Neonatal (cord blood) T cells can competently raise type 1 and 2 immune responses upon polyclonal activation

In the neonate, cellular immunity has generally been hypothesized as being incompetent. Accumulating evidence from several recent studies, together with our present report, contradicts this hypothesis. T-helper cell and T cytotoxic type 1 and 2 (Th1/Th2 and Tc1/Tc2, respectively) cytokine responses to polyclonal T cell receptor (TCR) activation were assessed in medium-term cultures of human cord blood T cells using intracellular cytokine staining, which could measure the frequencies of cytokine-producing cells. In this study, we examined the responses of cord blood CD4(+) and CD8(+) T cells in regard to the production of interferon (IFN)-gamma and interleukin (IL)-4 and compared the responses with those obtained from T cells of healthy adults. We found that the responses in cord blood T cells activated with TCR stimulation were comparable to those of their adult counterparts. Moreover, the Th/Tc cells that developed in cord blood were as competent as adult cells for both IFN-gamma and IL-4 secretion. In addition, IL-12 production, which is critical for both Th1 and Tc1 responses, was equally comparable in the two groups. The production of two major cross-regulatory cytokines, tumor necrosis factor-alpha and IL-10, was similarly comparable and not significantly different between the two groups. Taken together, these results indicate that, though naive, the neonatal T cell is competent to respond to TCR-mediated stimulation and to produce both type 1 and type 2 cytokines.     

Refinement in the production and purification of recombinant HCMV IE1-pp65 protein for the generation of epitope-specific T cell immunity

Human cytomegalovirus (HCMV) remains one of the most common opportunistic infections causing disease following stem cell transplantation, despite the availability of anti-viral therapies. Adoptive immunotherapy has the potential to further aid in counteracting chronic viral reactivation and subsequent disease by restoring viral immunity through the transfer of virus-specific T cells from transplant donors to their recipients. Our study refines the production and purification of a recombinant HCMV protein containing two of the most immunodominant antigens (IE1 and pp65) for the generation of polyclonal HCMV-specific T cells. In doing so, a 6x His-tagged IE1-pp65 protein was generated using a serum-free baculovirus/insect cell expression system and soluble IE1-pp65 protein was subsequently purified using Ni-NTA affinity chromatography under stringent conditions to obtain a highly pure product. The ability of the recombinant IE1-pp65 protein to elicit a functional T cell mediated immune response was demonstrated by the vigorous reactivation and expansion of HLA-A2-restricted pp65(495-503)-specific CD8+ T cells. This recombinant IE1-pp65 protein can potentially generate a multitude of HLA-restricted HCMV-specific T cells, providing a better alternative to using costly overlapping peptides or HCMV lysates for expansion of T cells for use in adoptive immunotherapy strategies.     

Group C adenovirus DNA sequences in human lymphoid cells.

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.     

DNA vaccines: influenza virus challenge of a Th2/Tc2 immune response results in a Th2/Tc1 response in the lung

For this study, we used DNA-based immunizations to elicit gamma interferon-producing (Tc1) or interleukin 4 (IL-4)-producing (Tc2) CD8 T cells to the influenza virus nucleoprotein. We examined the response of these cells to an intranasal viral challenge. Both the Tc2- and Tc1-biased responses were present in mice with predominantly IL-4-producing (Th2) CD4 T cells. After viral challenge, Tc1 cells underwent more efficient expansion than did Tc2 cells, and only Tc1 cells were detected at the site of infection. In contrast, the CD4 response remained IL-4 biased. However, only a limited number of CD4 cells appeared in the postchallenge lung, and these were strongly enriched for the Th1 phenotype. Thus, the type of memory T-cell response induced by DNA vaccination does not determine the type of response that will predominate at the site of an infection.     

Activation of Th1 immunity is a common immune mechanism for the successful treatment of hepatitis B and C: tetramer assay and therapeutic implications

Both chronic hepatitis B and C virus (HBV and HCV) infections respond ineffectively to current antiviral therapies. Recent studies have suggested that treatment outcomes may depend on the development of type 1 T helper (Th1) and Th2 cell responses. Specifically, activation of Th1 immunity may play a major role in successfully treating hepatitis B and C. This model was revisited herein by evaluating immune responses in 36 HBV and 40 HCV patients with or without treatment, in an attempt to find a common immune mechanism for successful treatment. The immune responses in all examined cases were studied by peripheral blood mononuclear cell (PBMC) proliferation and cytokine responses to viral antigens, cytotoxic T lymphocyte (CTL) responses, enzyme-linked immunospot (ELISPOT) assay, and tetramer staining of virus-specific CD8+ T cells. The overall results revealed that all responders among both HBV- and HCV-infected cases displayed significantly higher PBMC proliferation to viral antigens with a predominant Th1 cytokine profile. Furthermore, the Th1-dominant responses were associated with significant enhancement of CTL activities and were correlated with ELISPOT data, while non-responders responded more weakly. During therapy, the numbers of tetramer-staining, virus-specific CD8+ T cells showed greater increases in responders than in non-responders (p = 0.001). The frequencies determined by the tetramer assay were approximately 200-fold higher than data estimated by limiting-dilution analysis. In conclusion, activation of Th1 immunity accompanied by enhancement of CTL activity during therapy is a common immune mechanism for successfully treating hepatitis B and C, and therefore may have important therapeutic implications.     

Differential usage of cellular niches by cytomegalovirus versus EBV- and influenza virus-specific CD8+ T cells

Immunological memory provides long-term protection against reinfection or reactivation of pathogens. Murine memory T cell populations may be compressed following infections with new pathogens. Humans have to retain memory T cells directed against a variety of microbes for many decades. Under these circumstances, the effect of pathogens that mount robust T cell reactivity on the pre-existing memory directed against unrelated microbes is unknown. In this study, we studied peripheral blood memory CD8+ T cells directed against different viruses following primary CMV infection in renal transplant recipients. The entrance of CMV-specific CD8+ T cells expanded the Ag-primed CD8+ T cell compartment rather than competing for space with pre-existing memory T cells specific for persistent or cleared viruses. Neither numbers nor phenotype of EBV- or influenza-specific CD8+ T cells was altered by primary CMV infection. CMV-specific CD8+ T cells accumulated over time, resulting in increased total CD8+ T cell numbers. Additionally, they acquired a highly differentiated cytolytic phenotype that was clearly distinct from EBV- or influenza-reactive T cells. Thus, the human immune system appears to be flexible and is able to expand when encountering CMV. In view of the phenotypic differences between virus-specific T cells, this expansion may take place in cellular niches different from those occupied by EBV- or influenza-specific T cells, thereby preserving immunity to these pathogens.     

Characterization of tumor antigen peptide-specific T cells isolated from the neoplastic tissue of patients with gastric adenocarcinoma

Gastric cancer is a significant cause of morbidity and mortality worldwide. Surgical resection remains the primary curative treatment for gastric adenocarcinoma, but the poor (15–35%) survival rate at 5 years has prompted many studies for new therapeutic strategies, such as specific immunotherapy. The aim of this study was to analyze the functional properties of the T cell response to different antigen peptides related to gastric cancer in patients with gastric adenocarcinoma. To this purpose, we have cloned and characterized tumor-infiltrating T cells (TILs) isolated from the neoplastic gastric tissue samples. A T cell response specific to different peptides of gastric cancer antigens tested was documented in 17 out of 20 patients, selected for their HLA-A02 and/or -A24 alleles. Most of the cancer peptide-specific TILs expressed a Th1/Tc1 profile and cytotoxic activity against target cells. The effector functions of cancer peptide-specific T cells obtained from the peripheral blood of the same patients were also studied. The majority of peripheral blood peptide-specific T cells also expressed the Th1/Tc1 functional profile. In conclusion, in most of the patients with gastric adenocarcinoma, a specific type-1 T cell response to gastric cancer antigens was detectable and would have the potential of hamper tumor cell growth. However, in order to get tumor cell killing in vivo, the activity and the number of cancer peptide-specific Th1/Tc1 cells probably need to be enhanced by vaccination with the appropriate cancer antigenic peptides or by injection of the autologus tumor peptide-specific T cells expanded in vitro.     

T-cell potential of human adult and cord blood hemopoietic stem cells expanded with the use of aryl hydrocarbon receptor antagonists

Background aimsExpansion of hemopoietic stem cells (HSCs) in vitro is a potential strategy for improving transplant outcomes, but expansion methods tend to promote differentiation and loss of stem cell potential. Aryl hydrocarbon receptor antagonists (AhRAs) have recently been shown to protect HSC stemness during expansion; however, little is known of the T-cell regenerative capacity of AhRA-expanded HSCs. In this study, we confirm the protective effect of two commercially available AhRA compounds on HSCs from both cord blood (CB) and adult samples and assess the T-lymphocyte potential of the expanded cells.MethodsAdult mobilized peripheral blood and CB samples were purified to CD34⁺ cells, which were expanded in vitro with cytokines and AhRAs. After 14 d, CD34⁺ cells were re-isolated and then grown on in OP9Delta co-culture under conditions that allow T-lymphocyte differentiation. Cells were monitored weekly for T-lineage markers by flow cytometry.ResultsBoth AhRA compounds promoted maintenance of CD34 expression during 2 weeks of proliferation with growth factors, although adult cells proliferated markedly less than CB cells. AhRA-expanded CD34⁺ cells from CB differentiated to T cells on OP9Delta co-culture with the same rate and time course as untreated cells. Adult cells, by contrast, had reduced differentiation to T cells, with donor-dependent variable responses.ConclusionsThis study shows that whereas AhRA treatment is effective in CB samples, expansion of adult HSCs is less successful and reflects their inherent poor potential in T-cell generation.     

Expanded Regulatory T Cells in Chronically Friend Retrovirus-Infected Mice Suppress Immunity to a Murine Cytomegalovirus Superinfection

It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically Friend virus (FV)-infected mice decreased murine cytomegalovirus (mCMV)-specific CD8⁺ T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was found only in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.     

Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-γ production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens.       

Memory and effector T cells modulate subsequently primed immune responses to unrelated antigens

Memory and effector T cells modulate subsequently primed T cell responses to the same antigen. However, little is known about the impact of pre-existing memory and effector T cell immunity on subsequently primed immune responses to unrelated antigens. Here, we show that an antigen-primed first wave of Th1 and Th2 immunity enhanced or inhibited the subsequently primed T cell immunity to an unrelated antigen, depending on whether the second antigen was administered in the same or opposite type of adjuvant. The regulatory effects of the first wave of T cell immunity on the subsequent T cell responses to an unrelated antigen attenuated over time. Notably, following challenge with the second antigen, there was a mutual cross-regulation between the first and second wave of humoral responses to unrelated antigens. Thus, immunization with one antigen not only primes immune responses to that antigen, but also influences subsequently primed immune responses to unrelated antigens.     

CD4+ T cells, including Th17 and cycling subsets, are intact in the gut mucosa of HIV-1-infected long-term nonprogressors

During acute human immunodeficiency virus (HIV) infection, there is a massive depletion of CD4(+) T cells in the gut mucosa that can be reversed to various degrees with antiretroviral therapy. Th17 cells have been implicated in mucosal immunity to extracellular bacteria, and preservation of this subset may support gut mucosal immune recovery. However, this possibility has not yet been evaluated in HIV-1-infected long-term nonprogressors (LTNPs), who maintain high CD4(+) T cell counts and suppress viral replication in the absence of antiretroviral therapy. In this study, we evaluated the immunophenotype and function of CD4(+) T cells in peripheral blood and gut mucosa of HIV-uninfected controls, LTNPs, and HIV-1-infected individuals treated with prolonged antiretroviral therapy (ART) (VL [viral load]<50). We found that LTNPs have intact CD4(+) T cell populations, including Th17 and cycling subsets, in the gut mucosa and a preserved T cell population expressing gut homing molecules in the peripheral blood. In addition, we observed no evidence of higher monocyte activation in LTNPs than in HIV-infected (HIV(-)) controls. These data suggest that, similar to nonpathogenic simian immunodeficiency virus (SIV) infection, LTNPs preserve the balance of CD4(+) T cell populations in blood and gut mucosa, which may contribute to the lack of disease progression observed in these patients.     

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma

Recent data suggest a decreased prevalence of IFN‐γ‐producing T lymphocytes (Type 1 T cells) in tumor‐bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti‐tumor responses.     

HMGB1 and cord blood: its role as immuno-adjuvant factor in innate immunity

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4(+) T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.     

History of the clinical use of umbilical cord blood hematopoietic cells

The first cord blood (CB) transplant was performed in 1988 in a patient with Fanconi anemia. The donor was his HLA-identical sister who was known by pre-natal diagnosis to be HLA identical and not affected by the Fanconi mutation. The CB was collected and cryopreserved at birth. The transplant was successful without GvHD and the patient is currently alive and free of disease more than 15 years after transplant, with full hematologic and immunologic donor reconstitution. At the time of the first transplant, little was known about the biologic properties of CB cells and it was thanks to the pioneering work of H. E. Broxmeyer and E. A. Boyse, who studied the progenitor cell content of CB, and of A. D. Auerbach, who realized the pre-natal diagnosis of Fanconi anemia, that this transplant was possible. Since this first transplant, many questions have been answered but others are still open for further research. For example: would a single CB unit contain enough stem cells to permanently engraft children and adults? Would maternal cell contamination in fetal blood engraft and give severe GvHD? What are the immunologic properties of CB cells? How does it interfere with GvHD, GvL and immune reconstitution? Is the immune immaturity of CB lymphocytes able to overcome the HLA barrier and authorize HLA-mismatched transplants? Is it possible to establish CB banks for unrelated and related transplants? What would be the criteria for collection, quality control and cryopreservation?     

人外周血Th细胞和Tc细胞内IFN-γ及IL-4表达水平与白塞病的关系

目的检测BD患者外周血Th细胞和Tc细胞内INF-γ和IL-4的表达水平,探索BD患者体内的免疫状况。方法采用流式细胞术检测BD活动期患者(n=22),BD稳定期(n=6)以及健康体检者(n=22)外周血T细胞亚群INF-γ和IL-4的表达水平。结果 BD患者活动期外周血细胞Th细胞和Tc细胞经过刺激后胞内合成IFN-γ的水平明显高于正常人水平(Th细胞:22.45%±7.33%vs.16.32±4.96%,P<0.05;Tc细胞:36.74%±13.19%vs.28.67%±11.56%,P<0.05),稳定期患者Th细胞和Tc细胞体外合成IFN-γ的能力与正常人相比无明显差异;稳定期患者Th细胞和Tc细胞胞内合成IL-4的能力相对于正常人均无明显差异:(稳定期Th细胞:3.01%±1.38%vs.2.87%±1.46%,Tc细胞:1.14%±0.28%vs.1.07%±0.32%,P>0.05)。而活动期患者Th细胞和Tc细胞胞内合成IL-4的能力相对于正常人均存在明显差异:(Th细胞:3.54%±1.62%vs.2.87%±1.46%,Tc细胞:1.82%±0.43%vs.1.07%±0.32%,P<0.05);导致Th细胞和Tc细胞有偏向Th1及Tc1的趋势(Th细胞IFN-γ/IL-4:6.34±3.24 vs.5.69±4.72,p>0.05;Tc细胞IFN-γ/IL-4:20.19±13.65 vs.26.79±14.52,P>0.05)。结论 BD患者外周血INF-γ和IL-4的表达水平明显升高,并与疾病活动期存在一定程度相关,可能对白塞病患者的诊断具有重要的临床意义。     

Activation-induced cell death in human T cells is a suicidal process regulated by cell density but superantigen induces T cell fratricide

Repeated ligation of the TCR results in apoptosis (activation-induced cell death; AICD). Superantigens such as Staphylococcal enterotoxin B (SEB) are particularly efficient at inducing AICD in T cells. We investigated whether apoptosis in human T cell subsets was due to fratricide (killing of neighboring cells) or suicide (cell autonomous death). AICD of Th1, Th2, Tc1, and Tc2 effector cells was dramatically enhanced at low cell densities and could be observed in single cell microcultures. AICD was unaffected by adhesion molecules or neighboring cells undergoing AICD, confirming the predominance of a suicidal mechanism. However, SEB was able to induce fratricidal apoptosis of type 1, but not type 2 cells. Fratricide was also observed when unstimulated T cells were exposed to activated Tc1 effector cells. Thus, AICD is tightly regulated to allow clonal T cell expansion and memory cell generation, but superantigens may subvert this process by allowing T cell fratricide.     

Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.     

Fetal immune responses to Plasmodium falciparum antigens in a malaria-endemic region of Cameroon

Plasmodium falciparum infection during pregnancy can lead to the transplacental passage of malarial Ags that are capable of inducing acquired immune responses in the fetus. Studies have identified cytokines produced by malaria-specific cord blood (CB) T cells, but information on fetal B cells is limited. Thus, CB mononuclear cells from 120 Cameroonian newborns were cultured for 7 days in vitro and supernatants were assessed by ELISA for Abs to an extract of malarial schizonts (MA), recombinant apical merozoite Ag 1 (AMA-1), the 42-kDa C-terminal region of merozoite surface protein 1 (MSP-1(42)), a B epitope of ring-infected erythrocyte surface Ag (RESA), and the dominant B epitope of the circumsporozoite protein (CSP). Only 12% of supernatants contained IgM to MA but 78% had IgG to one or more malarial Ags, with 53% having IgG to AMA-1, 38% to MSP-1(42), 3% to RESA, and 0% to CSP. The Abs to AMA-1 and MSP-1(42) were predominantly IgG1 and IgG3. CB mononuclear cells were also tested for the ability to secrete cytokines in response to MA and a pool of conserved MSP-1 T cell epitopes. Among the Ag-reactive samples, 39.3% produced only Th2-type cytokines, whereas 60.6% produced a combination of Th1- and Th2-type cytokines. Although a Th2 bias was observed, the in utero cytokine environment was adequate to support isotype switching to cytophilic IgGs, the isotypes that are protective in adults. Because many infants living in a low transmission area are born with malaria-specific B and T cells, the influence of in utero priming on neonatal immunity merits further investigation.     

Overcoming the barriers to umbilical cord blood transplantation

Umbilical cord blood (UCB) transplantation (UCBT) has seen a marked increase in utilization in recent years, especially in the pediatric population; however, graft failure, delayed engraftment and profound delay in immune reconstitution leads to significant morbidity and mortality in adults. The lack of cells available for post-transplant therapies, such as donor lymphocyte infusions, has also been considered a disadvantage. To overcome the cell–dose barrier, the combination of two UCB units is becoming commonplace in adolescent and adult populations, and is currently being studied in pediatrics as well. In some studies, the use of two UCB units appears to have a positive impact on outcomes; however, engraftment is still suboptimal. A possible additional way to improve outcome and extend applicability of UCBT is via ex vivo expansion. Studies to develop optimal expansion conditions are still in the exploratory phase; however, recent studies suggest expanded UCB is safe and can improve outcomes. The ability to transplant across HLA disparities, rapid procurement time and decreased graft-versus-host disease (GvHD) seen with UCBT makes it a promising stem cell source and, while barriers exist, consistent progress is being made to overcome them.