scholarly journals Cord-Blood-Stem-Cell-Derived Conventional Dendritic Cells Specifically Originate from CD115-Expressing Precursors

Cancers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 181 ◽  
Author(s):  
Maud Plantinga ◽  
Colin G. de Haar ◽  
Ester Dünnebach ◽  
Denise A.M.H. van den Beemt ◽  
Kitty W.M. Bloemenkamp ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Treatment Success ◽  
Gene Set Enrichment Analysis ◽  
Long Term Memory ◽  
Vaccination Strategies

Dendritic cells (DCs) are professional antigen-presenting cells which instruct both the innate and adaptive immune systems. Once mature, they have the capacity to activate and prime naïve T cells for recognition and eradication of pathogens and tumor cells. These characteristics make them excellent candidates for vaccination strategies. Most DC vaccines have been generated from ex vivo culture of monocytes (mo). The use of mo-DCs as vaccines to induce adaptive immunity against cancer has resulted in clinical responses but, overall, treatment success is limited. The application of primary DCs or DCs generated from CD34+ stem cells have been suggested to improve clinical efficacy. Cord blood (CB) is a particularly rich source of CD34+ stem cells for the generation of DCs, but the dynamics and plasticity of the specific DC lineage development are poorly understood. Using flow sorting of DC progenitors from CB cultures and subsequent RNA sequencing, we found that CB-derived DCs (CB-DCs) exclusively originate from CD115+-expressing progenitors. Gene set enrichment analysis displayed an enriched conventional DC profile within the CD115-derived DCs compared with CB mo-DCs. Functional assays demonstrated that these DCs matured and migrated upon good manufacturing practice (GMP)-grade stimulation and possessed a high capacity to activate tumor-antigen-specific T cells. In this study, we developed a culture protocol to generate conventional DCs from CB-derived stem cells in sufficient numbers for vaccination strategies. The discovery of a committed DC precursor in CB-derived stem cell cultures further enables utilization of conventional DC-based vaccines to provide powerful antitumor activity and long-term memory immunity.

Expansion of human cord blood CD34+CD38−cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 102-110 ◽  
Author(s):  
Craig Dorrell ◽  
Olga I. Gan ◽  
Daniel S. Pereira ◽  
Robert G. Hawley ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Function ◽  
Cultured Cells ◽  
Genetic Manipulation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Large Expansion

Abstract Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2203-2203
Author(s):  
Sandeep Chunduri ◽  
Dolores Mahmud ◽  
Javaneh Abbasian ◽  
Damiano Rondelli
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
T Cell Response ◽  
Solid Organ ◽  
Cd34 Cells

Abstract Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.

Short-Term Ex Vivo Expansion of CD133+ Cells by Co-Culture with Mesenchymal Stem Cells: Role of SDF-1/CXCL12

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3471-3471
Author(s):  
Sarah Vaiselbuh ◽  
Jeffrey Michael Lipton ◽  
Johnson M. Liu
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Human Mesenchymal Stem Cells ◽  
Fold Increase ◽  
Feeder Layer ◽  
Short And Long Term

Abstract CD133 (prominin-1) is the first in a class of novel pentaspan membrane proteins identified in humans and mice, and studies have since confirmed the utility of CD133 as a marker of stem cells with hematopoietic and non-hematopoietic lineage potential. A number of human transplantation studies have documented hematopoietic reconstitution from CD133+ stem cells from mismatched donors, with a suggested advantage over standard grafts in avoidance of graft versus host disease. We have developed a novel hematopoietic culture system (Long-Term Stem Cell Culture or LTSCC) to investigate the potential of human mesenchymal stem cells (MSC) to form stroma that can support short- and long-term hematopoiesis derived from cord blood (CB)-derived CD133+ cells. In addition, we analyzed the effect of stromal derived factor-1 (SDF-1/CXCL12) on survival and short-and long-term colony-forming capacity of CD133+ hematopoiesis. LTSCC induced stroma-like changes in the MSC feeder layer, with adipocyte formation, thought to be needed for formation of stem cell niches, and supported long-term (>9 weeks) survival of CB-CD133+ cells. Cobblestone areas of active CD133-derived hematopoiesis were seen in LTSCC for up to 9 weeks of culture. SDF-1/CXCL12 acted as a survival factor for CB-CD133+ cells and induced a significant ex vivo cell expansion at weeks 3 and 4 of LTSCC (maximal 500-fold increase), while maintaining the capacity for CFU-Mix and BFU-E colony formation up to 7 weeks. Long-term hematopoiesis was assessed by enumeration of long-term culture initiating cells (LTC-IC). When SDF-1/CXCL12 was added to LTSCC, we found a significant increase in LTC-IC: 0.3% (+SDF-1/CXCL12) vs. 0.05% (-SDF-1/CXCL12). Finally, homing capacity, as defined by SDF-1/CXCL12-induced adhesion and migration of CB-CD133+ cells, was maintained and even increased during the first 3 weeks of LTSCC. In summary, MSC can be maintained in LTSCC medium, and this simplified feeder layer is able to provide niches for cobblestone area forming cells derived from CB-CD133+ cells. SDF-1/CXCL12 is critical to support the survival and expansion of CD133+ cells, either directly or indirectly by paracrinesignaled retention of CD133+ cells in contact with specialized MSC niches. We suggest that expansion of CD133+ cells from cord blood may be useful in clinical transplantation limited by insufficient numbers of stem cells.

The Effect of the Aged Niche on Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3844-3844
Author(s):  
Aysegul Verim
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Aged Mice ◽  
Effects Of Aging ◽  
Old Mice

Abstract Abstract 3844 Hematopoietic stem cells (HSCS) have the ability to self-renew and give rise to more HSCs or differentiate to produce all blood lineages. Due to their role in sustaining the hematopoietic system throughout life, it was thought that HSCs might be protected from the effects of aging that have an impact on differentiated cells. However, we and others found that aged HSCs were approximately three fold less active than young HSCs in competitive repopulating assays (Chambers et al. 2007; Plos Biology). Paradoxically, there was an expansion in the HSC pool and the population was more homogenous for stem cell marker expression (Sca-1 and c-Kit). Besides the diminished regenerative capacity of the HSC, there was also a skewing of lineage potential from lymphopoiesis toward myelopoiesis with age (Sudo et al. 2000; J Exp Med). While the effects of aging on HSC have been characterized, limited focus has been spent on what role the surrounding bone marrow environment (stem cell niche) plays in pathways dysregulated with age in the HSC. In order to understand age-related changes in the microenvironment, we performed a cytokine protein array in young versus old mice. From this study, we found an increase in two pro-inflammatory cytokines Mig and Rantes in aged mice, indicating the presence of an inflammatory microenvironment in aged mice. We also found that Rantes expression increases with age in the bone and lineage positive cells of the bone marrow. With enforced expression of Rantes, we saw a significant change in the differentiated progeny of HSCs 16 weeks after transplantation. There were significantly fewer (p<0.05) T cells and more myeloid cells in the Rantes over-expressing group. We also analyzed bone marrow for HSC and progenitor populations after 18 weeks of transplantation and saw significantly more LT-HSCs in the Rantes over-expressing group. We also tested effects of Mig and Rantes on LT-HSCs by ex-vivo treatment followed by functional transplantion assay. We saw the same lineage skewing phenotype after 16 weeks of transplantation (more myeloid cells and significantly less lymphoid cells with Rantes treatment), consistent with the over-expression study. When we transplanted old or young bone marrow cells into either old or young mice and compared either old-into-old with old-into-young, or young-into-young with young-into-old transplants, we observed a significant decrease in T cells and an increase in myleoid cells 16-weeks post-transplant when bone marrow cells were transplanted into old mice. The heterochronic transplantation results demonstrated that an old environment favors myeloid differentiation against T cell differentation. Since enforced expression of Rantes had the same lineage skewing phenotype with those transplants, one possible explanation would be that enforced expression of Rantes or ex-vivo treatment mimicks the old environment and leads to myeloid skewing phenotype. We are now testing Rantes knock-out stem cells for their ability to reconstitute blood. We are expecting to see a better engraftment and more T cells in the peripheral blood. Collectively, Rantes is a key inflammatory cytokine that contributes to myeloid skewing and functional deficits in HSCs of aged mice. Disclosures: No relevant conflicts of interest to declare.

Cord Blood CD34+ Stem Cell Sialyl Lewis X Levels and E-Selectin Binding Are Predictive Of Engraftment In Mice: Functional Separation Of Stemness and Homing To Improve Engraftment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 893-893 ◽  
Author(s):  
Patrick A Zweidler-McKay ◽  
Simon N Robinson ◽  
Michael W Thomas ◽  
JunJun Lu ◽  
Hong Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Sialyl Lewis X ◽  
Sialyl Lewis ◽  
Cord Blood Cd34 ◽  
Selectin Binding

Abstract Background Cord blood (CB) is an increasingly used alternative to bone marrow and mobilized peripheral blood as a source of hematopoietic tissue for transplantation. However, the relatively low cell dose and significantly delayed engraftment when compared to BM and mPB remain significant hurdles. A deficit in the homing of CB hematopoietic progenitor and stem cells (HPSC) to the hematopoietic microenvironment due to suboptimal expression and/or activity of homing molecules is thought to be responsible in part for the delayed engraftment seen with CB in patients. Sialyl Lewis X (sLeX) bearing cells can bind E-selectin, and we have previously reported that ex vivo fucosylation of CB HPSCs with fucosyltransferase VI or VII enhance the rapidity and magnitude of engraftment in mice. Here we explore the engraftment potential of endogenous sLeX bearing CD34+ CB HPSCs to determine if physiologic levels of E-selectin binding predicts engraftment in murine xenografts. Approach CB cells were sorted with CD34 and HECA-452 (anti-sLex) antibodies. sLeX-bearing CD34+ HPSCs (CD34+HECA+) and CD34+ HPSCs lacking sLeX (CD34+HECA-) were compared phenotypically for stem cell and differentiation markers, by gene expression profiling, E/P/L-selectin binding, colony-forming assays, and for multi-lineage engraftment into NOD-SCID-IL2Rg immune-deficient mice. Results Cord blood CD34+HECA+ cells represent 10-20% of CB MNCs and show no significant phenotypic differences from the CD34+HECA- cells in stem cell (CD133, CD90 (Thy-1), CD117 (c-kit), CD143/BB9) and differentiation (CD38, CD33, CD14, CD3, CD19) markers. In agreement, similar percentages of CD34+CD38- and CD34+CD38+ CB cells were found to be HECA+ (18% and 15% respectively, p=0.38), showing no significant bias toward the more immature CD34+CD38- phenotype. mRNA-seq expression analysis revealed relatively few differences in gene expression patterns, although CD34+HECA+ cells express higher levels of the gamma globin genes HBG1 and HBG2, the components of fetal hemoglobin. As predicted, CD34+HECA+ cells demonstrated significantly increased ability to bind recombinant E-selectin in vitro, with no differences in P- and L-selectin binding. Importantly, colony forming assays revealed a small (30%) disadvantage to the CD34+HECA+ cells revealing that the CD34+HECA+ CB cells do not have enriched stem cells activity by CFU assay. However, CD34+HECA+ cells demonstrated significantly higher rate and magnitude of engraftment when compared to CD34+HECA- cells in three independent NSG experiments (Figure 1). Indeed bone marrow, peripheral blood and splenic levels of human hematopoietic cells were consistently 3-5-fold higher in CD34+HECA+ injected mice than in CD34+HECA- injected controls (Figure 2). Multi-lineage engraftment data reveals higher levels of myeloid (CD33+/CD14+), B-lymphocytes (CD19+/CD20+) and platelets (6-14-fold, CD41a+/CD61+) in CD34+HECA+ cells, but interestingly lower levels of T-lymphocytes (CD3+). Finally, secondary transplants had equal magnitude of engraftment, indicating no bias in short- versus long-term HSPCs. Conclusions Data presented here supports the hypothesis that endogenous sLex levels on CD34+ cells is associated with enhanced engraftment rapidity and magnitude but that this is not reflective of an enriched stem cell fraction. Rather it appears to be an indicator of homing to the bone marrow through E-selectin binding. Functional separation of stemness and homing supports the approach to improve CB transplantation through decoration of CB cells with sLex via ex vivo fucosylation (see clinical trial abstract by Popat and Shpall) Disclosures: No relevant conflicts of interest to declare.

Expansion of human cord blood CD34+CD38−cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 102-110 ◽  
Author(s):  
Craig Dorrell ◽  
Olga I. Gan ◽  
Daniel S. Pereira ◽  
Robert G. Hawley ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Function ◽  
Cultured Cells ◽  
Genetic Manipulation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Large Expansion

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Umbilical Cord Blood Transplantation: A New Alternative Option

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 377-383 ◽  
Author(s):  
William Tse ◽  
Mary J. Laughlin
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Hematopoietic Stem Cell ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Alternative Source ◽  
Hematopoietic Stem

Abstract Allogeneic hematopoietic stem cell transplantation is a life-saving procedure for hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. However, wide application of this procedure is limited by availability of suitably HLA-matched adult donors. Umbilical cord blood (UCB) has being increasingly used as an alternative hematopoietic stem cell source for these patients. To date, over 6000 UCB transplant procedures in children and adults have been performed worldwide using UCB donors. Broader use of UCB for adult patients is however limited by the available infused cell dose. This has prompted intensive research on ex vivo expansion of UCB stem cells and UCB graft-engineering including accessory cells able to improve UCB engraftment and reconstitution and for tissue regenerative potential. Recently, two large European and North American retrospective studies demonstrated that UCB is an acceptable alternative source of hematopoietic stem cells for adult recipients who lack HLA-matched adult donors. UCB is anticipated to address needs in both transplantation and regenerative medicine fields. It has advantages of easy procurement, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity.

scholarly journals A novel method for efficient generation of antigen-specific effector T-cells using dendritic cells transduced with recombinant adeno-associated virus and p38 kinase blockade

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Leonardo Mirandola ◽  
Maurizio Chiriva-Internati ◽  
Robert Bresalier ◽  
Lucia Piccotti ◽  
Fabio Grizzi ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Dendritic Cells ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Effector T Cells ◽  
Cell Vaccine ◽  
Ovarian Cancer Cells ◽  
Long Term Memory

Abstract Background The inefficacy of standard therapeutic strategies for ovarian cancer is reflected by the enduring poor prognosis of this malignancy. Due to the potential for exquisite specificity, sensitivity and long-term memory, immunotherapy offers an alternative modality for durable control of the disease, provided appropriate antigens can be identified and presented in the right context. Methods We tested a novel dendritic cell vaccine formulation to reprogram autologous antigen-specific T-cells in vitro, in vivo in a murine model of ovarian cancer, and ex vivo using human cells from patients. Results We show that dendritic cells (DCs) treated with a p38 MAPK inhibitor and transduced with a recombinant adenovirus associated vector (AAV) expressing Sperm protein (Sp) 17 are highly effective in generating antigen-specific T-cell cytotoxic response against ovarian cancer cells. Additionally, these DCs enhanced the differentiation of effector T-cells while reducing the frequency of Foxp3+ T-reg cells in vitro. Conclusions This work provides a rationale for translation of pharmacologically reprogrammed DCs into clinical trials for prevention of tumor recurrence and progression in high-risk ovarian cancer patients.

scholarly journals Safety and feasibility of virus-specific T cells derived from umbilical cord blood in cord blood transplant recipients

2019 ◽  
Vol 3 (14) ◽  
pp. 2057-2068 ◽  
Author(s):  
Allistair A. Abraham ◽  
Tami D. John ◽  
Michael D. Keller ◽  
C. Russell Y. Cruz ◽  
Baheyeldin Salem ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Viral Disease ◽  
Viral Reactivation ◽  
Cell Transplant ◽  
Transplant Recipients ◽  
Cell Recovery

Abstract Adoptive transfer of virus-specific T cells (VSTs) has been shown to be safe and effective in stem cell transplant recipients. However, the lack of virus-experienced T cells in donor cord blood (CB) has prevented the development of ex vivo expanded donor-derived VSTs for recipients of this stem cell source. Here we evaluated the feasibility and safety of ex vivo expansion of CB T cells from the 20% fraction of the CB unit in pediatric patients receiving a single CB transplant (CBT). In 2 clinical trials conducted at 2 separate sites, we manufactured CB-derived multivirus-specific T cells (CB-VSTs) targeting Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV) for 18 (86%) of 21 patients demonstrating feasibility. Manufacturing for 2 CB-VSTs failed to meet lot release because of insufficient cell recovery, and there was 1 sterility breach during separation of the frozen 20% fraction. Delayed engraftment was not observed in patients who received the remaining 80% fraction for the primary CBT. There was no grade 3 to 4 acute graft-versus-host disease (GVHD) associated with the infusion of CB-VSTs. None of the 7 patients who received CB-VSTs as prophylaxis developed end-organ disease from CMV, EBV, or adenovirus. In 7 patients receiving CB-VSTs for viral reactivation or infection, only 1 patient developed end-organ viral disease, which was in an immune privileged site (CMV retinitis) and occurred after steroid therapy for GVHD. Finally, we demonstrated the long-term persistence of adoptively transferred CB-VSTs using T-cell receptor-Vβ clonotype tracking, suggesting that CB-VSTs are a feasible addition to antiviral pharmacotherapy.

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