Umbilical Cord Blood Transplantation Supported by Third Party Donor Cells: Rationale, Results and Applications

Abstract

Low incidence of GVHD provides the major rational for pursuing UCB stem cell transplant (UCB SCT). Considerable evidence also suggests a lower rate of recurrence after UCB SCT than after transplantation from adult donors. Recent advances in understanding of the human fetal immune development provide a rational underpinning for these clinical outcomes. The fetal immune system is geared toward maintaining tolerance to foreign antigens, particularly to the maternal antigens to which it is exposed throughout gestation. To this purpose it is dominated by a unique population of peripheral T regulatory cells which actively maintain tolerance. This and other features of the UCB lymphoid system explains the low incidence of GVHD and superior outcomes of UCB SCT with NIMA (non-inherited maternal antigens)-matched grafts. At the same time, highly sensitized maternal microchimeric cells are frequently detected in UCB and likely contribute to superior GVL effects and low rates of disease recurrence in IPA (inherited paternal antigen) matched UCB recipients.

But historically erratic and slow hematopoietic recovery after UCB SCT leads to increased early morbidity and mortality, excessive hospitalization and costs. This has held up the widespread utilization of UCB SCT in adults. Here we summarize recent data on UCB SCT with an emphasis on studies of co-infusion of adult CD34 selected hematopoietic stem cells with UCB SCT. This procedure, through transient engraftment of adult hematopoietic stem cells largely overcomes the problem of delayed engraftment. We also briefly discuss unresolved issues and possible future applications of this technology.

Introduction

Attempts at allogeneic transplantation were reported as early as 1957 (and among the small group of early recipients, at least one received fetal rather than adult bone marrow).¹ These early attempts faltered because of graft rejection and GVHD. It was not until the discovery of the HLA system and the recognition of its pivotal role in GVHD and graft acceptance that allogeneic transplantation became a feasible procedure.² Initially restricted to HLA-identical sibling pairs, it was rapidly expanded to unrelated donor transplantation. Refinements in HLA-typing over the past decade have led to the recognition that HLA-identical donorsare lacking for many.³ It is estimated that only 20% of African Americans, approximately 70% of Caucasians and intermediate percentages of subjects of other ethnicities have access to HLA-identical unrelated donors.⁴ The development of transplant methods that obviate the need for HLA-identity is, therefore, imperative. UCB (UCB) transplantation has considerable promise, because of its tolerance to the host environment and its potent GVL effects, features that will be briefly summarized in the light of current understanding of the fetal immune system. When supported by co-infusion of third party cells, hematologic reconstitution after UCB SCT is fast and reliable, removing one of the largest hurdles to successful UCB SCT.

Biological characteristics of UCB and implications for transplantation

The cellular composition of the UCB graft reflects the functional status of the fetus at full term gestation. UCB contains lymphoid and dendritic cells a well as cells of hematopoietic lineages.^5;6 In addition, many if not all, UCB units contain variable percentages of cells of maternal origin, a phenomenon called maternal microchimerism.^7–11 Important studies over the past years (reviewed by Mold and Mc Cune)^12;13 have led to a new understanding of the organization and function of the human fetal immune system.

The fetal immune system is geared toward tolerance to foreign antigens

From an immunological standpoint, pregnancy represents an extraordinary situation in which both fetus and mother are exposed to an immunologically foreign organism.¹² In this process, the fetus develops profound and long lasting tolerance to antigens to which it was exposed during gestation. Owen was the first to observe this, reporting on the immunological behavior of Freemartin cattle, genetically females, but in whom there is lifelong male chimerism due to transmission of cells from a male twin during pregnancy.¹⁴ Subsequent reports on tolerance to tissue grafts between fraternal twins, both in humans and other large mammals, established that tolerance to MHC antigens can occur upon intrauterine exposure between fraternal twins who share an intrauterine blood supply.¹² Owen was again the first to show that tolerance to non-inherited maternal antigens can occur during gestation when he studied the effects of fetal exposure to Rhesus antigens.¹⁵ During pregnancy, Rhesus negative women often develop anti-rhesus antibodies, the cause of hemolytic disease of their newborns. But those women who were daughters of rhesus positive mothers had been rendered tolerant during fetal life and rarely developed Rhesus antibodies during their own pregnancies. Claas et al extended this concept in a landmark 1988 paper when they reported that heavily transfused and multiply sensitized adults failed to raise antibodies to foreign HLA antigens that were present in their own mother (the non-inherited maternal antigen or NIMA). Presumably they had been rendered tolerant during fetal life to these antigens.¹⁶

The mechanisms of fetal tolerance remained long poorly understood and were attributed to immaturity of the fetal immune system.^17;18 But recent evidence has shown that the fetal immune system undergoes extensive development during gestation with waves (or so-called “layers”) of cells of different phenotypic profile and/or function succeeding each other.¹³ (Figure 1) This model of layered immune system development is reminiscent of the better known model of development of the erythroid system where early embryonic cells are replaced by cells containing fetal hemoglobin followed by another switch at the time of delivery toward adult hemoglobin development.¹⁹

Figure 1. Model for transition from fetal to adult hematopoiesis (reproduced from Mold and Mc Cune¹³).

Model for transition from fetal to adult hematopoiesis. Several potential scenarios could account for the layering of the adaptive immune sysytem during development. Based on the linear decline in the frequency of Treg cells in umbilical cord blood across the third trimester of development, we propose that a shift in HSC identity from fetal to adult occurs at some point during this time. Whether this occurs through (i) the de novo generation of adult HSC from an upstrean progenitor cell or (ii) from a direct conversion of fetal HSC to adult-type HSC remains unknown. Examples of different hematopoietic lineages that have been shown to arise during fetal development and after birth are listed below the figure. HSPC, hematopoietic stem and progenitor cells.

A complete description of this rapidly developing area is well beyond the scope of this review but recent data with potential relevance to UCB SCT are summarized below. The two best studied cell populations in this regard are CD4 T cells as well as B1 type B cells. T regulatory cells, CD4+,CD25+,FoxP3+ cells dominate the fetal immune system during mid gestation, with numbers declining toward adult levels by the time of delivery.^20,5;21 These T regulatory cells are thought to be essential in the development of fetal tolerance to maternal cells.^12;22 Recent work has shown that although fetal T reg cells are phenotypically similar to adult cells, they are functionally quite different and have a unique gene expression profile. Similarly, CD4+, CD25 negative fetal T-cells are functionally different from their adult counterparts. Complex transplant experiments support the model of fundamentally different T-cell populations in the fetus and the adult.²³ How many fetal T-cells persist at the time of delivery and possibly, even throughout childhood is still unknown. But it is likely that the powerful suppressive effect of fetal T-reg cells contributes at least partially to the suppression of GVHD reactions after UCB SCT.

B cell immunogenesis is similarly characterized by successive generations of different B-cell types. In mice two types of B cells are recognized, labeled B1 and B2 cells. B1 cells (CD5+ in mice) predominate during neonatal life and appear to derive from unique precursors.¹³ B cells in human UCB constitute about 11% of lymphocytes (as opposed to about 4% in adults) and a very high percentage of these B cells are CD5+ and CD27+.^24;25 These CD27+ cells in human UCB have recently been identified as the counterpart of murine B1 cells. ²⁵ B1 cells are important in innate immunity through secretion of “natural Ig” and through their ability to stimulate T cells. It is likely that they contribute to innate resistance to infections.²⁵ The rapid proliferation of CD5+ B cells after UCB SCT may therefore be an important, though still incompletely studied component of effective immune reconstitution.

Less is known about the ontogenesis of other components of the fetal immune system. Umbilical cord dendritic cells are hyporeactive upon stimulation with limited up-regulation of surface receptors, limited signaling and a bias against inducing CD4 Th1 responses.^5;12;26 The preferential Th2 responses may further diminish GVHD. Fetal NK cells have reduced function and cytolytic activity compared to adult NK cells.⁵

Tolerance induction is therefore the hallmark of the fetal immune system and at least partial persistence of the fetal immune system in the full term umbilical blood contributes to the unusually low rate of GVHD after UCB stem cell transplantation despite significant HLA disparity and to the tolerance to NIMA antigens that has recently been shown to occur after UCB SCT as it was previously shown in solid organ transplant. ²⁷ In an initial preliminary report, Van Rood et al had access to a set of UCB SCT recipients with complete HLA typing of the infant donor of the UCB unit, and of the mother of the infant. ²⁸ Most of the UCB recipients were HLA-mismatched with their donors at one or several antigens. In some cases, the HLA mismatch was identical to an HLA antigen present in the mother of the infant donor (NIMA match). In other cases, the mismatched HLA antigen was not present in the donor’s mother (no-NIMA match). (example, see Table 1A) They found that treatment related mortality and overall mortality was significantly lower in the recipients of a NIMA matched transplant, particular among recipients older than ten years of age. Acute and chronic GVHD were reduced among NIMA-matched transplant recipients but this reduction was not statistically significant. Neutrophil engraftment was more rapid among NIMA matched recipients and somewhat surprisingly there was also a trend toward lower relapse rate in the NIMA matched patients. The effect of NIMA matching on transplant related mortality was recently confirmed in a study where the outcome of 48 NIMA matched UCB SCTs were compared with that of 116 non-NIMA matched UCB SCTs in a case-control fashion. The groups were matched for important covariates. No effect of NIMA matching on relapse rate, acute or chronic GVHD or neutrophil recovery was found in this more recent analysis. But TRM was lower after NIMA-matched UCB SCTs compared with NIMA-mismatched UCB SCTs (relative risk, 0.48; P =.05; 18% versus 32% at 5 years). Consequently, overall survival was higher after NIMA-matched UCB SCT. The 5-year probability of overall survival was 55% after NIMA-matched UCB SCTs versus 38% after NIMA mismatched UCB SCTs (P = .04). Of note, the importance of NIMA matching may also extend to the setting of adult haplo-identical transplantation. Two groups have shown that transplant from a NIMA matched haplo-identical donor is associated with improved outcome mainly because of a lower incidence of graft vs host disease.^29;30

Table 1A.

Examples of NIMA match vs NIMA Mismatch

Recipient	A1	A24	B7	B65	DR0102	DR1501
NIMA MATCH
CB unit 1	A2	A24	B7	B65	DR0102	DR1501
Mother of Unit1	A1	A24	B57	B65	DR0102	DR1305
NIMA MISMATCH
CB unit 2	A2	A24	B7	B65	DR0102	DR1501
Mother of Unit 2	A3	A24	B7	B65	DR0102	DR1501

In this example two identical 5/6 HLA matched CBU are evaluated for use in a particular recipient. The mismatch between UCB recipient and donor is in the HLA A locus. UCB recipient HLA A1 and CBU HLA A2.

Case 1 NIMA match:The mother of CBU1 happens to have HLA A1. During gestation CBU was therefore exposed to HLA A1 and has become tolerant to NIMA HLA A1.

Case 2 NIMA mismatch:The mother of CBU2 does not have HLA A1. This CBU was therefore never exposed to HLA A1 and has not become tolerant to it.

All things being equal, transplant with CBU1 is expected to have superior outcome.

Maternal Microchimerism in the UCB may be responsible for GVL

Several observations point to a reduced rate of disease recurrence after allogeneic cord blood transplantation compared to adult related or unrelated donor transplantation. This was initially shown in double UCB SCT, which has been shown to have lower recurrence rates compared to adult transplant. ^31;32 (though the benefit of reduced rates of recurrence was largely offset by increased early TRM after UCB SCT). In some studies similar low relapse rates have been observed after single UCB SCT.^31;33 Some have attributed the increased GVL effects after UCB SCT to increased mismatching.^34;35 Indeed, preliminary evidence in children shows that more mismatching decreases the risk of disease recurrence. This mechanism could explain why relapse rates after UCB SCT are lower than those after transplant from HLA-identical donors. But haplo-identical adult grafts are even more HLA mismatched than UCB. Yet, in several studies, haplo-identical SCT is associated with higher rates of disease recurrence.^37;38 Others have proposed that Killer-cell immunoglobulin-like receptor (KIR)-ligand mismatching after UCB SCT may be associated with reduced relapse risk.³⁹ While this may be correct, KIR antigen mismatching is also frequent in matched related and unrelated donor SCT and KIR mismatching does not explain the differential relapse rates with these graft sources.

More recently Van Rood et al proposed a hypothesis based on the observation of frequent maternal microchimerism in the graft.⁴⁰ It has been known for decades that maternal cells can cross the placenta and colonize the fetus and vice versa(for review see Mold and Mc Cune).¹² These cells are most prevalent during mid gestation but can also be frequently detected in UCB. In contrast to the fetal immune system that invariably becomes tolerant to non-inherited maternal antigens, the maternal immune system becomes sensitized to the paternal HLA-antigens inherited by the fetus (the inherited paternal antigens or IPA). This phenomenon is the frequent cause of anti-HLA antibodies in parous women that can complicate platelet transfusions and contribute to graft rejection in stem cell and organ transplant.^41–43 Since sensitized maternal cells are present in virtually all UCB, van Rood et al reasoned that the small number of maternal cells rather than the majority fetal lymphoid cells are primarily responsible GVL effects after cord blood transplant. In an ingenious analysis, they compared the outcome of two sets of patients undergoing UCB SCT. In the large majority of cases, there was an HLA-mismatch between the mother of the UCB donor and the transplant recipient to which the mother was sensitized. They labeled this donor-recipient combination “shared IPA transplants”. In a minority of cases, labeled “no-shared IPA transplants” there was no IPA target in the recipient. This can happen when father and mother of the fetus share an HLA-haplotype; (example see Table 1B) this fetus can be HLA-identical or nearly HLA-identical to the mother. In other cases, the HLA- mismatch between recipient and cord blood was such that the IPA targets were absent in the recipient. The recurrence rate was significantly lowerin shared IPA transplants than in no-shared IPA transplants, presumably because of the anti-IPA mediated GVL effects in the IPA transplants. This study, while retrospective and with several limitations, provides a powerful and rational explanation for maternally mediated GVL effects in UCB SCT. It will be up to future investigators to prospectively validate these observations. Interestingly, in pediatric recipients of parental haplo-identical T-cell depleted SCT, there is a much better outcome in patients receiving SCT from the mother compared to those receiving paternal SCT, probably due to the fact that mothers have been previously sensitized to the child’s inherited paternal antigens.⁴⁴

Table 1B.

Example of IPA targeting vs no IPA targeting by maternal microchimerism.

Recipient	A1	A24	B7	B65	DR0102	DR1501
IPA Targeted
CB unit 1	A2	A24	B7	B65	DR0102	DR1501
Mother of Unit1	A1	A24	B57	B65	DR0102	DR1305
NOT IPA Targeted
CB unit 2	A2	A24	B7	B65	DR0102	DR1501
Mother of Unit 2	A3	A24	B7	B65	DR0102	DR1501

In this example the same identical 5/6 HLA matched CBU are evaluated for use in the same recipient. The mismatch between UCB recipient and donor is in the HLA A locus. UCB recipient HLA A1 and CBU HLA A2.

Case 1 IPA targeting: During gestation microchimeric maternal cells of CBU1 were sensitized to paternal HLA B7 and HLA DR 1501. These same IPA antigens are present in the recipient and are therefore targets of GVL activity.

Case 2 No IPA targeting: The mother of CBU2 happens to be homozygous to her child in HLA B and HLA DR. Therefore exposure to HLA B7 and HLA DR1501 dit not in this case lead to sensitization of maternal microchimeric cells.

All things being equal, transplant with CBU1 is expected to have superior outcome due to more potent GVL effects.

This also illustrates how one can simultaneously select UCB units to be NIMA matched and IPA targeted.

Hematopoietic potential of UCB-comparison with adult stem cells

Stem cells and progenitor cells are present in UCB, at much higher concentration than in adult peripheral blood and in concentrations that may exceed even those of adult bone marrow..^6;45 The phenotypic characteristics of UCB and adult bone marrow cells are remarkably similar though,⁴⁶ with the exception of a higher density of expression of CD34 on the cells and an increased expression of HLA DR on neonatal cells. Functionally, UCB has a higher proportion of cells with stem cell characteristics including the ability to re-plate and expand while maintaining primitive characteristics. They are also more proliferative in long term cultures and have increased migration capacity. Lastly, in contrast to adult bone marrow cells, they are less dependent on exogenous cytokines for establishing growth in SCID mouse models.⁴⁷ The mechanisms underlying the proliferative and expansive advantage of UCB over adult HSC include longer telomere length, more rapid exit from G0/G1 into cell cycle, increased cytokine sensitivity and/or paracrine cytokine effects. Gene expression analysis shows considerable differences in gene expression patterns between adult bone marrow and UCB with fetal gene expression regulated by the transcription factor Sox17. ⁴⁸ In murine competitive repopulation experiments, fetal or newborn blood tends to outcompete adult bone marrow competitors.⁴⁹ Rossler et al, directly compared the hematopoietic potential of human adult and fetal hematopoietic stem cells in a SCID HU model.⁵⁰ For this purpose immunodeficient disease (SCID) mice were implanted with human fetal bones. HLA mismatched CD34 selected cells from 2 human donors were injected simultaneously and 8–10 weeks later engraftment of myeloid and lymphoid cells was analyzed. Both adult bone marrow CD34 cells and cord blood CD34 cells could routinely engraft and differentiate into myeloid and B-lymphoid cells in this assay. After 8 to 10 weeks, they accounted for approximately 50% of cells circulating in the animals. When cord blood and adult bone marrow were injected simultaneously, the engraftment of cord blood cells was markedly superior to that of adult bone marrow cells. Cord blood engraftment was detected in 10 of 10 animals. Adult cells were detected in only five of ten and even in those, the adult cells constituted only a small fraction, less than 10% of all myeloid and B cells. But though HSC characteristics of UCB favor long term dominance, other work has shown that rate of engraftment is determined by the number of hematopoietic stem cells transplanted.⁵¹ In clinical practice, the usual measure of hematopoietic stem cells is the number of CD34+ cells in the graft. UCB grafts contain at least one log fewer CD34+ cells than adult CD34+ stem cell grafts. The low number of hematopoietic stem cells in UCB grafts explains the often slow and erratic hematopoietic recovery.

Clinical UCB SCT: Overcoming Limitations

The first UCB SCT was reported in 1989 and since then extensive experience has accrued with UCB SCT using both related and unrelated donors. Multiple aspects of this experience have been reviewed extensively by others.^52–54 We will here only briefly summarize those data with an emphasis on novel developments and areas of controversy. It is now well accepted that UCB SCT can result in durable engraftment and cure of leukemia. The incidence of acute and chronic GVHD is relatively low despite extensive HLA mismatching between donor and recipient.³⁴ Still, even though mismatching is much better tolerated with UCB SCT compared to adult mismatched SCT, matching cannot be completely ignored. Better matching grafts tend to produce better long term outcomes and recent data suggest that HLA-C matching further improves outcomes particularly among recipients that are 5/6 or 6/6 matched at HLA A,B and DR.⁵⁵ There is also considerable evidence that UCB SCT, particularly double UCB SCT, has a lower recurrence rate than adult SCT. For example the group from Minnesota compared the outcomes of myeloablative conditioning followed by double UBC SCT, matched related, matched unrelated and mismatched unrelated transplants for adults with hematologic malignancies.³² The risk of relapse was only 15 % recipients of double UCB (95% CI,9%-22%) significantly lower than in recipients of matched related donor SCT (43%, 95%CI, 35%-52%), adult matched unrelated donor SCT (37%, 95% CI, 29%-46%) or adult mismatched unrelated donor SCT (35%, 95% CI, 21%-48%). Similarly in parallel studies of haplo-identical vs double UCB stem cell transplant, recurrence rates were considerably lower after double UCB SCT. ³¹ Verneris et al suggested that the reduced rate of recurrence was limited to recipients of double UCB SCT vs single UCB SCT. Other investigators did not confirm this and the relative benefits of double vs single UCB SCT has been the topic of a recently completed and soon to be reported randomized study in children.³³

The limitations of UCB SCT are also increasingly clear and relate mostly to graft failure/rejection or delayed engraftment. Closely associated with this are the prolonged length of stay, increased expense and increased TRM after UCB SCT.^56;57 Recently, several groups have reported an association between the presence of donor specific antibodies (DSA) and graft rejection in HLA mismatched transplant and particularly in cord blood transplant,^{42;43;58–60} but there is considerable debate over the relative importance of this finding,⁶¹ over the exact mechanisms by which this occurs and over the management of transplant patients with positive DSA.^62;63 For many transplant programs there is now sufficient evidence to avoid grafts that are targeted by DSA.⁶⁴

Another area of debate centers on UCB quality and its assessment. As best studied by the Sloan Kettering group, it is clear that occasional cases of graft failure are due to poor graft quality.^65;66 Further investigation will be required to understand the best determinants and measures of cord quality to ensure engraftment. For an extensive discussion of this topic we refer to the recent review by Barker et al.⁶⁵

Less widely debated, is the issue of study endpoints and determinants of success. UCB studies, typically focus on engraftment usually defined as the first of three consecutive days of neutrophil recovery of ANC>0.5 ×10⁹/L. This may not be the best endpoint for studies. In consecutive studies, the Seattle group has shown that time to achievement of an ANC >0.1 ×10⁹/L is better correlated with survival after allogeneic transplant, regardless of graft type and that TRM rapidly increases if this milestone is not reached by day 16.^67;68 Rather than focusing on the median time to neutrophil recovery it may therefore be advisable in future studies to report the percentage of patients achieving an ANC >0.1 ×10⁹/L by day 16. Unfortunately this measure is not readily available in US labs, since differential counts are not usually performed in patients with ANC <0.5 ×10⁹/L. Platelet recovery is another very important determinant of long term outcome. Delayed platelet recovery is associated with increased length of stay, increased expense and in some studies it is an important correlate of TRM.^56;57;69 Platelet recovery after UCB SCT tends to be much more delayed than that after adult transplant.

Enhancing Hematopoietic recovery after UCB SCT

Initial efforts at improving outcomes of UCB SCT addressed the issue of cell dose by infusing several products.^70;71 Prior to double UCB, many patients were ineligible for UCB SCT because of lack of UCB units that were both well enough matched, and had an acceptable cell number. The problem was particularly dire among African American (AA) patients. There is obviously some genetic clustering; as a result UCB from AA newborns tend to be the best matched for AA recipients, but UCB from AA infants on average contain considerably fewer nucleated cells.⁷² By combining UCB units, an acceptable cell number could be reached. Though hematopoietic recovery remains unpredictable and often slow, transplant could now be offered to the majority of patients in need. It is now well established that in the majority of cases only one of the UCB units assures durable engraftment. What biological characteristic, if any, determines the winner graft remains a topic of debate.^73;74

Much effort has been devoted to expansion of UCB stem cells or subsets of such cells. This field has the promise of tissue engineering with generation of cell types of particular interest that potentially could be further manipulated in vitro to generate improved anti- leukemic effects. ⁷⁵ As far as cord blood is concerned, the primary goal of most trials is to enhance hematopoietic recovery. Several excellent recent reviews have summarized this field.^76;77 While faster than expected hematopoietic recovery has been observed, this does not occur reliably. In some studies, expanded UCB has been infused with non-expanded cells from another donor; in such cases durable engraftment is almost universally derived from the non-expanded cord. No study or expanded UCB has yet been able to show any impact on platelet recovery. An additional hurdle for UCB expansion is the time required for generating a sufficient number of cells to assure rapid recovery. This is expected to take several weeks, atime period not always permissible because of the aggressive nature of many hematologic malignancies. In order to circumvent this problem, efforts are ongoing to develop “off the shelf” products that might provide transient hematopoietic support.

Co-infusion of adult progenitor cells with UCB stem cells

A technically less demanding approach consists of the infusion of UCB cells with cytokine mobilized CD34+ selected donor peripheral blood stem cells from an adult, usually a mismatched relative. Fernandez et al reported in 2001 three patients with advanced hematologic malignancies who underwent UCB SCT after myeloablative conditioning supported by co-infusion of adult haplo-identical CD34+ cells.⁷⁸ In all patients adult donor hematopoiesis was transiently detected and caused rapid count recovery but disappeared over time. The same group has since performed 87 such transplants and detailed results were reported in 2009 on the 55 initial patients.^79;80 Four other groups have reported outcomes of cord blood transplant supported by co-infusion of adult, usually haplo identical cells.^81–84 As of July 2012, data on 206 patients have been presented and are summarized in Table 2.

Table 2.

Haplo Cord Summary of Studies.

Author	N	Age, Median (range)	Diagnosis	Conditioning	Adult donor relation R vs U	median time to ANC	time to plat	Fail both grafts	Fail cord only	aGVHD III-IV	cGVHD	Relapse rate	TRM	MFU (mo)	PFS	OS	Major Complications
Bautista, 200980 Sebrango 201097	55	34 (14–60)	AML 30%, ALL 40%, others 30%	Cy TBI rATG (41) BuCY rATG 11	R(38) U(17)	10 (9–36)	32 (13–98)	1	3	6	9 lim, 3 ext	CI 17% (8–35)	CI 35%		47% @ 5 yrs	56% @ 5 yr	7 CMV disease, 4 Toxoplasma, 4 HBV 1 PTLD
Gormley 201182 Childs 201298	12	19 (7–27)	SAA	Flu Cy, TBI 2 Gy eATG	R	10(10–38)		0	2	0	4 mild, 1 mod		1	14 (3–42)	11/12	11/12)
Lindemans 201283	11	12 (0.2–24)	non malig 8 malign 3	Bu Flu rATG (9), Treo, flu Campath (2)	R	12(9–15)	29 (14–300)	1		0				2	11 (.5–46)	73% (8/11)
Kwon 201281	20	39 (28–49)	AML 11, ALL 6, other 3	TBI co+rATG 2, BuCyFlu rATG 11	R(19) U(1)	14(9–28)	27 (9–84)	1	2	0	8% mod/sev	44% @ 1 yr		36(IQR 19–75)	47%	44%
Liu et al, updated from84	59	52 (10–69)	AML/MDS 39 ALL 10 NHL 7 CLL/MPD 3	Flu Mel rATG	R	11 (IQR 10—14)	21 (IQR 16–36)	3 prim 4 sec	6	3	6%	28%	29%	20 (2–54)	42% @ 1yr	53% @ 1 yr	5 PTLD 1 adeno 1 CMV dx

Abbreviations :rATG: rabbit antithymocyte globulin. CI: cumulative incidence. eATG (equine ATG). Ext: Extensive. Fail: graft failure. Flu: fludarabine. HBV: Hepatitis B virus. IQR: Interquartile range. Lim: limited. Mel: Melphalan.MFU:median follow up. Mod: moderate. OS: Overall Survival. PFS: Progression Free Survival. PTLD: Post transplant lymphoproliferative disease. R: related. Treo:Treosulfan. TRM: transplant related mortality. U: Unrelated.

In all studies the adult donor cells were T-cell depleted, usually by a CD-34 selection process. In all studies, ATG was part of the conditioning regimen, further T-cell depleting the adult donor and as a secondary effect, also depleting the cord blood unit. But there were also major differences. The NIH study was restricted to young patients with severe aplastic anemia.⁸² They used a non-myeloablative conditioning regimen. They were the only group to use equine-ATG (ATGAM®) as part of the conditioning regimen (as opposed to rabbit ATG –thymoglobulin® in all other studies). The Utrecht group in the Netherlands treated children, most of whom had metabolic disorders.⁸³ The largest experiences come from the Madrid group and from the University of Chicago.^80;84;85 Both studies treated adults with hematologic malignancies. The patients in Chicago were fifteen years older, (median age 51 vs 34 years) and more often had advanced hematologic malignancies. The Spanish group used myeloablative conditioning and the Chicago group used a fludarabine melphalan regimen. GVHD prophylaxis consisted of calcineurin inhibitors combined with steroids in Spain, and combined with mycophenolate mofetil in Chicago. Infection prophylaxis varied, with very intensive CMV prophylaxis routinely utilized in Chicago.^86;87

There were also differences in donor selection procedures. Most adult donors were haplo-identical relatives, but approximately one third of the Spanish patients received cells from completely mismatched unrelated donors. Cord blood selection procedures were similar, although the Chicago group allowed a lower UCB cell dose (average of 1.6X10^7 nucleated cells/kg recipient weight).

Regardless of the patient and donor selection or procedural differences, all groups observe rapid neutrophil recovery with a median time of 10 to 11 days. (figure 2) This early hematopoietic recovery is almost invariably generated by the adult graft. Median time to platelet recovery was also faster than what is generally reported after UCB SCT and ranged from 19 days in Chicago to 32 days in Madrid (figure 3) (compared to 38 days after reduced intensity conditioning and double UCB SCT in Minnesota).⁵⁷ In the large majority of patients initial hematopoiesis is over time replaced by UCB derived hematopoiesis (figure 4) Occasional failures of the UCB graft or of both grafts were observed in all series. In some cases graft failure was associated with donor specific antibodies (DSA). UCB graft failures were occasionally attributed to poor cord blood viability.⁸⁵ Of note, in the Chicago experience, an excessive dose of adult haplo-identical CD34 cells was associated with decreased UCB engraftment. The Madrid group addressed cord failures by infusion of a second UCB unit, which tended to engraft and outcompete the residing adult graft. In other centers, the adult graft was left unchallenged after cord blood failure. Such patients, a minority, had a high incidence of relapse and opportunistic infections.^84;85 The data from NIH are particularly provocative. Until recently, the outcome of UCB SCT was disappointing for patients with severe aplastic anemia. In their series eleven of twelve heavily transfused patients engrafted and had durable responses.⁸²

Figure 2. Time to Neutrophil Engraftment A: Madrid, B:Chicago (Reproduced from Sebrango et al.⁹⁷ and Liu et al.⁸⁴).

ANC-500: Time to neutrophil count of 500. CB ANC-500 Time to achieve 500 neutrophils from the UCB graft. CB full chimerism: Time to full Cord Blood Chimerism.

Figure 3.

Time to Platelet Recovery. A: Madrid Cumulative incidence of platelet count >20,000 per microliter and >50,000 per microliter, B:Chicago Cumulative incidence of platelet count >20,000 per microliter (Reproduced from Sebrango et al.⁹⁷ and Liu et al.⁸⁴)

Figure 4.

Evolution of chimerism in the Chicago series. A:unfractionated peripheral blood cells. B: CD3 cells (Reproduced from Liu et al.⁸⁴)

Other outcomes varied considerably; the incidence of opportunistic infections, particularly CMV disease and toxoplasmosis, was considerable in the Spanish cohort, but much less in other cohorts. This difference may be due to differences in infection prophylaxis, particularly CMV prophylaxis and in GVHD prophylaxis and treatment. The routine use of steroids for GVHD prophylaxis as practiced in Madrid may result in an increased propensity for infectious complications. Cases of EBV reactivation and EBV PTLD were observed in all series and close monitoring and pre-emptive treatment for this complication is warranted.

Incidence and severity of GVHD was generally low. In the Madrid series there were six cases of grade III–IV acute GVHD out of 55 recipients. None were observed in the other centers. There were three cases of extensive chronic GVHD in Spain, but none in other centers. Despite this low incidence of severe GVHD, the relapse rates for those with malignant disease were moderate. The cumulative incidence of recurrence was 17% at 1 year in Spain and 28% in Chicago.

Long term immune reconstitution was studied by the Chicago group as well as the Madrid group. The Chicago group assessed lymphocyte subsets, T-cell diversity, Cylex Immuknow assay (a measure of T cell responsiveness), and serological response to pneumococcal vaccination⁸⁸ (and manuscript submitted). NK-cell and B-cell reconstitution were extremely rapid occurring at 1 month and 3 months, respectively. T-cell recovery was delayed with a gradual increase in the number of T-cells, starting around 6 months post-transplantation and was characterized by a diverse polyclonal T-cell repertoire. Recovery of immunoglobulins and responsiveness to pneumococcal vaccination was observed. T-cell spectratype was often remarkably diverse. They concluded that immune reconstitution after haplo-cord transplantation was similar to that seen after cord blood transplantation despite infusion of much lower cord blood cell doses.

The Madrid group reported similar observations in their patients.⁸⁹ Natural killer and B cells recovered to normal values by six and nine months, respectively. This was somewhat slower than in the Chicago series, possibly because of routine use of post-transplant steroids for GVHD prophylaxis. Recovery of T cells was slower. Serial analyses of signal joint TCR excision circles showed very low levels by the third month after CBT, followed by recovery to levels persistently similar or higher than those observed before transplantation and in normal controls. In both the Chicago (figure 4) and Madrid series early T cell recovery derived from the adult donor followed by gradual replacement by cells of UCB origin. It is likely that most of the early B cells after haplo-cord transplant are also UCB derived. Of interest, the NIH group observed immediate UCB derived T-cell reconstitution, without detectable adult donor derived T cells at any timepoint. This strikingly different kinetic profile of immune reconstitution may be due to the use of a different formulation of ATG. Nobody has evaluated the origin of NK and B cells.

Conclusion, opportunities and unresolved issues

The characteristics of the umbilical cord grafts include potent hematopoietic potential, an immune system dominated by effectors of peripheral tolerance, but also by the presence of microchimeric maternal cells that are routinely sensitized to the recipient. These biological features help to explain the ability of UCB cells to outcompete adult grafts and cause minimal GVHD while exerting GVL effects that are most likely due to maternal cells. Future research may provide further evidence of these mechanisms, particularly of the GVL effects exerted by maternal cells. At present, these features provide a compelling rationale to continue clinical research to optimize the use of this unique stem cell source.

Slow and erratic hematopoietic recovery is the Achilles heel of UCB SCT and has prevented more widespread use in adults. It causes a heightened incidence of early mortality, prolonged length of hospital admissions, and significant costs. Co-infusion of adult cells as described here is the first method that is readily applicable and results in reliable and clinically relevant improvements in hematopoietic recovery. Highly encouraging results are now available from four centers with over 200 patients transplanted.

There are many opportunities for further development of this technology. For example, cord blood selection algorithms are currently heavily weighted toward maximizing cell doses at the expense of HLA matching. But our data suggest that for haplo-cord SCT, UCB cell dose has no effect on outcome. It should thus be possible to select cords based on other criteria that may be more important for long term outcomes such as HLA matching, NIMA matching, IPA targeting or even KIR mismatching. This concept will require prospective validation but, if confirmed, will have significant potential to improve long-term outcomes.

There is also the possibility of using haplo-cord SCT to support the infusion of rare cell populations that have important therapeutic implications. For example haplo cord transplant has already been used in the transplant of an HIV positive leukemia patient, where the UCB donor was CCR5 delta 32 mutated and therefore resistant to HIV.⁹³ At least in theory this approach could also be used for transplanting genetically modified UCB cells or to support transplant for sickle cell disease.

Many questions and issues remain to be resolved, some of which are briefly discussed. Graft rejection or failure has occurred in each series and constitutes a considerable risk. The quality and assessment of cord blood products is under continuing debate, and standards are developing. Adhering to rigorous standards of viability as proposed by the Sloan Kettering Group may prevent some issues. Donor specific antibodies have also recently been identified as risk factors for graft rejection, and exposure of the graft to donor specific antibodies (DSA) should be avoided if possible. Lastly, very little is known about the interplay between adult hematopoietic and UCB cells. The group from Chicago found a correlation between adult graft CD34 dose and persistence of the adult graft. Though very preliminary, these data are intriguing and suggest that the balance between UCB and adult graft could be manipulated by varying the adult cell dose (and possibly composition). Unresolved questions also linger around the choice of the adult donor and its fate. Is there a benefit to using related donors? Or might a totally mismatched adult, as the Spanish group has utilized in a third of their patients, be preferred for a temporary graft as this may enhance the likelihood of the cord blood unit predominating Do such donors cause more complications, or to the contrary, could they be selected in a fashion to optimize GVL effects, (for example by KIR mismatching)? Is T cell depletion necessary for the adult donor or would it be outcompeted by the umbilical cord even if were infused without manipulation? Data in this regard are extremely limited. Is the adult graft destined to irreversibly and completely disappear, or are there residual adult donor cells that persist in the blood or other recipient organs? And if so, what is their function, if any? In the Chicago series, a small fraction of residual adult donor T cells were often detected in the blood.

Other questions pertain to the conditioning regimen and particularly to the use of ATG which has been part of every single protocol, but also contributes to some of the side-effects, particularly to EBV reactivation.⁹⁴ Could ATG be omitted or replaced by other agents with less risk for EBV reactivation, for example by alemtuzumab?⁹⁵

The further development of this and other related transplant procedures combined with parallel advances in diagnostics and supportive care, may finally fulfill the promise of a donor for all.⁹⁶