrected c-statistic, 0.78; 95% confidence interval, 0.72 to 0.89). Exploratory analysis revealed that use of the prediction model could achieve a false-positive rate of 44% and a true-positive rate of 97%. CONCLUSIONS: A guideline-recommended strategy for IMS selects all patients with true nodal disease and most patients without nodal disease for IMS. Our prediction model appears to maintain (within a margin of error) the sensitivity of a guideline-recommended invasive staging strategy and has the potential to reduce the use of invasive procedures.

Challenges in Predicting Recurrence After Resection of Node-Negative Non-Small Cell Lung Cancer.

BACKGROUND: One in 5 patients with completely resected early-stage non-small cell lung cancer will recur within 2 years. Risk stratification may facilitate a personalized approach to the use of adjuvant therapy and surveillance imaging. We developed a prediction model for recurrence based on five clinical variables (tumor size and grade, visceral pleural and lymphovascular invasion, and sublobar resection), and tested the hypothesis that the addition of several new molecular markers of poor long-term outcome (vascular endothelial growth factor C; microRNA precursors 486 and 30d) would enhance prediction. METHODS: We performed a retrospective cohort study of patients with completely resected, node-negative non-small cell lung cancer from 2011 to 2014 (follow-up through 2016) using the Lung Cancer Biospecimen Resource Network. Cox regression was used to estimate the 2-year risk of recurrence. Our primary measure of model performance was the optimism-corrected C statistic. RESULTS: Among 173 patients (mean tumor size, 3.6 cm; 12% sublobar resection, 32% poorly differentiated, 16% lymphovascular invasion, 26% visceral pleural invasion), the 2-year recurrence rate was 23% (95% confidence interval, 17% to 31%). A prediction model using five known risk factors for recurrence performed only slightly better than chance in predicting recurrence (optimism-corrected C statistic, 0.54; 95% confidence interval, 0.51 to 0.68). The addition of biomarkers did not improve the model's ability to predict recurrence (corrected C statistic, 0.55; 95% confidence interval, 0.52 to 0.71). CONCLUSIONS: We were unable to predict lung cancer recurrence using a risk-prediction model based on five well-known clinical risk factors and several biomarkers. Further research should consider novel predictors of recurrence to stratify patients with completely resected early-stage non-small cell lung cancer according to their risk of recurrence.

Multispectral Optical Tweezers for Biochemical Fingerprinting of CD9-Positive Exosome Subpopulations.

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.

Pretreatment with bone marrow-derived mesenchymal stromal cell-conditioned media confers pulmonary ischemic tolerance.

OBJECTIVE: Mesenchymal stromal cell-based therapies have demonstrated efficacy in treating a variety of diseases. Despite the potential benefits, there are still significant hurdles that need to be overcome for clinical use. We describe a cell-free-based immunotherapy approach for inducing pulmonary ischemic tolerance by using mesenchymal stromal cell-conditioned media. METHODS: In our well-established lung ischemia-reperfusion model, we pretreated with mesenchymal stromal cell-conditioned media 30 minutes before injury. To determine the degree of lung injury, we assessed for changes in lung vascular permeability, proinflammatory cytokines and cellular infiltrates in bronchoalveolar lavage, and histopathology. Macrophage and T-cell subsets were assessed by immunohistochemistry. RESULTS: Pretreatment with mesenchymal stromal cell-conditioned media conferred protection against lung ischemia-reperfusion injury. This protection is characterized by a significant reduction in proinflammatory cytokines, a decrease in infiltrating inflammatory cells, and increases in M2-like macrophages and regulatory T cells. CONCLUSIONS: Cell-free mesenchymal stromal cell-conditioned media therapy confers pulmonary ischemic tolerance. This therapy uses paracrine factors that provide beneficial protective effects by immunomodulating the inflammatory response in resident and infiltrating cell subsets.

Validating the use of short interfering RNA as a novel technique for cell-specific target gene knockdown in lung ischemia-reperfusion injury.

OBJECTIVE: Short interfering RNA is an effective method for target gene knockdown. However, concerns surround the design, administration, efficacy, specificity, and immunostimulatory potential. Although uptake by alveolar macrophages has been demonstrated, studies have not examined its use in lung ischemia-reperfusion injury. We describe the validation of short interference RNA as a novel technique for cell-specific target gene knockdown in our model of lung ischemia-reperfusion injury. METHODS: Dose-response experiments were performed, and 3 distinct sequences of toll-like receptor-4, toll-like receptor-2, and myeloid differentiation factor-88 short interference RNA were tested for efficacy of knockdown. Saline, lipid vector, and noncoding short interference RNA controls were used. Similar experiments were performed in primary cultures of resident pulmonary cells. Target protein knockdown was assessed by Western blot. Rat serum and cell culture media were assessed for interferon and cytokine production. Biotin labeling was used to assess short interference RNA uptake. RESULTS: Target protein expression was significantly reduced using short interference RNA. However, toll-like receptor-4 knockdown was isolated to alveolar macrophages, and biotin labeling confirmed toll-like receptor-4 short interference RNA localization to alveolar macrophages. There was significant knockdown of toll-like receptor-4 expression in cultured cells treated with toll-like receptor-4 short interference RNA. There was no significant change in interferon production after short interference RNA treatment. There was effective target protein knockdown with each sequence used. CONCLUSIONS: Short interference RNA is a valid method for achieving target protein knockdown in alveolar macrophages and is an important tool in the evaluation of its role in the development of lung ischemia-reperfusion injury.

Vascular endothelial growth factor C complements the ability of positron emission tomography to predict nodal disease in lung cancer.

OBJECTIVE: Vascular endothelial growth factors (VEGFs) C and D are biologically rational markers of nodal disease that could improve the accuracy of lung cancer staging. We hypothesized that these biomarkers would improve the ability of positron emission tomography (PET) to predict nodal disease among patients with suspected or confirmed non-small cell lung cancer (NSCLC). METHODS: A cross-sectional study (2010-2013) was performed of patients prospectively enrolled in a lung nodule biorepository, staged by computed tomography (CT) and PET, and who underwent pathologic nodal evaluation. Enzyme-linked immunosorbent assay was used to measure biomarker levels in plasma from blood drawn before anesthesia. Likelihood ratio testing was used to compare the following logistic regression prediction models: ModelPET, ModelPET/VEGF-C, ModelPET/VEGF-D, and ModelPET/VEGF-C/VEGF-D. To account for 5 planned pairwise comparisons, P values <.01 were considered significant. RESULTS: Among 62 patients (median age, 67 years; 48% men; 87% white; and 84% NSCLC), 58% had fluorodeoxyglucose uptake in hilar and/or mediastinal lymph nodes. The prevalence of pathologically confirmed lymph node metastases was 40%. Comparisons of prediction models revealed the following: ModelPET/VEGF-C versus ModelPET (P = .0069), ModelPET/VEGF-D versus ModelPET (P = .1886), ModelPET/VEGF-C/VEGF-D versus ModelPET (P = .0146), ModelPET/VEGF-C/VEGF-D versus ModelPET/VEGF-C (P = .2818), and ModelPET/VEGF-C/VEGF-D versus ModelPET/VEGF-D (P = .0095). In ModelPET/VEGF-C, higher VEGF-C levels were associated with an increased risk of nodal disease (odds ratio, 2.96; 95% confidence interval, 1.26-6.90). CONCLUSIONS: Plasma levels of VEGF-C complemented the ability of PET to predict nodal disease among patients with suspected or confirmed NSCLC. VEGF-D did not improve prediction.

Differential toll-like receptor activation in lung ischemia reperfusion injury.

OBJECTIVE: The requirement for toll-like receptors (TLRs) in lung ischemia reperfusion injury (LIRI) has been demonstrated but not fully characterized. Previously, we reported that TLR-4 is required by alveolar macrophages but not pulmonary endothelial or epithelial cells for development of LIRI. Additionally, we demonstrated differential patterns of mitogen-activated protein kinase (MAPK) activation and cytokine release in these cell types during LIRI. Here, we sought to determine whether these differences in activation responses are related to cell-specific TLR activation requirements. METHODS: Primary cultures of alveolar macrophages, pulmonary endothelial, and immortalized epithelial cells were pretreated with TLR-2 or TLR-4 short interference RNA (ribonucleic acid) before hypoxia and reoxygenation. Cell lysates and media were analyzed for receptor knockdown, MAPK activation, and cytokine production. Rats were pretreated with TLR-2 or TLR-4 short interference RNA before lung ischemia reperfusion and changes in lung vascular permeability were assessed. RESULTS: Knockdown of TLR-2 in alveolar macrophages did not affect MAPK phosphorylation or cytokine secretion. Conversely, TLR-2 knockdown in pulmonary endothelial and epithelial cells demonstrated significant reductions in extracellular signal-regulated kinase 1/2 activation and cytokine secretion. The lung permeability index in LIRI was decreased by TLR-4 but not TLR-2. CONCLUSIONS: Differential TLR signaling and MAPK activation in response to LIRI seem to be cell specific. Short interference RNA provides an outstanding tool for examination of the underlying mechanism.

Role of toll-like receptor-4 in lung ischemia-reperfusion injury.

BACKGROUND: Toll-like receptor-4 has been implicated in modulating ischemia-reperfusion injury in cardiac, hepatic, renal, and cerebral models. However, its role in lung ischemia-reperfusion injury is unknown. We hypothesize that toll-like receptor-4 has a key role in initiating the inflammatory cascade in lung ischemia-reperfusion injury. METHODS: We used toll-like receptor-4 specific short interference RNA to achieve toll-like receptor-4 knockdown in rats prior to undergoing ischemia and reperfusion. Lungs were explanted and studied for protein expression and markers of lung injury. Additional animals were evaluated for cellular uptake of toll-like receptor-4 short interference RNA. Toll-like receptor-4 short interference RNA localized to the alveolar macrophage. RESULTS: In animals pretreated with toll-like receptor-4 short interference RNA, toll-like receptor-4 expression and mitogen-activated protein kinase phosphorylation were suppressed. Markers of lung injury including permeability index, myeloperoxidase content, and bronchoalveolar lavage inflammatory cell counts were all reduced with toll-like receptor-4 knockdown. CONCLUSIONS: Toll-like receptor-4 is critical in the development of lung ischemia-reperfusion injury and its activation in the alveolar macrophage may be the initiating step.

Simultaneous transplantation of hematopoietic stem cells and a vascularized composite allograft leads to tolerance.

BACKGROUND: We have previously demonstrated that tolerance to a vascularized composite allograft (VCA) can be achieved after the establishment of mixed chimerism. We test the hypothesis that tolerance to a VCA in our dog leukocyte antigen-matched canine model is not dependent on the previous establishment of mixed chimerism and can be induced coincident with hematopoietic cell transplantation (HCT). METHODS: Eight dog leukocyte antigen-matched, minor antigen mismatched dogs received 200 cGy of radiation and a VCA transplant. Four dogs received donor bone marrow at the time of VCA transplantation (group 1), whereas a second group of four dogs did not (group 2). All recipients received a limited course of postgrafting immunosuppression. All dogs that received HCT and VCA were given donor, third-party, and autologous skin grafts. RESULTS: All group 1 recipients were tolerant to their VCA (>62 weeks). Three of the four dogs in group 2 rejected their VCA transplants after the cessation of immunosuppression. Biopsies obtained from the muscle and skin of VCA from group 1 showed few infiltrating cells compared with extensive infiltrates in biopsies of VCA from group 2. Compared with autologous skin and muscle, elevated levels of CD3+ FoxP3+ T-regulatory cells were found in the skin and muscle obtained from the VCA of HCT recipients. All group 1 animals were tolerant to their donor skin graft and promptly rejected the third-party skin grafts. CONCLUSION: These data demonstrated that donor-specific tolerance to all components of the VCA can be established through simultaneous nonmyeloablative allogeneic HCT and VCA transplantation protocol.

Tolerance to vascularized composite allografts in canine mixed hematopoietic chimeras.

BACKGROUND: Mixed donor-host chimerism, established through hematopoietic cell transplantation (HCT), is a reproducible strategy for the induction of tolerance toward solid organs. Here, we ask whether a nonmyeloablative conditioning regimen establishing mixed donor-host chimerism leads to tolerance of antigenic vascularized composite allografts. METHODS: Stable mixed chimerism was established in dogs given a sublethal dose (1-2 Gy) total body irradiation before and a short course of immunosuppression after dog leukocyte antigen-identical marrow transplantation. Vascularized composite allografts from marrow donors were performed after a median of 36 months (range, 4-54 months) after HCT. RESULTS: All marrow recipients maintained mixed donor-host hematopoietic chimerism and accepted vascularized composite allografts for periods ranging between 52 and 90 weeks; in turn, marrow donors rejected vascularized composite allografts from their respective marrow recipients within 18 to 29 days. Biopsies of muscle and skin of vascularized composite allografts from mixed chimeras showed few infiltrating cells compared with extensive infiltrates in biopsies of vascularized composite allografts from marrow donors. Elevated levels of CD3+ FoxP3+ T-regulatory cells were found in skin and muscle of vascularized composite allografts of mixed chimeras compared with normal tissues. In mixed chimeras, increased numbers of T-regulatory cells were found in draining compared with nondraining lymph nodes of vascularized composite allografts. CONCLUSIONS: These data suggest that nonmyeloablative HCT may form the basis for future clinical applications of solid organ transplantation and that T-regulatory cells may function toward maintenance of the vascularized composite allograft.

Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation.

BACKGROUND: It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function. METHODS: The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb. RESULTS: One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells. CONCLUSION: These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.

Mesenchymal stromal cells fail to prevent acute graft-versus-host disease and graft rejection after dog leukocyte antigen-haploidentical bone marrow transplantation.

Mesenchymal stromal cells (MSCs) have been shown to have immunosuppressive effects in vitro. To test the hypothesis that these effects can be harnessed to prevent graft-versus-host disease (GVHD) and graft rejection after hematopoietic cell transplantation (HCT), we administered a combination of 3 different immortalized marrow-derived MSC lines (15-30 × 106 MSCs/kg/day, 2-5 times/week) or third-party primary MSC (1.0 × 106 MSCs/kg/day, 3 times/week) to canine recipients (n = 15) of dog leukocyte antigen-haploidentical marrow grafts prepared with 9.2 Gy of total body irradiation. Additional pharmacological immunosuppression was not given after HCT. Before their in vivo use, the MSC products were shown to suppress alloantigen-induced T cell proliferation in a dose-dependent, major histocompatibility complex-unrestricted, and cell contact-independent fashion in vitro. Among 14 evaluable dogs, 7 (50%) rejected their grafts and 7 engrafted, with ensuing rapidly fatal acute GVHD (50%). These observations were not statistically different from outcomes obtained with historical controls (n = 11) not given MSC infusions (P = .69). Thus, survival curves for MSC-treated dogs and controls were virtually superimposable (median survival, 18 vs 15 days, respectively). Finally, outcomes of dogs given primary MSCs (n = 3) did not appear to be different from those given clonal MSCs (n = 12). In conclusion, our data fail to demonstrate MSC-mediated protection against GVHD and allograft rejection in this model.

A novel monoclonal antibody specific for canine CD25 (P4A10): selection and evaluation of canine Tregs.

A monoclonal antibody (mAb), P4A10, was made to the canine interleukin-2 receptor alpha chain (IL-2Ralpha; p55; Tac antigen; CD25) to facilitate studies of canine regulatory T-cells (Treg). By non-reduced Western blot, P4A10 bound to a 55kDa protein, the size of human IL-2Ralpha. In flow cytometry assays, it reacted with a minor population of circulating dog CD3(+)CD4(+) T-cells and the majority (>60%) of in vitro PMA-Ionomycin (PMA-IO)-activated canine CD3(+) T-cells. P4A10 recognized a hematopoietic cell population enriched for FoxP3+ cells as measured by flow cytometry. The P4A10-selected fractions of T-cells had significantly increased copy numbers of CD25, FoxP3, IL-10, and TGFbeta as detected by RT-PCR (reverse transcriptase-PCR) compared to the negative fractions. The P4A10-selected cells inhibited (3)H (tritiated) thymidine incorporation in a mixed leukocyte reaction (MLR) containing responders of the same origin. P4A10-selected T-cells from fresh peripheral blood mononuclear cells had less FoxP3 (p=0.07) by qRT-PCR (quantitative RT-PCR) and were less suppressive (p=0.01) than in vitro alloantigen-activated Treg. The mAb P4A10 is specific for canine CD25 and can be used to facilitate studies of CD25+FoxP3+ Treg in this clinically relevant large animal model.