Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy.

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1 × 1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene ( NR3C1 ) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Allogeneic BK Virus-Specific T Cells for Progressive Multifocal Leukoencephalopathy.

JC virus, the cause of progressive multifocal leukoencephalopathy (PML), and the BK virus are genetically similar and share sequence homology in immunogenic proteins. We treated three immunosuppressed patients with PML with ex vivo-expanded, partially HLA-matched, third-party-produced, cryopreserved BK virus-specific T cells. The immunosuppression in these patients was due to the conditioning regimen for cord-blood transplantation in one patient, a myeloproliferative neoplasm treated with ruxolitinib in another, and acquired immunodeficiency syndrome in the third. After T-cell infusion in two of the patients, alleviation of the clinical signs and imaging features of PML was seen and JC virus in the cerebrospinal fluid (CSF) cleared. The other patient had a reduction in JC viral load and stabilization of symptoms that persisted until her death 8 months after the first infusion. Two of the patients had immune reconstitution syndrome. Donor-derived T cells were detected in the CSF after infusion. (Funded by the M.D. Anderson Cancer Center Moon Shots Program and the National Institutes of Health; ClinicalTrials.gov number, NCT02479698 .).

Cytokines Produced by Dendritic Cells Administered Intratumorally Correlate with Clinical Outcome in Patients with Diverse Cancers.

Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice.Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection.Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01).Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes. Clin Cancer Res; 24(16); 3845-56. ©2018 AACR.

Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplantation.

Relapse has emerged as the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT). To test the hypothesis that natural killer (NK) cells can decrease the risk of leukemia relapse, we initiated a phase 1 dose-escalation study of membrane-bound interleukin 21 (mbIL21) expanded donor NK cells infused before and after haploidentical HSCT for high-risk myeloid malignancies. The goals were to determine the safety, feasibility, and maximum tolerated dose. Patients received a melphalan-based reduced-intensity conditioning regimen and posttransplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. NK cells were infused on days -2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 105/kg to 1 × 108/kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients engrafted with donor cells. Seven patients (54%) developed grade 1-2 acute GVHD (aGVHD), none developed grade 3-4 aGVHD or chronic GVHD, and a low incidence of viral complications was observed. One patient died of nonrelapse mortality; 1 patient relapsed. All others were alive and in remission at last follow-up (median, 14.7 months). NK-cell reconstitution was quantitatively, phenotypically, and functionally superior compared with a similar group of patients not receiving NK cells. In conclusion, this trial demonstrated production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, and was associated with significantly improved NK-cell number and function, lower viral infections, and low relapse rate posttransplant.

Ex Vivo Mesenchymal Precursor Cell-Expanded Cord Blood Transplantation after Reduced-Intensity Conditioning Regimens Improves Time to Neutrophil Recovery.

We previously showed the safety of using cord blood (CB) expanded ex vivo in cocultures with allogeneic mesenchymal precursor cells (MPC) after myeloablative conditioning with faster recovery of neutrophils and platelets compared with historical controls. Herein, we report the transplantation outcomes of 27 patients with hematologic cancers who received 1 CB unit expanded ex vivo with MPCs in addition to an unmanipulated CB (MPC group) after reduced-intensity conditioning (RIC). The results in this group were compared with 51 historical controls who received 2 unmanipulated CB units (control group). The analyses were stratified for 2 RIC treatment groups: (1) total body irradiation 200 cGy + cyclophosphamide + fludarabine) (TCF), and (2) fludarabine + melphalan (FM). Coculture of CB with MPCs led to an expansion of total nucleated cells by a median factor of 12 and of CD34+ cells by a median factor of 49. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 12 days in the MPC group, as compared with 16 days in controls (P = .02). The faster neutrophil engraftment was observed in both RIC groups. The cumulative incidence of neutrophil engraftment on day 26 was 75% with expansion versus 50% without expansion in patients who received FM as the RIC regimen (P = .03). Incidence of neutrophil engraftment was comparable in MPC and control groups if treated with TCF (82% versus 79%, P = .40). Transplantation of CB units expanded with MPCs is safe and effective with faster neutrophil engraftment even after RIC regimens.

Phase I study of cord blood-derived natural killer cells combined with autologous stem cell transplantation in multiple myeloma.

Multiple myeloma (MM) is a disease with known immune dysregulation. Natural killer (NK) cells have shown preclinical activity in MM. We conducted a first-in-human study of umbilical cord blood-derived (CB) NK cells for MM patients undergoing high dose chemotherapy and autologous haematopoietic stem cell transplantation (auto-HCT). Patients received lenalidomide (10 mg) on days -8 to -2, melphalan 200 mg/m2 on day -7, CB-NK cells on day -5 and auto-HCT on day 0. Twelve patients were enrolled, three on each of four CB-NK cell dose levels: 5 × 106 , 1 × 107 , 5 × 107 and 1 × 108 CB-NK cells/kg. Ten patients had either high-risk chromosomal changes or a history of relapsed/progressed disease. There were no infusional toxicities and no graft-versus-host disease. One patient failed to engraft due to poor autologous graft quality and was rescued with a back-up autologous graft. Overall, 10 patients achieved at least a very good partial response as their best response, including eight with near complete response or better. With a median follow-up of 21 months, four patients have progressed or relapsed, two of whom have died. CB-NK cells were detected in vivo in six patients, with an activated phenotype (NKG2D+ /NKp30+ ). These data warrant further development of this novel cellular therapy.

Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method.

BACKGROUND AIMS: Umbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination. METHODS: In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. RESULTS: Using Sepax, we observed higher recovery of total nucleated cells (TNC), CD34+ cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34+ viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 - 175 minutes versus manual method: 180 - 290 minutes). The use of DNAse and MgCl2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. DISCUSSION: We optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system.

Haploidentical Natural Killer Cells Infused before Allogeneic Stem Cell Transplantation for Myeloid Malignancies: A Phase I Trial.

Allogeneic stem cell transplantation is an effective treatment for high-risk myeloid malignancies, but relapse remains the major post-transplantation cause of treatment failure. Alloreactive natural killer (NK) cells mediate a potent antileukemic effect and may also enhance engraftment and reduce graft-versus-host disease (GVHD). Haploidentical transplantations provide a setting in which NK cell alloreactivity can be manipulated, but they are associated with high rates of GVHD. We performed a phase I study infusing escalating doses of NK cells from an HLA haploidentical-related donor-selected for alloreactivity when possible-as a component of the preparative regimen for allotransplantation from a separate HLA-identical donor. The goal of infusing third-party alloreactive NK cells was to augment the antileukemic effect of the transplantation without worsening GVHD and, thus, improve the overall outcome of hematopoietic transplantation. Twenty-one patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or chronic myelogenous leukemia refractory or beyond first remission received a preparative regimen with busulfan and fludarabine followed by infusion of apheresis-derived, antibody-selected, and IL-2-activated NK cells. Doses were initially based on total nucleated cell (TNC) content and later based on CD56(+) cells to reduce variability. CD56(+) content ranged from .02 to 8.32 × 10(6)/kg. IL-2, .5 × 10(6) units/m(2) subcutaneously was administered daily for 5 days in the final cohort (n = 10). CD3(+) cells in the NK cell product were required to be < 10(5)/kg. Median relapse-free, overall, and GVHD-free/relapse-free survival for all patients enrolled was 102, 233, and 89 days, respectively. Five patients are alive, 5 patients died of transplantation-related causes, and 11 patients died of relapse. Despite the small sample size, survival was highly associated with CD56(+) cells delivered (P = .022) and development of ≥ grade 3 GVHD (P = .006). There were nonsignificant trends toward higher survival rates in those receiving NK cells from KIR ligand-mismatched donors and KIR-B haplotype donors. There was no association with disease type, remission at time of transplantation, or KIR content. GVHD was not associated with TNC, CD56(+), or CD3(+) cells infused in the NK cell product or the stem cell product. This trial demonstrates a lack of major toxicity attributable to third-party NK cell infusions delivered in combination with an HLA-compatible allogeneic transplantation. The infusion of haploidentical alloreactive NK cells was well tolerated and did not interfere with engraftment or increase the rate of GVHD after allogeneic hematopoietic transplantation. Durable complete remissions occurred in 5 patients at high risk for disease recurrence. This approach is being further developed in a phase I/II trial with ex vivo-expanded NK cells to increase the NK cell dose with the objective of reducing relapse and improving the outcome of allogeneic hematopoietic transplantation for AML/MDS.

Enforced fucosylation of cord blood hematopoietic cells accelerates neutrophil and platelet engraftment after transplantation.

Delayed engraftment is a major limitation of cord blood transplantation (CBT), due in part to a defect in the cord blood (CB) cells' ability to home to the bone marrow. Because this defect appears related to low levels of fucosylation of cell surface molecules that are responsible for binding to P- and E-selectins constitutively expressed by the marrow microvasculature, and thus for marrow homing, we conducted a first-in-humans clinical trial to correct this deficiency. Patients with high-risk hematologic malignancies received myeloablative therapy followed by transplantation with 2 CB units, one of which was treated ex vivo for 30 minutes with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose to enhance the interaction of CD34(+) stem and early progenitor cells with microvessels. The results of enforced fucosylation for 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. The median time to neutrophil engraftment was 17 days (range, 12-34 days) compared with 26 days (range, 11-48 days) for controls (P = .0023). Platelet engraftment was also improved: median was 35 days (range, 18-100 days) compared with 45 days (range, 27-120 days) for controls (P = .0520). These findings support ex vivo fucosylation of multipotent CD34(+) CB cells as a clinically feasible means to improve engraftment efficiency in the double CBT setting. The trial is registered to www.clinicaltrials.gov as #NCT01471067.

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56(+)/CD3(-)) and less than 1% CD3(+) cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Cord-blood engraftment with ex vivo mesenchymal-cell coculture.

BACKGROUND: Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS: We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS: Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS: Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).

Effect of spaceflight on ability of monocytes to respond to endotoxins of gram-negative bacteria.

Astronauts live and work in relatively crowded, confined environments on the Space Shuttle and the International Space Station. They experience a unique set of stressors that contribute to a diminishment of many immune responses. This study investigated the ability of the shuttle crew members' monocytes to respond to gram-negative endotoxin that they could encounter during infections. Blood specimens were collected from 20 crew members and 15 control subjects 10 days before launch, 3 to 4 h after landing, and 15 days after landing and from crew members during their annual medical examination at 6 to 12 months after landing. When challenged with gram-negative endotoxin, the crew member's monocytes collected at all three time points produced lower levels of interleukin-6 (IL-6) and IL-1beta and higher levels of IL-1ra and IL-8 compared to those of control subjects. Cytokines were assessed by measuring the number of cells positive for intracellular cytokines. These values returned to normal 6 to 12 months after landing, except for IL-1ra, which was still higher (five- to sixfold) than in controls. This phenomenon was accompanied by an increased expression of Toll-like receptor 4 and decreased expression of CD14 on the crew members' monocytes at all time points. There were also increased levels of the lipopolysaccharide binding protein in the plasma of the crew members 3 to 4 h and 15 days after landing. This study shows that spaceflight-associated factors (in-flight and preflight) modulate the response of monocytes to gram-negative endotoxins.

Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing.

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.

Changes in monocyte functions of astronauts.

As part of the systematic evaluation of the innate immune system for long duration missions, this study focused on the antimicrobial functions of monocytes in astronauts participating in spaceflight. The study included four space shuttle missions and 25 astronauts. Nine non-astronauts served as controls. Blood specimens were collected 10 days before launch, within 3h after landing, and again 3 days after landing. The number of monocytes did not differ significantly over the interval sampled in both the astronaut or control groups. However, following 5-11 days of spaceflight, the astronauts' monocytes exhibited reductions in ability to engulf Escherichia coli, elicit an oxidative burst, and degranulate. The phagocytic index was significantly reduced following spaceflight when compared to control values. This reduction in phagocytosis was accompanied by changes in the expression of two surface markers involved in phagocytosis, CD32 and CD64. Levels of cortisol, epinephrine, and norepinephrine after spaceflight did not increase over preflight values.

Changes in neutrophil functions in astronauts.

Exploration class human spaceflight missions will require astronauts with robust immune systems. Innate immunity will be an essential element for the healthcare maintenance of astronauts during these lengthy expeditions. This study investigated neutrophil phagocytosis, oxidative burst, and degranulation of 25 astronauts after four space shuttle missions and in nine healthy control subjects. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch, immediately after landing, and 3 days after landing. The number of neutrophils increased by 85% at landing compared to preflight levels. The mean values for phagocytosis of Escherichia coli and oxidative burst capacity in neutrophils from astronauts on the 5-day mission were not significantly different from those observed in neutrophils from the control subjects. Before and after 9- to 11-day missions, however, phagocytosis and oxidative burst capacities were significantly lower than control mean values. No consistent changes in degranulation or expression of surface markers were observed before or after any of the space missions. This study indicates that neutrophil phagocytic and oxidative functions are affected by factors associated with space flight and this relationship may depend on mission duration.