A major benefit of allogeneic hematopoietic cell transplantation (HCT) in the treatment of malignancies is the graft-versus-tumor (GVT) effect conferred by lymphocytes contained within the graft. However, lymphocytes can also induce the potentially lethal complication of graft-versus-host disease (GVHD). We have previously reported a method of generating large numbers of ex vivo activated and expanded T cells with antitumor activity after culture with interferon-y, cross-linking antibodies to CD3, and interleukin-2. Murine splenocytes expanded under these conditions are a heterogeneous population of which approximately 20% to 60% of cells express natural killer (NK)-cell markers (NK1.1 and DX5) and display major histocompatibility complex (MHC)-unrestricted antitumor activity. Here we demonstrate the in vivo antitumor activity of this population of expanded CD8+ NK-T cells when transplanted across MHC barriers into tumor-bearing hosts. In cotransfer studies with purified allogeneic hematopoietic stem cells, expanded CD8+ NK-T cells confer GVT activity with minimal to no GVHD. In vitro studies show that, although expanded NK-T cells lyse normal allogeneic bone marrow cells, they preferentially mediate cytolysis against tumor targets. These cells persist in the peripheral circulation of host animals for at least 3 weeks posttransfer. GVT activity is dependent on perforin, but not on Fas-ligand. We conclude that expanded CD8+ NK-T cells may serve as a valuable adjuvant population for allogeneic HCT because they mediate GVT effects with minimal GVHD.

Abstract:To determine whether exercise increases endothelial progenitor cells (EPCs) in patients with peripheral vascular disease, we developed a multi-parameter flow cytometry assay to rigorously assess EPCs and mature endothelial cells (ECs) in control subjects and patients with peripheral artery disease (PAD) subjected to graded exercise. Blood was collected from young healthy subjects (n ϭ 9, mean age 33 years), older healthy subjects (n ϭ 13, mean age 66 years), and older subjects with PAD (n ϭ 15, mean age 69 years) before and 10 minutes after exercise. White blood cells were isolated and stained with a five-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33, PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion. Viable, low, side scatter singlets that were CD3-, 19-, and 33-negative were counted. While baseline levels of EPCs and ECs were similar among all subjects, young healthy subjects demonstrated significantly greater (p Ͻ 0.05) levels of progenitor cells (PCs) than older healthy and PAD subjects. Levels of EPCs and ECs tended to increase in all subjects after exercise; however, increases in PCs were only observed in young healthy and PAD subjects. Further, trends in the magnitude of change of subsets with exercise were most similar between young and PAD subjects. Our findings suggest that aging may reduce baseline circulating levels of PCs, but not EPCs or ECs, and that exercise-induced mobilization of subsets may differ depending on age and presence of PAD.

Methods: Blood was collected from young healthy subjects (N = 9, mean age 33 6 8 years), older healthy subjects (N = 13, mean age 66 6 8 years), and older subjects with PAD (N = 15, mean age 69 6 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3, -CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later.Results: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings.Conclusions: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status. q

ImportanceIn patients with severe aortic valve stenosis at intermediate surgical risk, transcatheter aortic valve replacement (TAVR) with a self-expanding supra-annular valve was noninferior to surgery for all-cause mortality or disabling stroke at 2 years. Comparisons of longer-term clinical and hemodynamic outcomes in these patients are limited.ObjectiveTo report prespecified secondary 5-year outcomes from the Symptomatic Aortic Stenosis in Intermediate Risk Subjects Who Need Aortic Valve Replacement (SURTAVI) randomized clinical trial.Design, Setting, and ParticipantsSURTAVI is a prospective randomized, unblinded clinical trial. Randomization was stratified by investigational site and need for revascularization determined by the local heart teams. Patients with severe aortic valve stenosis deemed to be at intermediate risk of 30-day surgical mortality were enrolled at 87 centers from June 19, 2012, to June 30, 2016, in Europe and North America. Analysis took place between August and October 2021.InterventionPatients were randomized to TAVR with a self-expanding, supra-annular transcatheter or a surgical bioprosthesis.Main Outcomes and MeasuresThe prespecified secondary end points of death or disabling stroke and other adverse events and hemodynamic findings at 5 years. An independent clinical event committee adjudicated all serious adverse events and an independent echocardiographic core laboratory evaluated all echocardiograms at 5 years.ResultsA total of 1660 individuals underwent an attempted TAVR (n = 864) or surgical (n = 796) procedure. The mean (SD) age was 79.8 (6.2) years, 724 (43.6%) were female, and the mean (SD) Society of Thoracic Surgery Predicted Risk of Mortality score was 4.5% (1.6%). At 5 years, the rates of death or disabling stroke were similar (TAVR, 31.3% vs surgery, 30.8%; hazard ratio, 1.02 [95% CI, 0.85-1.22]; P = .85). Transprosthetic gradients remained lower (mean [SD], 8.6 [5.5] mm Hg vs 11.2 [6.0] mm Hg; P < .001) and aortic valve areas were higher (mean [SD], 2.2 [0.7] cm2 vs 1.8 [0.6] cm2; P < .001) with TAVR vs surgery. More patients had moderate/severe paravalvular leak with TAVR than surgery (11 [3.0%] vs 2 [0.7%]; risk difference, 2.37% [95% CI, 0.17%- 4.85%]; P = .05). New pacemaker implantation rates were higher for TAVR than surgery at 5 years (289 [39.1%] vs 94 [15.1%]; hazard ratio, 3.30 [95% CI, 2.61-4.17]; log-rank P < .001), as were valve reintervention rates (27 [3.5%] vs 11 [1.9%]; hazard ratio, 2.21 [95% CI, 1.10-4.45]; log-rank P = .02), although between 2 and 5 years only 6 patients who underwent TAVR and 7 who underwent surgery required a reintervention.Conclusions and RelevanceAmong intermediate-risk patients with symptomatic severe aortic stenosis, major clinical outcomes at 5 years were similar for TAVR and surgery. TAVR was associated with superior hemodynamic valve performance but also with more paravalvular leak and valve reinterventions.

Purpose: To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression. Experimental Design: Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived from Ewing's family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFTcell lines was done with either freshly isolated or ex vivo activated and expanded Tcells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging. Results: EFTcell lines express 5-to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines.Treatment of EFTcell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo. In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas). Conclusions: CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.