Following conditioning [chemotherapy and/or total body irradiation (TBI)] and infusion of the UCB graft, there is an initial period of aplasia during which time the donor UCB HSC and early hematopoietic progenitors engraft, differentiate, and proliferate within the BM environment. The total nucleated cell (TNC) dose and/or CD34⁺ cell dose within the UCB unit(s) (per recipient body weight) are important contributing factors to the probability and rate of neutrophil and platelet engraftment (6, 18). UCB grafts contain a lower TNC dose compared to BM and PBSCH and, therefore, the time to neutrophil engraftment (defined as the first of three consecutive days with a neutrophil count >0.5 × 10⁹/l) is prolonged, with a median time of approximately 30 days for UCB, 21 days for BM harvests, and 14 days for PBSCH (19). Similarly, platelet recovery in UCB transplantation is also prolonged with a median time to engraftment (defined as the first of three consecutive days with an unsupported platelet count >20 × 10⁹/l) ranging from 50 to 100 days. Other important factors influencing engraftment are the degree of HLA-matching, the intensity of the conditioning regimen, and the type of immunosuppression used for GvHD prophylaxis (20).

In keeping with allogeneic HSCT using BM or PBSCH, lymphocyte reconstitution following UCB transplantation typically begins with rapid recovery of natural killer (NK) cells (21) (Figure 1). UCB contains both main NK-cell populations found in PB, i.e., CD16⁺CD56^dim and CD16⁻CD56^bright, although differences in their phenotype, maturity, and function have been reported (22, 23). UCB also contains precursor NK-cell populations, including CD16⁺CD56⁻ cells (24). UCB NK cells have lower cytotoxicity, although their function can be enhanced following interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12) or interleukin-15 (IL-15) stimulation (22). They also have lower expression of critical adhesion molecules, e.g., CD2, CD54, and L-selectin, and higher expression of inhibitory receptors including killer-cell immunoglobulin-like receptor (KIR) and NKG2A/CD94 (23). Post-UCB transplant, NK-cell recovery initially occurs through expansion of CD16⁻CD56^bright cells with total NK-cell numbers returning to normal values within the first 3 months (21, 25, 26). Beyond this, UCB transplantation is then associated with an increase in both the absolute number and proportion of CD16⁺CD56^dim cells compared to BM transplantation (27). Total NK-cell numbers may temporarily exceed the normal range seen in the healthy population, possibly through a compensatory expansion that occurs during periods of profound T-cell lymphopenia (28). This pattern has been reported in both children and adults, following single and double UCB transplants [NK cells are derived from the predominant unit in the case of double UCB transplantation] and can last up to 9 months post-transplantation (21, 25, 26).

In allogeneic HSCT, T-cell reconstitution typically occurs in two phases (Figure 1). The first involves early allo-antigen driven homeostatic proliferation of memory T cells, contained either within the graft or, in the setting of T-cell depleted grafts, from residual host T cells escaping pre-transplant conditioning therapy (“thymic-independent”). This, however, produces a restricted T-cell population with limited T-cell receptor (TCR) repertoire against infection. Homeostatic proliferation also occurs faster in CD8⁺ T cells compared to CD4⁺ T cells, producing a reversal of the normal CD4:CD8 T-cell ratio (9, 19). In contrast to BM and PBSCH, UCB mainly contains antigen-inexperienced naïve T cells. Early T-cell reconstitution can therefore only occur via the more stringent in vivo priming, activation, and proliferation of the limited naïve T-cell repertoire contained within the graft. The immaturity of UCB T cells is also associated with reduced effector cytokine expression (IFNγ, TNFα) and reduced expression transcription factors involved in T-cell activation (NFAT, STAT4, and T-bet) (11). Consequently, longitudinal studies of immune reconstitution in UCB transplantation have consistently demonstrated profound early T-cell lymphopenia with impaired functional immunity and limited responses to viral infections, in keeping with a primary immune response (9, 28–30).

For long-term effective immune reconstitution with a broad T-cell repertoire, a second T-cell expansion phase is necessary involving thymic production of new naïve T cells (“thymic-dependent”). Hematopoietic progenitors, produced from the engrafted HSC within the BM, enter the thymus to form early T-cell progenitors (ETPs). During T-cell development in the thymus, double positive thymocytes (CD4⁺CD8⁺) are exposed to self-MHC on the thymic cortical epithelial cells. Only those thymocytes that bind to self-MHC with appropriate affinity will be “positively” selected to continue their development into single positive T cells; CD4⁺ T cells interact with MHC Class II molecules, CD8⁺ T cells interact with MHC Class I molecules. Double positive thymocytes that bind too strongly or too weakly to self-MHC undergo apoptosis. As the thymocytes pass through the thymic medulla they are then exposed to self-antigens presented in association with self-MHC molecules. Thymocytes that bind to self-antigens are removed by “negative” selection, thus preventing the production of autoreactive T cells (31). The presence of naïve T cells with markers of recent thymic emigration, i.e., T-cell receptor rearrangement excision DNA circles (TRECs), usually begins around 3–6 months post-UCB transplant (32, 33). However, the timing and effectiveness of thymopoiesis can be impaired by age-related thymic atrophy and/or thymic damage from conditioning therapy and GvHD. Escalon and Komanduri reported a longer delay in the recovery of thymopoiesis, as measured by TREC, in UCB transplantation compared to other HSC sources, possibly due to the limited dose of lymphoid progenitors within the UCB grafts (30). As a consequence, T-cell reconstitution was delayed with a median time to recovery of approximately 9 months for CD8⁺ cytotoxic T cells and 12 months for CD4⁺ helper T cells (25). Similarly, in a retrospective Eurocord analysis of 63 children transplanted with related and unrelated UCB grafts, the median time to T-cell reconstitution was 8 months for CD8⁺ T cells and 12 months for CD4⁺ and total T cells (21). Factors favoring T-cell recovery were HLA-matched UCB, higher nucleated cell dose, and positive recipient cytomegalovirus (CMV) serology prior to transplantation. Conversely, the presence of acute GVHD delayed T-cell recovery. Interestingly, in a recent Eurocord study of children with severe combined immunodeficiency (SCID) transplanted with either UCB (n = 74) or haploidentical grafts (n = 175), there were no significant difference in T-cell recovery (total T cells and CD4⁺ T cells) between the groups at any time, although the UCB transplant recipients had significantly faster recovery of total lymphocyte counts (34).

Over the last few years, the use of double cord blood transplants has significantly increased, mainly in adults following reduced-intensity conditioning (RIC) regimens. In this setting, although two CBUs are initially transplanted, only one provides prolonged engraftment, i.e., the “predominant” unit. Few studies have reported data on T-cell recovery after double CBT. Ruggeri and colleagues reported outcomes, infection rates, and immune reconstitution after 35 double UCB transplants in recipients with high-risk hematological diseases (25). Lymphocyte subset analyses were performed at 3, 6, 9, and 12 months post-transplant and demonstrated reduced T and B cell counts until 9 months. Recovery of thymopoiesis, as measured by TRECs, was also impaired until 9 months post-transplant. Somers and colleagues also analyzed engraftment kinetics in leukocyte subsets following non-myeloablative double UCB transplants (26). CD4⁺ T cells, CD8⁺ T cells, and NK cells all showed early engraftment predominance by day 11, followed by predominance in myeloid cells by day 18. Based upon these findings, it is proposed that T cells and/or NK cells from the predominant unit may elicit an early immune response against the second unit.

B cell reconstitution occurs over the first 6 months post-UCB transplantation, although full recovery of immunoglobulins takes longer (Figure 1). In 63 children given related or unrelated UCB transplants, the median time to B cell recovery was 6 months (21). PB CD19⁺CD28^highCD38^high transitional B cells are first detectable at 1–2 months post-transplant. Over the next 3–6 months, total B cell numbers gradually rise with an associated increase in the proportion of mature CD19⁺CD28^intCD38^int mature B cells to approximately 90% by 9 months (35). As reported with NK cells, total B cell numbers may also expand above the normal range during the period of T-cell lymphopenia before returning to normal values when T-cell recovery occurs (28). At the same time, levels of immunoglobulins to commonly encountered antigens typically rise to normal levels by the end of the first year. Recovery of B cell function, as measured by the time to discontinuation of intravenous immunoglobulin replacement therapy, was compared following UCB (n = 74) and haploidentical transplants (n = 175) in children with SCID (34). At 3 years after transplantation, 45% of the UCB transplant recipients had discontinued immunoglobulins, compared with 31% of the haploidentical recipients (P = 0.02). Other factors associated with improved B cell function were the absence of pre-transplantation infections and use of myeloablative conditioning (MAC).

In UCB transplantation, the cell dose (TNC dose and/or CD34⁺ dose) and HLA-matching of the graft are important factors for successful engraftment in both pediatric and adult patients (3, 5, 7). Therefore, in 2009, Eurocord published recommendations for selection of CBUs for transplantation (6). In summary, when a single CBU (6/6 or 5/6 HLA-matched) does not contain sufficient number of cells (TNC >2.5 × 10⁷/kg upon freezing; >2.0 × 10⁷/kg on thawing), double cord blood transplantation should be considered, aiming for a combined TNC dose >3.0 × 10⁷/kg. Even higher doses are recommended if the single CBU is only 4/6 HLA-matched (TNC >3.5 × 10⁷/kg upon freezing; >2.5 × 10⁷/kg on thawing for malignant disorders; TNC >4.0 × 10⁷/kg upon freezing; >3.5 × 10⁷/kg on thawing for non-malignant disorders). UCB transplants using two units from different donors were first reported in 2001 by the Minneapolis group in an attempt to increase cell dose infused in adults and older children (46). Both units contribute to early engraftment, although eventually, one unit predominates (47). In an analysis of 23 double UCB transplants following MAC, hematopoiesis was observed from a single donor in 76% patients at day 21 and 100% patients by day 100 (48). Likewise, on review of 81 patients with sustained chimerism after receiving a double UCB transplant using a non-myeloablative regimen, single donor chimerism was detectable in 57, 81, and 100% patients at day 21, 100, and 365, respectively (47). Double UCB transplants show high rates of engraftment (85–100%) with the median time to neutrophil engraftment ranging from 9 to 33 days depending on the conditioning regimen and/or the use of granulocyte colony stimulating factor (GCSF) (26, 47, 49). Interestingly though, a significant difference in the rate of engraftment has not been demonstrated between patients receiving one or two CBUs (47, 50). However, a lower relapse risk was found in patients receiving two CBUs for acute leukemia (CR1/CR2), possibly through an enhanced graft-versus-leukemia (GvL) effect (50). Recently, Ruggeri and colleagues reported the outcomes of 35 double cord blood transplants in recipients with high-risk hematological diseases (25). The cumulative incidence of neutrophil recovery was 86%, acute GvHD 47%, and first viral infection 92%. Immune recovery was delayed with reduced T and B cell counts and compensatory expansion of NK-cells observed until 9 months post-transplant, followed by the appearance of new thymic precursors.

An alternative approach being used to increase cell dose in UCB transplantation is ex vivo expansion of cord blood. As well as increasing the total number of HSC cells for long-term engraftment, this may also increase the number of committed progenitors to reduce the initial period of neutropenia. Ex vivo expanded CB can then be given alone or in combination with an unmanipulated unit. In this setting, although the expanded unit improves early hematopoietic recovery, it is the unmanipulated unit that usually provides long-term engraftment (51). UCB expansion has been achieved using several different methods. The first is liquid culture in which isolated CD34⁺ or CD133⁺ HSC are expanded in the presence of selected cytokines and growth factors, including stem cell factor (SCF), thrombopoietin (TPO), GCSF, and/or FMS-like tyrosine kinase 3 ligand (FLT-3-L) (52, 53). The optimal milieu of cytokines and growth factors remains uncertain but several groups have shown improved expansion by the addition of IL-3 and/or IL-6 (54). Shpall and colleagues performed a feasibility study in which CD34⁺ cells were isolated from a fraction (40–60%) of the CBU and expanded in liquid culture with SCF, GCSF, TPO, and megakaryocyte growth and differentiation factor (52). The remainder of the unit was then infused with the expanded cells following MAC. The median TNC dose infused was 0.99 × 10⁷/kg and the median time to engraftment was 28 days (range, 15–49 days) for neutrophils and 106 days (range, 38–345 days) for platelets. Using a modification to this approach, a Phase I/II trial was performed in which CD133⁺ cells were isolated from a portion of the CBU and expanded in liquid cultures with SCF, FLT-3-L, IL-6, TPO, and the copper chelator TEPA (55). The median TNC fold expansion was 219 (range, 2–260). Both expanded and unexpanded cells were infused with a median TNC of 1.8 × 10⁷/kg. Nine of the 10 patients engrafted with a median time to neutrophil and platelet engraftment of 30 days (range, 16–46 days) and 48 days (range, 35–105 days), respectively. Delaney and colleagues reported results from a Phase I trial using an immobilized Notch ligand Delta-1 in addition to SCF, FLT-3-L, TPO, IL-3, and IL-6 (56). Ten patients with high-risk leukemia were treated with a myeloablative double UCB transplant in which one unit was expanded using this protocol. The average fold expansion was 562 (range, 146–1496) for TNC and 164 (range, 41–471) for CD34⁺ cells. Nine of the 10 patients engrafted with a median time to neutrophil engraftment of 16 days (range, 7–34 days). However, in contrast to other reported studies, there was a predominance for donor CD33⁺ and CD14⁺ cell engraftment from the expanded unit.

The second expansion method uses co-culture with a supporting network of mesenchymal stromal cells to provide a hematopoietic microenvironment that supports HSC proliferation (57). de Lima and colleagues reported the results of 31 patients receiving 2 CBUs, 1 of which was expanded ex vivo with mesenchymal stem cells (MSC). This ex vivo culture system expanded TNC and CD34⁺ cells by a median factor of 12.2 and 30.1, respectively, and the median TNC dose infused was 8.34 × 10⁷/kg (51). Of the 24 patients who received ex vivo expanded cells, 23 achieved neutrophil engraftment, at a median time of 15 days (range, 9–42 days), and 18 had sustained platelet engraftment, at a median time of 42 days (range, 15–62 days). Both compared favorably to 80 CIBMTR historical controls that received unmanipulated double UCB transplants only [neutrophil engraftment 24 days (range, 12–52 days), P < 0.001; platelet engraftment 49 days (range, 18–264 days), P = 0.03]. In addition, while the expanded CBU improved early hematopoietic recovery, in all cases, the unmanipulated unit provided long-term engraftment.

HSC expansion has also been achieved using a continuous perfusion culture system in which cells are supplied with fresh culture media and gaseous exchange (58, 59). In a Phase I study, Jaroscak and colleagues expanded a portion of a CBU using a continuous perfusion culture device and infused these expanded cells 12 days after the remainder of the original unit (58). The median fold increase in TNC was 2.4 (range, 1.0–8.5). Twenty-one of the 26 patients attained neutrophil engraftment with a median time of 22 days (range, 13–40 days). The median time for platelet engraftment was 71 days (range, 39–139 days; n = 16). Ongoing early phase clinical trials of all three methods are in progress.

Another strategy being used to improve engraftment following UCB transplantation is the combined use of cord blood and haploidentical transplants (60, 61). While the haploidentical graft provides early engraftment, it is the UCB graft that usually provides long-term engraftment. Sebrango and colleagues reported the results of 55 combined UCB/haploidentical transplants for high-risk myeloproliferative and lymphoproliferative disorders (60). The maximum cumulative incidence of neutrophil and platelet engraftment was 96 and 78% with a median time to recovery of 10 and 32 days, respectively. Full UCB chimerism was achieved in 50 patients [cumulative incidence 91% (95% CI, 84–99%)] with a median time of 57 days (range, 11–186 days). Immune reconstitution analysis showed NK-cell recovery occurred within 3 months, at a time when patients had dual UCB/haploidentical chimerism. However, B and T cells recovered by 6 months and 1 year, respectively when full UCB chimerism had been attained. Liu and colleagues transplanted 45 patients using a RIC regimen with an unrelated UCB graft and CD34⁺ selected cells from a haploidentical donor (61). The cumulative incidence of neutrophil engraftment was 95% at day 50 with a median time to recovery of 11 days. The cumulative incidence of platelet engraftment was 83% at day 100 with a median time to recovery of 19 days. The median percentage of PB cells of UCB origin was 10, 78, and 95% at day 30, 100, and 180, respectively. Conversely, the median percentage of PB cells from the haploidentical graft was 86, 22, and 2% at the corresponding times. The cumulative incidence of acute and chronic GvHD was 25 and 5%, respectively, with non-relapse mortality (NRM) at 1 year 38%, relapse 30%, and overall survival 55%.

To improve HSC engraftment following UCB transplantation, direct intrabone infusion of cord blood cells is currently being investigated. In a Phase I/II study, 32 consecutive patients with acute leukemia received UCB transplants with intrabone infusion between 2006 and 2008 (62). No complications occurred during administration. The median time to neutrophil and platelet engraftment was 23 days (range, 14–44 days; n = 28) and 36 days (range, 16–64 days; n = 27), respectively, and all engrafted patients showed full donor chimerism from day 60 onward. Sixteen patients were alive and in remission with a median follow-up of 13 months. Okada and colleagues demonstrated in a Phase I study that intrabone infusion of unwashed cord blood following a RIC regimen was also well tolerated (63). In 10 patients, there were no injection related complications and the median time to neutrophil recovery was 17 days. Saglio and colleagues showed that intrabone injection was also well tolerated in children (64). In a recent Eurocord retrospective comparison of single unit intrabone UCB transplants (n = 87) with double unit intravenous UCB transplants (n = 149), intrabone infusion was associated with improved neutrophil engraftment by day 30 (76 vs. 62%, P = 0.014) and improved platelet engraftment by day 180 (74 vs. 64%, P = 0.003). Intrabone infusion was also associated with a lower incidence of acute GvHD and had a trend toward improved disease-free survival (DFS) (65). These results are encouraging and further clinical trials are currently ongoing to evaluate engraftment kinetics and immune reconstitution following intrabone infusion (66).

Factors that promote homing of UCB HSC to the BM niche may also improve engraftment. Stromal-derived factor-1 (SDF-1) (CXCL12) is produced by BM stromal cells and binds to its receptor, CXCR4, on the surface of HSC, pre-B lymphocytes, and T cells. SDF-1 levels are increased following HSCT conditioning and the infused HSC follow the SDF-1 gradient toward the BM niche. Once engrafted, SDF-1 may also promote HSC proliferation and survival (67, 68). Many factors increase the sensitivity of CXCR4 on HSC to SDF-1, including complement (C3a), hyaluronic acid, VCAM-1, fibrinogen, and thrombin. Therefore, ex vivo priming of UCB HSC with these molecules may promote homing and engraftment of the HSC (69, 70). Inhibition of the membrane bound extracellular peptidase (CD26), which cleaves SDF-1, also enhances long-term engraftment in UCB CD34⁺ cells in NOD/SCID/beta 2 microglobulin null mice (71, 72). Fucosylation of ligands expressed on HSC is also required for their interaction with selectins expressed in the BM microvasculature. In NOD-SCID interleukin-2Rγ (null) mice, Robinson and colleagues demonstrated that only fucosylated UCB CD34⁺ were responsible for engraftment and that ex vivo fucosylation improved UCB engraftment rates (73). All these pre-clinical studies show encouraging results and, as such, further investigation is warranted to determine whether these techniques can improve HSC engraftment in clinical UCB transplantation.

Until recently, CBUs for transplantation were selected using cell dose (TNC and/or CD34⁺) and HLA-matching at HLA-A and -B (antigen) and HLA-DRB1 (allele). However, the importance of enhanced HLA-matching has now been recognized. In 2011, Eapen and colleagues retrospectively reviewed the results from 803 single UCB transplants (leukemia/MDS), analyzing the impact of HLA typing at HLA-A, -B, and -C (intermediate resolution) and HLA-DRB1 (allelic) (74). Neutrophil recovery (day 28) was inferior in UCB transplants mismatched at three or more HLA-loci (70, 64, 64, 54, and 44% with zero, one, two, three, and four mismatches, respectively). In addition, TRM was higher when CBU were mismatched at two or more HLA-loci [HR 3.27 (P = 0.006); HR 3.34 (P = 0.005); HR 3.51 (P = 0.006) for two, three, and four mismatches]. Compared to fully matched transplants (8/8), CBU mismatched at HLA-C had higher TRM [HR 3.97 (P = 0.018)]. TRM was also higher in UCB transplants with a single HLA-mismatch at HLA-A, -B, or -DRBI and mismatched at HLA-C compared to transplants with a single HLA-mismatch at HLA-A, -B, or -DRBI but matched for HLA-C [HR 1.70 (P = 0.03)]. Additional matching for HLA-C was therefore recommended. More recently, a joint CIBMTR and Eurocord study analyzed the effect of high resolution (allele) typing at HLA-A, -B, -C, and -DRB1 on the outcomes of 1658 MAC single UCB transplants (75). Neutrophil recovery (day 28) was inferior in transplants mismatched at three or more alleles compared to fully matched CB [odds ratio (OR) 0.56 (95% CI, 0.36–0.88) P = 0.01; OR 0.55 (95% CI, 0.34–0.88) P = 0.01; OR 0.45 (95% CI, 0.25–0.82), P = 0.009 for three, four, and five allelic mismatches]. NRM was also associated with the degree of HLA-mismatching. Single HLA-allele mismatches at HLA-A, -C, or -DRB1 had higher NRM [HR 3.05 (1.52–6.14), P = 0.02; HR 3.04 (95% CI, 1.28–7.20), P = 0.01; HR 2.93 (95% CI, 1.38–6.25), P = 0.005, respectively]. Importantly, CBUs with TNC <3.0 × 10⁷/kg were associated with significantly higher NRM, independent of HLA-matching. Therefore, Eapen and colleagues proposed that single UCB transplants should have a minimum TNC dose of 3.0 × 10⁷/kg. The best HLA-allele matched CBU should then be selected. However, units with three or more HLA-allele mismatches should only be used with caution due to increased graft failure and higher NRM.

In UCB transplantation, screening for donor specific anti-HLA antibodies (DSA) should also be considered. In a retrospective analysis of 386 MAC single UCB transplants performed for hematological malignancies, 89 patients had anti-HLA antibodies, 20 with specificity against the CBU (76). In multivariate analysis, neutrophil and platelet recovery were significantly worse in these 20 patients compared to the antibody negative group [RR 0.23 (0.09–0.56), P = 0.001; RR 0.31 (95% CI, 0.12–0.81), P = 0.02, respectively]. Similarly, in 73 double UCB transplants, the presence of DSA was associated with increased graft failure (5.5 vs. 18.2 vs. 57.1% for none, single, or dual DSA positivity; P = 0.0001) and a longer median time to neutrophil recovery [29 days (any DSA) vs. 21 days (no DSA), P = 0.04] (77). More recently, a retrospective Eurocord analysis on the impact of DSA in 294 RIC UCB transplants was performed. 21% recipients had anti-HLA antibodies of which 14 (5%) had donor specificity. Day 60 neutrophil engraftment (44 vs. 81%, P = 0.006) and 1 year TRM (46 vs. 32%, P = 0.06) were inferior in the presence of DSA (78). In 70 children receiving single UCB transplants, the presence of antibodies to major-histocompatibility-complex Class I-related chain A antigen (MICA) was also associated with delayed platelet engraftment [HR 4.2 (95% CI, 1.02–17.08), P = 0.04] (79). While not all studies have found this association between DSA and engraftment, possibly due to use of lower thresholds for DSA detection, it is recommended that potential recipients be screened for anti-HLA antibodies before UCB transplantation (79, 80). CBUs for which the recipient has high levels of DSA should then be avoided. The full implication of DSA at lower levels remains less clear and further evaluation is required.

The use of RIC regimens in all forms of allogeneic HSCT has increased over the last decade. RIC regimens are less myeloablative but provide sufficient immunosuppression to allow donor engraftment. Disease eradication is then dependent upon the donor-derived T cells recognizing residual tumor as “non-self,” producing an immune mediated graft-versus-tumor (GvT) response (81). RIC regimens have less toxicity and lower TRM, therefore allowing transplantation to be performed in older patients and/or in those with other significant co-morbidities that would otherwise prevent HSCT. RIC regimens are now commonly used in both pediatric and adult UCB transplants (47, 82). However, immune reconstitution following RIC UCB transplantation has not been extensively reported. Geyer and colleagues monitored immune subset recovery in 88 consecutive UCB transplants of which 49 had MAC and 39 had RIC (83). In this series, no significant difference was observed in T, B, or NK-cell recovery or immunoglobulin reconstitution, although the two groups were not evenly matched for other potential confounding factors, e.g., disease type and status at transplant.

To improve engraftment and limit GvHD, many UCB transplant regimens also use in vivo T-cell depletion with anti-thymocyte globulin (ATG). ATG is a polyclonal antibody, prepared in rabbits or horses, raised against thymocyte antigens (84). Although infused into the recipient pre-transplant, the half-life of ATG is such that it reduces T cells in both the recipient and infused grafts, producing profound T-cell depletion and delaying immune reconstitution. Chiesa and colleagues recently published a study measuring early immune reconstitution in 30 pediatric UCB transplants without in vivo T-cell depletion (85). In keeping with T-depleted transplants, NK-cell recovery was rapid with a median time to recovery of 1 month (1–3 months). However, in contrast to ATG-based protocols, T-cell recovery was faster with a rapid “thymic-independent” expansion, broader T-cell repertoire with virus-specific responses, and rapid conversion from naïve to central memory phenotype. In particular, CD4⁺ T-cell recovery was faster with a median count of 0.6 × 10⁹/l at 2 months post-UCB transplant. Although CD8⁺ T-cell recovery was delayed, it was still faster than previously reported for T-depleted UCB transplants. Similarly, B cell reconstitution was also faster. Twenty-nine of the 30 patients engrafted with the median time to neutrophil engraftment of 22 days (range, 13–38 days) and platelet engraftment of 42 days (range, 17–123 days). The cumulative incidence of grade II–IV acute GvHD was 50% but chronic GvHD was relatively low at 14%. In a similar review in adult patients, T-cell reconstitution was measured in 72 double UCB transplants (52 myeloablative and 20 non-myeloablative) without ATG (86). In this group, the median CD4⁺ T-cell count was 0.27 × 10⁹/l at 4 months. Again, CD4⁺ T-cell recovery was faster than that seen in similar series of single and/or double UCB transplants using ATG. Four patients had graft failure and the median time to neutrophil engraftment was 23 days (range, 11–43 days) in the myeloablative group and 11 days (range, 7–36 days) in the non-myeloablative group. The cumulative incidence of acute GvHD at day 100 was 43% (95% CI, 33–56%) and survival at 1 year was 68% (95% CI, 57–79%). Therefore, both studies show promising results with potentially improved immune reconstitution compared to T-depleted UCB transplants although this has to be balanced against the risk of GvHD. In addition, as recognized by the authors, the limitations of comparing results from different retrospective series must be considered and further studies will be necessary.

Mesenchymal stem cells are multipotent undifferentiated stromal cells with capacity to self renew and/or differentiate into mesenchymal cells including chondrocytes, osteocytes, adipocytes, cardiomyocytes, and neurons. They are present in PB, BM, UCB, and non-hematopoietic tissues including fat, muscle, and UC connective tissue, e.g., Wharton’s jelly, although their exact function in vivo remains unclear. MSC are a heterogeneous population that lack hematopoietic markers (CD45/CD34/CD14) but express the antigens SH-3/SH-4 (CD73), Thy-1 (CD90), and Endoglin (CD105) (173). However, there is considerable phenotypic variation between MSC obtained from different sources and there is no universal marker allowing specific isolation of these cells.

In relation to UCB transplantation, MSC have low immunogenicity and potent immunosuppressive function that may be useful for improving engraftment and preventing GvHD. MSC do not express Class II MHC molecules or co-stimulatory molecules and, thus, do not elicit allo-antigenic responses. They can also suppress T and NK-cell proliferation, cytokine secretion, and B cell function (174–176). Possible mechanisms include cell-contact dependent and independent responses including IL-10, TGFβ, nitric oxide, and PGE2 and induction of Tregs (174, 177–179). Pre-clinical murine studies showed that co-transplantation of MSC with UCB CD34⁺ cells in NOD/SCID mice improved engraftment (180–182). In addition, UC MSC supported ex vivo expansion of UCB HSC in long-term cultures (183). In 2009, MacMillan and colleagues performed a Phase I/II study of ex vivo expanded haploidentical BM-derived MSC in pediatric unrelated UCB transplants (184). Eight patients received MSC [median dose 2.1 × 10⁶/kg (range, 0.9–5.0)] in addition to UCB [median TNC 3.1 × 10⁷/kg (range, 2.0–12.4)], with three patients receiving an additional infusion of MSC on day 21. There were no harmful side effects related to infusion of the MSC. All patients achieved neutrophil engraftment at a median time 19 days (range, 9–28 days). Six patients achieved platelet engraftment at a median of 53 days (range, 36–98 days). Rates of engraftment, GvHD, and survival were comparable to equivalent historical group demonstrating the safety and feasibility of this approach. In a similar pilot study, nine patients received MAC followed by UCB transplants with co-infusion of BM-derived MSC and T-depleted HSC from a third-party donor (185). All patients achieved neutrophil engraftment at a median of 12 days (range, 10–31 days) with full CB chimerism at a median of 51 days (range, 20–186 days). The maximum cumulative incidence of platelet engraftment was 88% (95% CI, 70–100%) at a median of 32 days (range, 13–97 days). However, there was no difference in engraftment rates compared to a control group of 46 transplants from the same center not receiving MSC. In addition, they reported no difference in recovery of lymphocyte populations although their data were not published. Bernardo and colleagues reported similar findings in 13 pediatric UCB transplants using paternal MSC with no difference in engraftment or rates of rejection compared to 39 matched historical controls (186). Recently, a Phase I/II study of UCB transplants with UC-derived MSC has been performed (187). Five patients received ex vivo expanded MSC obtained from Wharton’s jelly without any adverse events. Neutrophil engraftment [median 11 days (range, 7–13 days)] and platelet engraftment [median 32 days (range, 22–41 days)] were significantly faster than in nine control patients not receiving MSC. There was no significant difference in total lymphocyte recovery. However, other studies have demonstrated that co-infusion of MSC at the time of UCB transplantation has a negative effect on thymopoiesis and TREC reconstitution, and was associated with reduced survival (188). Therefore, the full implications of co-infusion of MSC with UCB transplantation on engraftment and immune reconstitution require further clarification.

In murine BMT models, MSC have also reduced GvHD, although the timing, dose, and frequency appeared critical as greatest effect was seen when the cells were given into a pro-inflammatory environment with high levels of IFNγ. MSC were first administered to a HSCT patient in 2004 when a 9-year-old boy with refractory GvHD, following a haploidentical transplant, was given BM-derived MSC with complete resolution of symptoms. In 2006, Ringden and colleagues reported a Phase I study in which eight patients with steroid-refractory grade III–IV acute GvHD were given MSC at a median dose of 1 × 10⁶/kg (range, 0.7–9.0) (189). There were no acute effects related to the infusions and six out of eight patients had complete resolution of all symptoms. Overall survival was reported to be better than in a similar group of 16 patients not receiving MSC. In a multi-center EBMT Phase II study, 55 HSCT patients received ex vivo expanded BM-derived MSC for steroid-refractory severe acute GvHD (190). Thirty had a complete response and nine had a partial response with median time from infusion to CR of 18 days (range, 3–63 days). Both TRM and overall survival were better in the complete responders. In a multi-center Phase III study, 244 patients with steroid-refractory grade II–IV acute GvHD were randomized to receive third-party MSC (eight infusions of 2 × 10⁶/kg). For liver and gastrointestinal GvHD, the overall response rate at 28 days was higher in the MSC group. However, no significant difference was found in the rate of durable responses (>28 days). MSC have also been used in first-line treatment of GvHD. In a Phase II study, 31 patients with de novo grade II–IV acute GvHD were randomized to receive third-party MSC at 2 or 8 × 10⁶/kg in addition to standard corticosteroid therapy. Complete responses were seen in 77% patients and 16% showed a partial response although there was no difference between the low and high dose cohorts. To date, few published studies have examined the use of MSC to treat GvHD following UCB transplantation. In the study by Gonzalo-Daganzo and colleagues using third-party MSC and UCB HSC at the time of transplantation, no difference was observed in acute GvHD (185). However, in this study, two patients who developed steroid-refractory GvHD were subsequently treated with therapeutic infusions of MSC and both had complete resolution of symptoms. In Bernardo and colleagues study, using co-transplantation of UCB and parental MSC, those patients receiving MSC had less grade III–IV acute GvHD (0 vs. 26%, P = 0.05) although there was no significant difference for grades II–IV (186). Therefore, the use of MSC to prevent a/or treat GvHD following UCB transplantation may have a role but further investigation is required.

Many cytokines and growth hormones involved in normal thymic function are currently under investigation in pre-clinical studies to determine whether they improve thymopoiesis and immune reconstitution post-HSCT. However, to date, few of these have been specifically tested in the setting of UCB transplantation.

Interleukin-7 is a 25-kDa glycoprotein growth factor produced by the BM stroma and thymic epithelial cells (TEC) and is important for T-cell development. IL-7 binds to its cognate receptor (IL-7R) on immature thymocytes, promoting their differentiation and proliferation into immature T lymphocytes. Mutations of the IL-7 gene or IL-7R produce severe immunodeficiency syndromes (191). In murine allogeneic BMT models, IL-7 increased thymopoiesis and peripheral expansion of recent thymic emigrants and mature T cells, as well as increasing B cells, NK cells, NKT cells, monocytes, and macrophages (192–194). Importantly, in these studies, GvHD was not exacerbated although GvL was maintained (192). However, other similar models have shown increased GvHD using IL-7 (195). In non-BMT human studies, administration of rhIL-7 increases CD4⁺ and CD8⁺ T cells with a preferential expansion of naïve T cells and an increased TCR repertoire (196, 197). More recently, Perales and colleagues reported results from a Phase I trial of rhIL-7 (CYT107) after T-cell depleted allogeneic HSCT (198). Twelve patients were treated with escalating doses of rhIL-7 (10–30 μg/kg). IL-7 produced an increase in effector memory T cells with an associated increase in viral specific T cells and enhanced TCR diversity.

Interleukin-2 is a 15-kDa soluble cytokine produced by activated T cells and is critical for their differentiation and proliferation into effector cells. It also promotes proliferation and expansion of B and NK cells and increases cytotoxic activity and production of effector cytokines. IL-2 binds via its cell surface receptor containing the IL-2Rα subunit (CD25), IL-2Rβ subunit (CD122), and the common gamma chain (CD132). As with IL-7, deficiency in IL-2 or its receptor leads to profound immune dysregulation with chronic infection and severe autoimmunity (199). In HSCT, several groups have shown the IL-2 administered after chemotherapy or DLI may reduce relapse and increase responses in refractory disease, possibly through an enhanced GvL response (200, 201). However, low dose IL-2 produces enhanced Treg proliferation with increased thymic Treg output while having little effect on conventional T cells (202). Therefore, such strategies may improve T-cell homeostasis post-HSCT, preventing GvHD and/or thymic damage. Clinical trials using IL-2 in T-cell depleted double UCB transplants for refractory AML are currently in recruitment (203).

Interleukin-15 is a 14- to 15-kDa glycoprotein belonging to the same family as IL-2 and IL-7. It is expressed by monocytes, macrophages, and dendritic cells and binds to the IL15Rα subunit, IL-2/15Rβ subunit (CD122), and the common gamma chain (CD132). Functionally, it causes proliferation of T cells, B cells, and NK cells and is the primary survival growth factor for NK cells. In murine BMT models, post-transplant IL-15 administration increased donor-derived CD8⁺ T cells, NK cells, and NKT cells with enhanced NK and T-cell function (204). IL-15 was able to enhance the GvL effect but also increased GvHD in T-replete BMT.

Sex steroids have important effects on myeloid and lymphoid recovery following HSCT. The sex steroid hormones (androgen, estrogen) impair thymic function and cause apoptosis in thymic stromal cells and developing thymocytes following puberty. Conversely, reducing sex steroid levels, either via castration or by modifying the hypothalamic–pituitary–gonadal axis, increases thymopoiesis by reducing the rate of apoptosis and increasing the proliferation of TECs. When a luteinizing hormone releasing hormone agonist (LHRHa) is administered in a continuous fashion at high doses, LHRH receptors become desensitized and there is a subsequent reduction in the production of follicle stimulating hormone (FSH) and luteinizing hormone (LH). In turn, this leads to a reduction in the production of sex steroids (205, 206). Using this approach in a murine BMT model produced a significant increase in myeloid and lymphoid progenitors and enhanced thymic reconstitution with increased peripheral T cells (205). GvHD was not increased but GvL was maintained. Furthermore, combined use of keratinocyte growth factor (KGF) and LHRHa increased thymopoiesis with enhanced reconstitution on naïve CD4⁺ and CD8⁺ T cells, reduced homeostatic expansion, and produced a broader T-cell repertoire (207).

Growth hormone (GH) and its mediator insulin-like factor-1 (IGF-1) may also improve thymopoiesis. GH is a 22-kDa protein produced by the pituitary gland, under the control of the hypothalamus via the production of growth hormone releasing hormone (GHRH) and growth hormone inhibiting hormone (GHIH). It is also produced by thymocytes and TEC and acts locally with its corresponding receptor (GHR) in an autocrine fashion. GH induces proliferation and cell growth within the thymus and increases thymic size, cellularity, and TCR repertoire when administered to GH-deficient mice (208). It also accelerates T-cell and immune recovery when given in murine T-depleted HSCT (209). Furthermore, GH may also protect against the effects of radiation, increasing production of HSC and promoting recovery of leukocytes, T, B, and NK cells in murine models (210). IGF-1 is a 7-kDa protein, which is mainly produced by the liver in response to GH but is also produced by other cell types in an autocrine fashion. IGF-1R is expressed on thymocytes, T cells, and TECs and binding of IGF-1 to its receptor increases the proliferation of thymocytes and peripheral T cells (211). Administration of IGF-1 in murine HSCT models also increased in vivo thymic precursor populations, donor-derived T cells as well as pro-, pre-, and mature B cells, and myeloid cells (212).

Keratinocyte growth factor is a 28-kDa protein produced by mesenchymal stromal cells and mature thymocytes. Several groups have shown that KGF protects mucosal, cutaneous, and epithelial cells from cytotoxic and irradiation induced injury (213, 214). In murine BMT models, pre-treatment with KGF protected the TECs and increased donor-derived thymocytes, peripheral naïve T cells, and production of IL-7 (215). KGF induces p53 and NF-kappa pathways in immature TECs, promoting proliferation and differentiation into mature TECs (216). In a murine UCB transplant model, KGF pre-treatment increased day 35 thymic outputs with higher T-cell and NKT-cell numbers within the spleen and increased the proportion of TREC (217). However, in human clinical trials, although KGF (Palifermin) reduced mucositis, it has not been shown to have any significant impact on lymphocyte reconstitution, GvHD, infectious complications, or overall survival (218–221).

Finally, other cellular pathways currently being investigated to improve thymopoiesis post-HSCT are the tyrosine kinases, Flt-3 and c-Kit, and the tumor suppressor gene, p53. FLT-3-L, produced in the thymus and expressed on the surface of perivascular fibroblasts, is upregulated following irradiation and increases proliferation of Flt-3 positive thymic precursors (222). Flt-3-L also promotes thymocyte maturation and homeostatic expansion of peripheral T cells following HSCT (223). Stem cells factor (SCF) also promotes early thymocyte development through its receptor tyrosine kinase, c-Kit. Pre-clinical studies in mice using the tyrosine kinase inhibitor, Sunitinib, suggest that pre-treatment of the recipient may promote improved donor-derived thymopoiesis by blocking c-Kit in the host’s early thymic progenitors and, thus, improving accessibility to the thymic niche (224). p53 is a tumor suppressor gene that activates DNA repair, arrests cell growth, and induces apoptosis in response to cell damage. Given the degree of epithelial damage following conditioning chemotherapy and/or irradiation, it has been proposed that temporary inhibition of p53 may reduce thymic apoptosis and promote T-cell recovery following HSCT. In murine HSCT models, Kelly and colleagues demonstrated that temporary inhibition of p53 using the small molecule pifithrin-β (PFT-β) prevented damage in TECs (225). Moreover, when combined with KGF, thymic function was improved post-HSCT with higher numbers of donor-derived naïve CD4⁺ and CD8⁺ T cells and enhanced responses against Listeria monocytogenes.