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Stem Cell Transplantation (Cord Blood Transplants)
Nelson J. Chao, Stephen G. Emerson, and Kenneth I. Weinberg
Allogeneic stem cell transplantation is an ac- In Section II, Dr. Stephen Emerson describes
cepted treatment modality for selected malignant the historical efforts associated with expansion
and non-malignant diseases. However, the ability of hematopoietic stem cells, specifically with
to identify suitably matched related or unrelated cord blood cells. These efforts to expand cord
donors can be difficult in some patients. Alterna- blood cells continue with novel methods. More-
tive sources of stem cells such as cord blood over, a better understanding of stem cell biology
provide a readily available graft for such patients. and signaling is critical if we are to be able to
Data accumulated over the past several years effectively expand these cells for clinical use. An
have demonstrated that the use of cord blood is alternative, more direct, approach to expanding
an accepted source of stem cells for pediatric stem cells could be achieved by specific genetic
patients. Since the cell numbers of hematopoi- pathways known or believed to support primitive
etic progenitors in cord blood is limited and the HSC proliferation such as Notch-1 receptor
collection can occur only in a single occasion, activation, Wnt/LEF-1 pathway induction,
its use in adult patients can be more problem- telomerase or the Homeobox (Hox) gene prod-
atic. Here, new developments in the use of cord ucts. The clinical experience with the use of
blood for adults and studies aimed at expansion expanded cord blood cells is also discussed.
of cord blood cells and immune reconstitution In Section III, Dr. Kenneth Weinberg de-
are described. scribes immune reconstitution or lack thereof
In Section I, Dr. Nelson Chao describes the following cord blood transplantation. One of the
early data in cord blood transplantation in adult hallmarks of successful hematopoietic stem cell
patients. The patient outcomes are reviewed and transplantation is the ability to fully reconstitute
analyzed for various factors such as cell dose, the immune system of the recipient. Thus, the
HLA typing, and patient selection that could have relationship between stem cell source and the
contributed to the final outcome of these adult development of T lymphocyte functions required
patients. Myeloablative as well as nonmyelo- for protection of the recipient from infection will
ablative approaches are presented. Discussion of be described, and cord blood recipients will be
the various benefits and risks are presented. compared with those receiving other sources of
More recent data from multiple single institu- stem cells. T cell development is described in
tions as well as larger registry data comparisons detail, tracking from prethymic to postthymic
are also provided. Analyses of these studies lymphocytes with specific attention to umbilical
suggest methods to improve on the outcome. cord blood as the source of stem cells. Moreover,
These newer data should lead to a logical a discussion of the placenta as a special micro-
progression in the use of cord blood cells in environment for umbilical cord blood is pre-
adult patients. sented. Strategies to overcome the immunologi-
cal defects are presented to improve the outcome
of these recipients.
354 American Society of Hematology
I. CORD BLOOD TRANSPLANTS: eages and the enrollment of high-risk patients with ad-
HOW CLOSE ARE WE TO USING THIS IN ADULTS? vanced disease all contributed to this caution. Recently,
older allogeneic recipients have been treated success-
Nelson J. Chao, MD* fully following a variety of less intense nonmyelo-
ablative conditioning regimens. On the basis of these
Umbilical cord blood transplantation (UCBT) has re- encouraging observations, it has been hypothesized that
cently been explored in an increasing number of adult a reduced-intensity preparative regimen would allow
patients. The relative ease of procurement and the lower- engraftment of the UCB stem cells. While the total num-
than-anticipated risk of severe acute graft-versus-host bers of mononuclear cells are limited, the progenitor
disease (GVHD) has made UCBT an appealing alterna- content and the proliferative potential of cord blood
tive to bone-marrow–derived hematopoietic stem cells. cells are high. This nonmyeloablative approach could
The use of reduced-intensity or nonmyeloablative pre- diminish some of those concerns raised with the use of
parative regimens to allow engraftment of UCBT broad- ablative regimens. This review will summarize the avail-
ens the scope of patients who may benefit, including able data on the use of UCB as an alternative source of
older and medically infirm patients without a matched hematopoietic stem cells for allogeneic transplantation
donor. This section will summarize the available data for adult patients.
on the use of UCB as alternative source of hematopoi-
etic stem cell transplantation in adult patients. Myeloablative Preparative Regimens
The first known attempt at UCBT occurred 34 years In comparison to the published studies on pediatric pa-
ago when a young 16-year-old boy with acute lympho- tients, the clinical data on the use of UCBT in adult
blastic leukemia received cord blood units from 8 dif- patients are relatively limited, although growing7-14
ferent donors. He was receiving 6-mercaptopurine and (Table 1). As can be gleaned from the composite data,
prednisone and did not receive a preparatory regimen. these patients received standard total body irradiation
One unit engrafted as demonstrated by red cell antigens or busulfan-based myeloablative regimens. GVHD pro-
and the graft lasted for 38 days.1 However, it was the phylaxis consisted of cyclosporine alone or in combi-
success of the allogeneic UCBT approximately 16 years nation with steroids or methotrexate. The degree of HLA
ago in a child with Fanconi anemia that opened a new disparity was not associated with outcome in any of
source of allogeneic hematopoietic stem cells.2 UCB is these studies, although the numbers in each were small.
now accepted as an alternative source for hematopoi- However, in a recent review update with 861 unrelated
etic stem cells for transplantation in children. Unre- UCBT recipients from the Placental Blood program of
lated UCB offers many practical advantages as an alter- the New York Blood Center, which includes 181 (21%)
native source of stem cells, including: (1) the relative patients with age ≥ 18 years and 170 patients (20%)
ease of procurement and availability (given the ability weighing ≥ 60 kg, Rubinstein et al15 have demonstrated
to store fully tested and HLA-typed cord blood avail- in a multivariate analysis that HLA match is an inde-
able for immediate use); (2) the absence of risk for pendent predictor of event-free survival.
mothers and donors; (3) the reduced likelihood of trans- The median age varied considerably from a low of
mitting infections, particularly cytomegalovirus; (4) po- 29 to a high of 48 years with a range of 15 to 58 years.
tential reduced risk of GVHD; (5) less stringent criteria Age is an important factor in the outcome, with the
for HLA matching for donor-recipient selection (with younger patients generally doing better than the older
the potential of finding donors for minority popula- patients. The median weight for these studies also var-
tions); and (6) absence of donor attrition. UCB banks ied from a low of 51 to 70 kg. As the cell dose of the
have been established for related and unrelated UCBT UCB is given per recipient weight, the patients with
with about 100,000 units currently available.3-6 lower body weights would have a higher chance of re-
While the clinical data are encouraging for pediat- ceiving a higher cell dose per cord blood unit. The range
ric patients, the limited number of hematopoietic stem of mononuclear cells/kg recipient weight ranged from
cells in UCB has been a cause for caution for its use in 1.5 to 2.43 × 107/kg. The total numbers of CD34+ cells
adult patients. Moreover, the use of myeloablative pre- has been correlated with both engraftment and the speed
paratory regimens with their known toxicities together of engraftment.
with the known delays in engraftment of all cell lin- The diseases for which these patients were trans-
planted consisted primarily of hematological malignan-
cies. The differences in outcome can be partially attrib-
* Duke University, 2400 Pratt Street, Suite 1100, Durham NC uted to the proportion with more favorable disease sta-
27705-3976 tus at transplantation. For example, most of the data
Hematology 2004 355
356
Table 1. Studies of unrelated umbilical cord blood (UCB) transplantation in adults.
Rocha et al7,9 Cornetta et al11
Laughlin et al7 Sanz et al8 (Eurocord) Goldberg et al10 (COBLT) Ooi et al12 Ooi et al13 Long et al14
Number of patients 68 22 108 19 34 18 13 57*
Median Age (range, years) 31.4 (17.6–58.1) 29 (18–46) 26 (15–53) 48 (20–59) 34.5 (18.2–55) 43 (21–52) 40 (20–51) 31 (18–58)
Median weight (range, kg) 69.2 (40.9–115.5) 70 (41–85) 60 (35–110) 69 (52–126) NA 55.2 (NA) 51 (43–68) 70 (46–110)
Diseases (n)
Hematological malignancies 54 21 96 18 32 18 13 50
Bone marrow failure syndrome/MDS 13 1 12 1 2 6
Number of HLA loci disparity (n)
0 2 1 6 4 1 0 0 2
1 18 13 38 8 10 4 4 8
≥ 2 48 8 64 7 23 14 9 47
Preparative regimen (n) TBI-based/ATG Thio/Bu/Cy/ATG NA Cy/TBI/ATG Cy/TBI TBI/Ara-C/Cy Cy/TBI/Ara-C Cy or Mel with
(n = 51) (n = 21) (n = 13) (n = 27) (n = 17) (n = 13) TBI or BU/
Bu-based/ATG Thio/Flu/ATG Mel/TBI/ATG Bu/Mel TBI/Ara-C/Flu ATG
(n = 14) (n = 1) (n = 4) (n = 7) (n = 1) n = 41
Others/ATG (n = 4) Bu/Cy (n = 2) TAI/Cy/ATG (n = 1)
GVHD prophylaxis CSA/Pred (n = NA) CSA/Pred (n = 22) CSA/Pred (n = 77) CSA/Pred (n = 13) NA CSA (n = 2) CSA/ MTX (n = 13) CSA (n = 1)
CSA (n = NA) Others (n = 31) FK506/Pred (n = 6) CSA/MTX (n = 16) CSA/Pred (n = 56)
Median time to engraftment (range,days)
ANC > 500/µLµ 27 (13–59) 22 (13–52) 32 (13–60) 28 (NA) 28.5 (13–55) 23 (16–41) 22.5 (19–35) 26 (12–55)
µ
Platelet > 20,000/µL 58 (35–142) 69 (49–153) 129 (26–176) 56 (NA) NA 49 (31–263)** 49 (30–164)** 84 (35–167)
Probability of myeloid engraftment 90% by 42 days 100% at 60 days 81% by day 60 75% by day 60 72% at day 60 94% 92% 80% at day 50
GVHD (n) ( probability, %)
Acute Grade II-IV 33 (60%) 16 (NA) 44 (38) NA 11 (38%) 10 (55%) 9**** 17 (41%)
Acute Grade III-IV 11 (20%) 7 (NA) 27 (NA) NA 6 (21%) 1 (6%) – 9 (22%)
Chronic/ patients at risk 12/33 9/10 15/58 NA NA 14/18 8/11 8/25
Median cell dose (range)
N.C. infused (x 107/kg) 1.6 (0.6–4) 1.71 (1.01–4.96) 1.71(0.2–6) 1.8 (0.4–5.3) 1.73 (1.11–3.75) 2.51 (1.16–5.29) 2.43 (2.09–4.06) 1.5 (0.54–2.78)
CD34+ cells infused (x 105/kg) 1.2 (0.2–16.7) 0.79 (0.27–2.60) NA NA NA NA NA 1.37 (0.02–12.45)
Therapy-related mortality (%) 50% at 100 day 43% at 100 day 54% at 100 day NA NA 6% 0% 56%
American Society of Hematology
Survival (%) NA NA 27% at 1 year 20% for good risk 30% at 180 days 76% at 2 years 76.2% at 2 years 19% at 3 years
and 21% for poor risk
at 1 year
Event-free survival (%) 26% at 40 monthsa 53% at 1yearb 21% at 1 yearc NAd NAe 77% at 2 yearsf NAg 15% at 3 yearsh
Findings and Comments:
a EFS is better in patients receiving graft with CD34+ cells >1.2 x 105/kg. b Patients under the age of 30 had significantly better survival. c100 day TRM is lower in patients with disease in chronic phase or remission,
N.C.dose infused ≥ 2.0 x 107/kg and transplant performed after January 1998. d 10 patients received ex vivo expanded UCBT. e The high mortality and relapse reflects the poor risk patients enrolled. . f All de novo AML.
g All except 1 patients received UCB transplant as an upfront treatment and all patients received > 2 x 107 N.C. per weight, perhaps due to smaller size of patients (median weight 51 kg). h CD34+ cell dose infused
correlated with rate of platelet recovery and age > 31 years was significant predictor of poorer event-free survival.
*19 of the 57 patients were included in the study published by Laughlin et al.7
** platelet > 50,000/µL
*** All had MDS-related secondary acute myeloid leukemia
**** 9 of 12 evaluable patients had acute acute graft-versus-host disease.
Abbreviations: NA, not available; TRM, therapy-related mortality; N.C., nucleated cell dose; TAI , thoraco-abdominal radiation; Ara-C, cytarabine arabinoside; TBI, total body irradiation; ATG, antithymocyte globulin;
Bu, bulsulfan; Cy, cyclophosphamide; Thio, thiotepa; Flu, fludarabine; Mel, melphalan; Pred, prednisone; CSA, MTX, methotrexate; GVHD, graft-versus-host disease; MDS, myelodysplastic syndrome
from Sanz et al8 or Ooi et al13 included recipients with appears that a UCB graft that contains at least 2 × 107
chronic myeloid leukemia (CML) in first chronic phase nucleated cells/kg and 1.7 × 105 CD34+ cells/kg is ac-
or acute leukemia in first remission or de novo acute ceptable for adult recipients.4-6 It is hoped that the ad-
myeloid leukemia or untreated myelodysplastic syn- vantage of a lower GVHD without any apparent in-
drome. In contrast, the less favorable survival in the crease in relapse will offset the adverse impact of re-
other studies reflects the poor-risk subjects enrolled into duced cell dose on survival. As in the pediatric setting,
the study. TRM remains the main obstacle for success in adult
The probability of myeloid engraftment ranged from recipients, although several investigators have demon-
72% to 100% by day +60; however, some patients were strated that the TRM is not different from that seen
censored to this event if they died prior to the set date with matched unrelated donors. With the profound in-
for the definition of engraftment. The median times to fluence of UCB cell dose (both nucleated cell dose and
engraftment of both neutrophils and platelets varied as CD34+ cell dose) on engraftment, survival and prob-
well with approximately 1 month to reach 500 neutro- ably TRM in the adult setting, future research should
phils/mL and 2–4 months to reach platelet counts also focus on increasing the cell dose of the UCB graft.
> 20,000/µL. Again, the pace of recovery is likely to Possible methods to increase cell dose include ex vivo
reflect the UCB stem cell dose as well as the amount of expansion, infusion of multiple cord blood units or use
prior therapies a patient could have received. As com- of possible additional cells such as mesenchymal stem
pared to the pediatric patients, the incidence of severe cells or improving HLA matching.
grade III-IV acute GVHD was low (approximately 20%)
given that the majority of the patients received HLA Nonmyeloablative Regimens
mismatched unrelated grafts. Of note, the HLA match- The nonmyeloablative stem cell transplantation (NST)
ing in general was done serologically for HLA-A and- regimen was proposed initially based on the rationale
B and molecularly for HLA-DR. Thus it is likely that that the therapeutic benefit of an allogeneic transplan-
the mismatching would have been greater than reported tation is partially related to the crucial immune-medi-
if molecular matching had been performed. Therapy- ated graft-versus-malignancy effect. The concept of
related mortality (TRM) was high, reaching approxi- graft-versus-malignancy as the pivotal therapeutic com-
mately 50% in most series. The overall survival again ponent of allogeneic transplantation is supported by the
varied considerably, reflecting the risk factors outlined observation from clinical studies that (1) patients with
above. Comparative studies of UCB transplantation acute and chronic GVHD have a reduced risk of re-
versus unrelated bone marrow have been performed from lapse; (2) patients with syngeneic BMT and after T cell–
two registry datasets. These two studies have demon- depleted allotransplant have a higher incidence of re-
strated that if there is a cord blood unit that is at least a lapse compared with other allogeneic donors; and (3)
4/6 HLA match with sufficient cells, the outcomes are patients with a relapsed malignancy after allogeneic
similar following UCB transplantation to matched or transplantation can be re-induced into complete remis-
one antigen mismatched unrelated donor bone marrow sion without any chemotherapy by donor lymphocyte
grafts.21,22 infusion (DLI). NST, with its reduced-intensity pre-
Thus, UCB contains sufficient numbers of hemato- parative regimen, makes allogeneic transplantation ap-
poietic stem cells to achieve engraftment in adult pa- plicable to patients with relative contraindications to
tients with lower than anticipated risk of severe acute myeloablative regimens given its lower rate of TRM
GVHD, even when HLA-disparate grafts are infused. with fewer infections and less GVHD. However, the
The use of UCB allows allografting to be offered to a preparative regimen does not contribute significantly
greater proportion of patients, many of whom do not to the antimalignancy effect; thus, the risk of disease
have a matched sibling or unrelated donor to allow al- progression after transplantation remains higher com-
logeneic therapy as the only chance to cure the under- pared with the myeloablative approach.
lying disease. The results thus far suggest that UCBT NST using UCB provides an opportunity for im-
can result in long-term disease-free survival in many of munotherapy for older patients, sicker patients and also
these patients. Similar to the pediatric series, clinical patients without suitable donors who are not eligible
experience in the adult patients has also documented for this potentially curative approach. However, there
the importance of graft cell dose in determining en- is increased concern about graft rejection using this ap-
graftment and survival. The critical “threshold dose,” proach given that there are, on average, two logs fewer
below which engraftment and survival become signifi- cells infused than would be considered for a standard
cantly inferior, remains to be defined in a larger study matched sibling or unrelated donor transplant (Table
with a longer follow-up. Based on current results, it 2). The clinical outcome of 2 patients with malignant
Hematology 2004 357
Table 2. Summary of results of nonmyeloablative unrelated umbilical cord blood transplantation in adult patients.
Investigator Barker et al18 McSweeney et al19 Chao et al20
Number of patients 43 5 13
Age, years: median (range) 49.5 (22–65) 64.5 (25–78) 49 (19–62)
Diagnosis HM HD; AML; CLL; NHL NHL; MDS; AML; MEL; ALL
Preparative regimen F + Bu + TB I (200cGy (n = 21) F + TBI (200cGy) F + C + ATG (n = 10)
F + C + TBI (200cGy) (n = 22) F + C + ATG + TBI (200cGy) (n = 3)
GVHD prophylaxis CYA + MMF CYA + MMF CYA + Pred (n = 8)
CYA + MMF (n = 5)
Cell Dose
×
N.C. (× 107/kg): median (range) 2.6 (1.6–3.8)† 1.1 (1.75–1.3) 2.07 (1.07–5.53)
3.2 (1.1–5.1)‡
×
CD34 (× 106/kg): median (range) 3.7 (1.1–8.1)† NA (0.01–0.04) 1.3 (0.5–9.6)
4.3 (1.1–10.3)‡
×
CD3 (× 106/kg): median (range) 0.06 (0.02–0.15)† NA (1.4–3.3) 4.6 (2.02–22.82)
0.05 (0.02–0.12)‡
Engraftment (n) C.I. = 76%† 2 8/12
C.I. = 94%‡
Median days to ANC 26 (12–30)† NA 12 (6–24)
µ
> 500/µL (range) 9.5 (5–28)‡
Median days to platelet NA 14 (6–61)
µ
> 20,000/µL (range)
Grade II-IV aGVHD / cGVHD (n) C.I. for aGVHD = 44% 1/NA 1/1
C.I. for cGVHD = 21%
Outcome O.S. : 39% at 1 year Alive: n = 3 O.S.: 43% at 1 year
DFS.: 31% at 1 year PR: n = 1 DFS: 43% at 1 year
Abbreviations: Gd, grade; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; N.C., nucleated
cell; ANC, absolute neutrophil count; NHL, non-Hodgkin’s disease; MDS, myelodysplastic syndrome; AML, acute myeloid leukemia;
MEL, melanoma; ALL, acute lymphoblastic leukemia; HD, Hodgkin’s disease; CLL, chronic lymphocytic leukemia; THAL, thalas-
semia; WAS, Wiskott–Aldrich syndrome; NBL, neuroblastoma; HM, hematological malignancies; F, fludarabine; ATG, antithymocyte
globulin; C, cyclophosphamide; TBI, total body irradiation; Bu, busulphan; CYA, cyclosporin A; MMF, mycophenolate mofetil; PDN,
prednisolone; FK506, tracrolimus; NA, not available; C.I., cumulative incidence; PR, partial remission; OS, overall survival; DFS,
disease-free survival
† The results refer to patients given F + Bu + TBI 200cGy as conditioning regimen.
‡ The results refer to patients given F + C + TBI 200cGy as conditioning regimen.
lymphoma using this novel approach was first reported potential for robust engraftment using this approach.
by investigators at Duke University.16,17 In that study, 2 The favorable outcome demonstrates the feasibility of
patients with relapsed lymphoma who had no matched the mismatched unrelated UCB cells, even with the NST
siblings, partially matched family members, or matched regimens.
unrelated donors successfully underwent nonmyelo- The largest experience with transplantation using a
ablative conditioning therapy followed by infusion of reduced-intensity regimen was evaluated by investiga-
4 and 6 matched, unrelated donor UCB cells, respec- tors from the University of Minnesota on a cohort of
tively, at the nucleated cell dose of 2.9 and 6.5 × 107/ high-risk patients with hematological malignancies.18
kg, respectively. The conditioning regimens consisted In their study, unrelated UCB graft with a median nucle-
of fludarabine 30mg/m2 and cyclophosphamide 500 mg/ ated cell dose of 3.7 × 107 per kg (range, 1.6–6.0 × 107
m2 daily for 4 days with antithymocyte globulin 30 /kg) was infused into 43 patients (median age 49.5 years;
mg/kg per day for 3 days. Cyclosporine and predniso- range, 22–65 years), after receiving 2 types of condi-
lone were given for acute GVHD prophylaxis. Both tioning regimens: fludarabine 200 mg/m2, total body
patients had 100% donor engraftment by the third month irradiation (TBI) 200 cGy and busulfan 8 mg/kg (Flu/
of transplant and remained in remission 6 and 12 months Bu/TBI) for the initial 21 subjects; fludarabine 200 mg/
following transplantation. Extension of these data to m2, TBI 200 cGy and cyclophosphamide 50 mg/kg (Flu/
11 other patients confirms the low morbidity and the Cy/TBI) for the subsequent 22 subjects. All patients
358 American Society of Hematology
received GVHD prophylaxis with cyclosporine and able to expand effectively in the periphery, and the de-
mycophenolate mofetil. The median time to neutrophil velopment of new T cells through the thymus is also
recovery of more than 0.5 × 109/L was 26 days (range, accelerated compared to the rate of development in those
12–30 days) for the Flu/Bu/TBI recipients, but only receiving ablative regimens. Alternatively, the lower
9.5 days (range, 5–28 days) for the Flu/Cy/TBI recipi- incidence of GVHD in NST may also play an impor-
ents. The cumulative incidence of engraftment for Flu/ tant role in the preservation of the peripheral and cen-
Bu/TBI and Flu/Cy/TBI recipients was 76% and 94%, tral niches for T cell development.
respectively. Despite the use of 1–2 HLA-antigen mis-
matched graft in 93% of the recipients, the cumulative Future Directions for UCB Transplantation
incidence of grade II–IV GVHD and grade III–IV in Adults
GVHD for the entire cohort of patients were 44% and While the use of NST signifies an advancement in the
9%, respectively. The disease-free survival of these high- field of transplantation and immunobiolology, several
risk subjects was also favorable: 24% at 1 year for Flu/ unresolved questions remain:
Bu/TBI recipients and 41% at 1 year for Flu/Cy/TBI 1. What is the optimal UCB cell dose required for
recipients. A similar approach and data have also been adult patients and can we increase it?
reported by investigators in University of Colorado 2. What is the optimal NST conditioning regimen and
Health Sciences Center.19 GVHD prophylaxis?
Immune recovery in 5 recipients of UCB trans- 3. Is the incidence of GVHD similar in ablative and
plant following the NST regimen were compared to NST regimens?
recovery in adult recipients of UCB following a myelo- 4. Does the incidence of infection differ in ablative
ablative regimen by investigators at Duke University.20 versus NST regimens and can one improve immune
Recipients of NST regimens had a more rapid and ro- recovery?
bust recovery of myeloid and lymphoid cells. The T 5. What is the difference in overall efficacy between
cell repertoire in UCB recipients treated with the NST myeloablative and NST UCBT?
regimen was markedly more diverse and robust com-
pared with the repertoire in those receiving the Conclusion
myeloablative regimen at similar time points. T cell UCB is a viable alternative to bone marrow and periph-
receptor excision circles (TRECs), which are generated eral blood as a source of stem cells capable of hemato-
within the thymus and identify new thymic emigrants poietic reconstitution for adults, when related or unre-
and those that have not divided, were detected 12 months lated marrow donor is not available. UCBT following
after transplantation in the NST recipients. This com- NST preparative regimen is an exciting new approach
pared favorably to the delayed detection of TRECs at that provides an option for patients who are otherwise
18–24 months among recipients of myeloablative regi- excluded from conventional hematopoietic stem cell
mens. Thus, in adults receiving an NST preparatory transplantation, including elderly or medically infirm
regimen, the quantitative and qualitative recovery of T patients with no matched sibling donor. Preliminary
cells occurs through rapid peripheral expansion. The results have shown that such an approach can be associ-
ability of patients receiving NST transplantation to re- ated with timely engraftment with full donor chimer-
cover within a few months suggests that the peripheral ism. Comparison between myeloablative and NST ap-
“niches” in which T cells can proliferate are preserved proaches will be needed before this therapy can be con-
in these patients compared to those receiving sidered for younger patients eligible for myeloablative
myeloablative regimens. Moreover, the presence of transplant. At the moment, the use of NST UCBT can-
TREC-positive cells within 1 year suggests that thymic not be encouraged outside of a clinical trials or selected
recovery is likewise accelerated in NST compared with patients. The future challenge will be to develop strat-
recipients of myeloablative regimens. The favorable egies to optimize the chance of early and durable en-
results of T cell recovery following the NST suggest graftment, as well as to minimize the risk of GVHD
that it may be possible to have an excellent outcome and transplant-related death.
with an unrelated mismatched UCB transplantation in
adult patients. Patients have a rapid recovery of T cells
with a complex diversity. The primary difference be-
tween the recipients of ablative and NST regimens was
the extent of physiologic damage caused by the prepa-
ratory regimen. When the damage is relatively mild, as
in nonmyeloablative regimens, the donor T cells are
Hematology 2004 359
II. EXPANSION OF UMBILICAL CORD BLOOD CELL shorten UCB transplant nadirs by providing increased
PROGENITORS AND STEM CELLS: BIOLOGY AND numbers of stem and progenitor cells to patients by
APPLICATION TO CLINICAL TRANSPLANTS expanding the numbers of hematopoietic stem cells
(HSCs) and progenitor cells, in the laboratory, prior to
Stephen G. Emerson, MD, PhD* transplant. This approach should succeed if the delay in
engraftment is due to a paucity of HSC and progenitor
The close correlation between preclinical assays and in numbers, but would fail if the delay were due to an
vivo clinical biology that is observed with bone mar- uncharacterized-but-insurmountable qualitative defect
row stem cells and with hematopoietic growth factors in UCB stem and progenitor cells.
such as erythropoietin (Epo), granulocyte colony-stimu-
lating factor (G-CSF) and granulocyte-macrophage Stem Cells, Stem Cell Assays, and Engraftment
colony-stimulating factor (GM-CSF) has been very re- Attempts to characterize UCB expansion protocols have
assuring and has accelerated the development and im- relied directly on the assays used to measure the cell
provement of clinical hematology practice. Clinical ex- populations that are targets for expansion. Over the past
perience with UCB transplants, in contrast, has proved 20 years, these assays have become more sophisticated,
much more paradoxical and problematic. Early labora- and clinical investigators have progressively adjusted
tory measurements showed that the density of both their clinical trial designs based on more recent, better
CD34+ cells and clonogenic progenitor cells was ex- assays. Pluripotent hematopoietic stem cells are very
tremely high in UCB.1 Similarly, the proliferative ca- rare, representing only 1 cell in 2–10 × 106 BM cells,
pacity of progenitor cell differentiation in laboratory perhaps slightly more frequent in UCB. There is no
assays and of UCB stem cells transplanted into immu- phenotypic assay, to date, that directly identifies and
nodeficient mice was measurably higher than for pro- measures these rare cells. Rather, all efforts to measure
genitors and stem cells from bone marrow (BM) or HSCs rely on functional assays. The easiest and most
peripheral blood (PB).2,3 But when comparable num- popular assays have been colony-forming cell (CFC)
bers of UCB progenitors (1–5 × 106/kg) and stem cells assays, which measure cells that are partially differen-
are transplanted as are delivered to patients with stan- tiated between HSCs and mature blood cells. These cells,
dard BM or PB grafts, engraftment is far slower, and which correlate closely but inexactly with CD34+ cell
graft failure is more likely.4 Given that opportunistic numbers, produce easily detectable blood cell colonies
infection and organ failure rise exponentially with time in 2 weeks in the laboratory. But careful cell fraction-
to engraftment, and that overall long-term survival cor- ation studies have shown that the intermediate-matu-
relates closely with platelet engraftment, this delay has rity cells detected in these assays contribute only very
proved to be a major barrier to adoption of what would slightly, and temporarily, to hematopoietic recovery after
otherwise be an overwhelmingly attractive stem cell transplantation, since they don’t produce enough cells
source. that survive long enough after transplantation; it is only
One of the striking lessons of BM and peripheral the immature pluripotent stem cell population that con-
stem cell (PSC) transplant is that time to engraftment tributes to sustained engraftment.5,6 So, although CD34+
correlates inversely with progenitor and stem cell dose, cell numbers in an unmanipulated graft may correlate
with most graphed comparisons showing hyperbolic with clinical time to engraftment after transplantation,
curves with asymptotic limits at 7–8 days for neutro- that is likely because the ratio of intermediate progeni-
phil engraftment and 10–12 days for platelet engraft- tor cells to very primitive stem cells is fairly constant
ments, both achieved with PSC stem and progenitor between clinical samples, and therefore CD34+ cell num-
cell doses of > 5 × 106 CD34+ cells per kg. Providing bers indirectly reflect primitive stem cell numbers trans-
lower doses of early hematopoietic cells results in in- planted. This assumption, however, may not be true
creasingly longer nadirs, but providing higher doses following many ex vivo expansion procedures, in which
does not further accelerate hematopoietic recovery. progenitors may be greatly expanded but primitive stem
Based on these well-documented observations, the ques- cells may not.7
tion has been raised whether one could significantly Better functional assays for primitive stem cells,
developed in the late 1980s and 1990s, include the long-
term culture initiating cell (LTCIC), cobblestone area
forming cells (CAFC), and best of all the human to
* Abramson Cancer Center; Univeristy of Pennsylvania,
Departments of Medicine, Pathology, and Pediatrics, 510 immunodeficient NOD/SCID mouse (NOD/SRC) re-
Maloney Bldg., 36th & Spruce Streets, Philadelphia PA 19104- populating cell assay.8 In evaluating UCB expansion
4283 protocols, it is important to determine what popula-
360 American Society of Hematology
tions were measured and specifically whether LTCIC, effect reflects quantitative increases in HSCs or a quali-
CAFC or NOD/SRC were identified as being expanded. tative improvement in their behavior is not certain.12
This may be the basis of the reported enhancement of
UCB Expansion Ex Vivo: Cytokine Cocktails UCB engraftment achieved by supplementing UCB
When UCB progenitors were first detected, investiga- grafts with mesenchymal stem cells. UCB HSCs are
tors noted that the progenitor cells formed colonies in selectively responsive to interleukin (IL)-6 stimula-
agar more rapidly than did BM or PSC progenitors, tion,13 and recent studies suggest that combining UCB
and that a higher fraction of UCB colonies could be CD34+ cell selection with IL-6/soluble IL-6 receptor/
replated to form secondary colonies. These data on UCB SCF/Flt-3l/Tpo stimulation induces NOD/SRC expan-
progenitors, together with their much higher frequency sion 4–5X. Overall, it appears that UCB stem cell ex-
in the UCB mononuclear cell fraction, suggested that pansion may be possible with ex vivo cytokine cock-
they were more proliferative than their adult counter- tails, but conditions that can generate such HSC expan-
parts. In the early 1990s Moore and Hoskins applied sions, as opposed to the expansion of more mature pro-
the techniques of PB CD34+ cell isolation followed by genitor populations, may have not yet been used in clini-
culture in high concentrations of multiple hematopoi- cal trial settings.
etic growth factors first pioneered by Haylock et al to
UCB, with prima facie very promising results. After Stem Cell Expansion: Hox Gene Products
removal of more mature CD34– cells, clonogenic pro- Individual cytokines are known to trigger many intra-
genitors could be expanded 50- to 100-fold, while cellular signal transduction pathways, which presum-
slightly more primitive ∆ cells could be multiplied 10– ably activate multiple gene transcription pathways.
20X, all in 2 weeks.9 Subsequently, many investigators Thus, in retrospect, combinations of cytokines might
confirmed that UCB progenitor populations could be well be expected to have multiple effects on HSCs that
readily expanded, and some showed that LTCIC could might not permit UCB stem cell expansion. An alterna-
also be expanded 10X, particularly if combinations of tive, more direct, approach to expanding stem cells
low-dose cytokines were combined with perfusion con- might focus on the specific genetic pathways known or
ditions that simultaneously removed the secreted prod- believed to support primitive HSC proliferation. Over
ucts of maturing blood cells and supplied continuous the past decade several such pathways and transcription
fluxes of cytokines.10 Of note, however, no assays for factors have been proposed, including Notch-1 recep-
primitive NOD/SRC repopulating cells were measured tor activation, Wnt/LEF-1 pathway induction and
in these studies, so it is not known to what extent, if telomerase. One of the most promising intracellular tar-
any, true HSC were expanded in these protocols. When gets for HSC expansion are Homeobox (Hox) gene
Lewis et al assayed UCB NOD/SRC repopulating cells products. These transcription factors, which were first
in parallel with CFC and LTCIC during ex vivo cytokine identified as master switch transcriptional regulators of
expansion cultures, they found that while CFC were in- early development, were discovered a decade ago to be
creased 20–25X, LTCIC were only increased 40%, and expressed in the most primitive HSCs. When cloned
NOD/SCR were maintained but not increased at all.11 and overexpressed in mouse HSCs, several of these Hox
True UCB NOD/SRC stem cell expansion may be genes induce HSC proliferation, in addition to more or
achievable by very judicious combinations of HSC pu- less disruption in differentiation.
rification and cytokine selection. SDF-1 treatment in- One of these Hox genes, HoxB4, has proven to be
creases the engraftment of UCB HSCs, but whether this the most interesting to date. HoxB4 is highly expressed
in primitive HSCs, and its expression declines with
differentiation. Overexpression of HoxB4 mRNA
Table 3. Currently available assays of hematopoietic progenitor
and stem cells in umbilical cord blood.
by retroviral infection results in HSCs that expand
over 100X if transplanted in very low numbers
Ease and Rapidity for Correlation with (analogous to the clinical HSC scenario).14 Of even
Assay Clinical Application Stable Engraftment more interest, recent experiments suggest that the
Colony assays +/– Indirect, unmanipulated grafts only same effect can be achieved by treating purified
CD34+ +++ Indirect, unmanipulated grafts only HSCs with a modified form of soluble HoxB4 pro-
LTCIC – Possibly direct tein (TAT-HoxB4), which increases intracellular
NOD/SRC – Likely direct HoxB4 protein levels for several hours only.15
While this approach has thus far only been applied
Abbreviations: LTCIC, long-term culture-initiating cell; NOD/SRC, to murine HSCs, and not to human BM or UCB
human to immundeficient NOD/SCID mouse repopulating cell assay HSC expansion, the application to human UCB
Hematology 2004 361
expansion could be very direct. ment to ANC > 500 was seen on day 24–33, and plate-
More recently, Zhu et al have discovered that the let engraftment to > 50,000 at approximately day 90.
protein NF-Y is a normal transcriptional activator for These results, while disappointing, highlight the
multiple Hox genes, as well as telomerase, Notch-1 and importance of building clinical trial design based upon
LEF-1.16 When overexpressed in murine HSCs by as the most precise and appropriate preclinical data. The
little as twofold, NF-Ya increases HSC numbers by 20X cytokine cocktails employed in the three published stud-
following transplantation. Application of TAT-NF-Y ies included some but not all of the cytokines required
protein transduction to UCB HSC expansion thus of- to achieve UCB HSC expansion in vitro, as measured
fers the attractive possibility of activating multiple HSC by our best NOD/SRC assays: One trial used SCF and a
expansion pathways simultaneously. The absolute and Tpo analogue, but not Flt3-l, and the other two used
relative efficiencies of these protein treatments for ex- Flt3-l, but not Tpo or SCF, and neither included IL-6
panding human UCB HSCs will need to be evaluated or soluble IL-6 receptor. In addition, a lesser but possi-
directly in NOD/SCID mouse xenotransplants, and then bly contributing variable was that in all three trials the
in appropriate Phase I clinical trials. ex vivo expanded cells were added 10–12 days later,
Similar approaches using other soluble, revers- thus mitigating any accelerating effect they might have
ible, biochemical stimulators of stem cell transcrip- otherwise have achieved.
tion programs, including perhaps copper chelation, It must be pointed out that these trial design limita-
have also been proposed and are under preclinical tions were known to the clinical investigators, but the
and clinical trial. lack of availability of clinical grade cytokines, and pos-
sibly also HSC selection devices, prevented the optimal
Clinical Experience with Ex Vivo cocktails and culture conditions from being employed.
Expanded Hematopoietic Cells These difficulties would be surmounted if simpler ex-
Clinical trials of ex vivo culture UCB cells have begun pansion conditions, requiring few added reagents and/
to be reported, but their interpretation should be tem- or devices while still supporting HSC expansion, could
pered by the knowledge that the conditions of culture be devised and employed.
used to date have not been shown to support USC am-
plification, only CFU expansion. At least three trials What if UCB HSC Numbers Are Not Rate
evaluating the combination of unmanipulated with ex Limiting? Exploring the Null Hypothesis
vivo expanded UCB grafts have been published to date; Another possibility that must be considered is that in-
in all three studies expanded cells were added on day creasing UCB numbers will not, by itself, accelerate
10–12 to supplement unmanipulated cells. One study engraftment. Perhaps there is a qualitative “immatu-
involved the use of a static culture system in defined rity” defect that prevents rapid engraftment. If so, then
media while the other two used a perfusion culture ex- is there a way to circumvent this defect: providing a
pansion system. Although no toxicities were seen in maturation effect through some combination of in vivo
these studies, no significant acceleration of neutrophil cytokine treatment, or perhaps ex vivo cell activation
or platelet engraftment was seen.17-19 Neutrophil engraft- targeted at maturation? Viewed through this perspec-
tive, one can envision manipulations of the
non-HSC components of the UCB graft, in-
Table 4. Ex vivo approaches to progenitor and stem cell expansion. cluding the mature hematopoietic cells, the
immune cells, antigen-presenting cells, and
Evidence for Application to
Protocol HSC Expansion Clinical Trials mesenchymal stromal cell components of the
CD34 + selected cells, + Limited trials, without best graft. Interactions between HSC and non-
high-dose cytokines combinations due to cytokine HSCs could form the basis for potentially
availability. No nadir reduction. improved engraftment in mixed graft settings
as well.20 Each of these manipulations could
Low-dose cytokines, ? In trials, without best cytokine be evaluated by comparing engraftment in
continuous perfusion combinations due to cytokine
availability. No nadir reduction.
NOD/SCID mice, prior to clinical transplanta-
tion trials.
In summary, experience with hematopoi-
Direct transcriptional +++ Future
activation of HSC cycling: etic cell expansion to date supports the fun-
HOXB4, telomerase, NF-Y damental principle of experimental hematol-
ogy, that the best clinical results follow from
Abbreviations: HSC, hematopoietic stem cells the most careful, comprehensive and creative
362 American Society of Hematology
preclinical trials in the proper in vitro and in vivo mod- T lymphocyte functions required for protection of the
els. For the future, this experience suggests concentrat- HSCT recipient from infection. In particular, the unique
ing on ex vivo manipulations that can be shown to di- aspects of UCBT, compared to either BMT or mobi-
rectly increase HSC numbers following NOD/SCID en- lized peripheral blood stem cell transplantation
graftment and/or accelerate the pace of engraftment in (PBSCT), will be described. Factors influencing the
these same models. development of adequate immune function after HSCT
include those related to the donor progenitor cell popu-
III. IMMUNE RECONSTITUTION AFTER UMBILICAL lations, pre-formed lymphocytes in the donor product,
CORD BLOOD CELL TRANSPLANTATION the host microenvironment, pharmacological interven-
tions, and antigenic exposure. All clinically relevant
Kenneth I. Weinberg, MD* stem cell sources include both progenitor cells and pre-
formed lymphocytes (Figure 1). Prethymic progeni-
One of the events necessary for the success of hemato- tors may include HSC, common lymphoid progenitors
poietic stem cell transplantation (HSCT), regardless of (CLP), committed T progenitors (CTP), whose T lym-
the source of stem cells, is the development of a func- phoid progeny depend on the host thymus for their de-
tional immune system from donor-derived cells. The velopment.2
production of adequate granulocytes, platelets, and red
blood cells usually occurs rapidly after HSCT. In con- Development of Thymic Progenitors
trast, the ability to produce lymphocytes, especially T Upon entry to the thymus, progenitor cells undergo
lymphocytes, is delayed. As a result, serious infection expansion; differentiation events including V(D)J re-
in the first 1–2 years after HSCT occurs in about 50% combination to generate functionally rearranged TCR
of uncomplicated transplants from histocompatible sib- genes; and both positive and negative selection events
ling donors, and up to 80%–90% of recipients of that cull potential T lymphocytes that would either be
matched unrelated donor (MUD) marrow transplants unresponsive to antigenic peptides presented by self-
or histocompatible recipients who developed GVHD.1 major histocompatibility complex (MHC) antigens or
Infections attributable to poor lymphocyte function in- overly responsive to either MHC or self antigens. The
clude viral pathogens such as the herpes group (cytome- ultimate fate of negatively selected cells is apoptosis.
galovirus [CMV], herpes simplex virus [HSV], vari- Since most thymocytes are fated to die, maintenance of
cella zoster virus [VZV], human herpes viruses [HHV]
6 and 8), respiratory viruses (respiratory syncytial vi-
rus [RSV], parainfluenza viruses), adenovirus, and en-
teroviruses (Coxsackie and ECHO viruses), as well as
EBV-associated lymphoproliferative disorders. Other
infections that may result from inadequate cellular im-
munity include fungal infections such as Pneumocystis
carinii, Candida, and Cryptococcus. Increased suscep-
tibility to Aspergillus infection may reflect defects in
innate immunity. Susceptibility to some bacterial in-
fections, notably those caused by encapsulated organ-
isms (Staphylococcus pneumoniae, Haemophilus
influenzae), is determined by the inability to produce
antipolysaccharide antibodies.
The emphasis of this review will be the relation-
ship between stem cell source and the development of
Figure 1. Two pathways for generation of T lymphocytes
* Children’s Hospital of Los Angeles, 4650 Sunset Boulevard, after hematopoietic stem cell transplantation (HSCT).
Box 62, Los Angeles CA 90027 Shown at top is the adoptive transfer of post-thymic T cells
which then undergo expansion in the host, resulting in a limited
Acknowledgments: Supported by NIH grants R01 HL54729, repertoire of mature T lymphocytes that can rapidly confer
HL70005, AI50765; P50 HL54850; M01 RR00043. The antigen-specific responses, including graft-versus-host
disease (GVHD). Below is shown the pathway for differentia-
thoughtful input of Gay Crooks, Bruce Blazar, Robertson
tion of pre-thymic progenitors by migration into the recipient
Parkman, Nelson Chao, Wes Brown, Hisham Abdel-Azim and thymus, resulting in the slow generation of a diverse repertoire
Dan Douek is greatly appreciated. that is host-tolerant.
Hematology 2004 363
thymic output depends on continual expansion and dif- cell development. For example, in patients with severe
ferentiation of the immature prothymocytes, which lack combined immune deficiency (SCID) receiving CD34+
expression of the TCR invariant CD3 complex, and the haploidentical HSCT, the appearance of functional
co-receptors CD4 and CD8 (“triple negative,” TN). mature T lymphocytes in the peripheral blood is 90–
The major signals for expansion of immature 120 days after transplant, a time period similar to that
prothymocytes are two cytokines, interleukin-7 (IL-7) required for normal prenatal immune ontogeny.
and c-kit ligand (KL), which are produced by thymic
epithelial cells (TEC). Murine studies have demonstrated Post-Thymic T Lymphocytes
that pre-HSCT radiotherapy and chemotherapy kill TEC, In contrast to prethymic progenitors, post-thymic T cells
resulting in defective intrathymic IL-7 production. have already undergone maturation and have antigenic
Early studies demonstrated that the thymic microenvi- and functional specificities that were conferred by the
ronment is a target of GVHD, and more recent studies donor’s thymus. Donor-derived post-thymic T cells ex-
have shown that TEC organization and function is ab- press a fixed TCR and thereby are programmed to re-
normal in experimental GVHD models. In non-HSCT spond to a set of antigenic determinants. Although adop-
studies, aging also results in TEC damage and decreased tive transfer of mature T lymphocytes had long been
intrathymic IL-7 production. Thus, thymic function may thought to be a relatively static process, recent studies
be adversely affected by several phenomena commonly have indicated that mature T lymphocyte populations
associated with clinical HSCT-cytotoxic therapies given are dynamic and regulated by interactions with other T
pre-HSCT, GVHD, and recipient aging. Clinical stud- lymphocytes as well as the host microenvironment.
ies have demonstrated that decreased capacity for pro- Mature T lymphocytes may undergo activation if they
duction of new T lymphocytes is associated with GVHD encounter a stimulating antigen and the proper set of
and increasing recipient age.3,4 The effects of pre-trans- co-stimulatory signals. Activation of T lymphocytes
plant conditioning on thymopoiesis has been more dif- leads to acquisition of functional properties such as cy-
ficult to assess because of the complexity of the prob- tolytic capacity or cytokine secretion. Activated T cells
lem of dose intensity as well as the effects of previous may proliferate and mature into memory cells, undergo
therapy for malignancies. Any assessment of the effect apoptosis (activation-induced cell death [AICD]), or
of stem cell source, e.g., UCB, on immune reconstitu- be anergized. Expansion of activated T lymphocytes is
tion must take into account the function of the thymic dependent on production of IL-2 by helper T lympho-
microenvironment. cytes and expression of high affinity IL-2 receptors (IL-
The biology of prethymic progenitors is also highly 2R) by the responsive helper and cytotoxic cells.
relevant to the outcome of HSCT. The lack of pre- Recent studies have demonstrated that naive T lym-
existing TCR rearrangements means that donor-derived phocytes in a lymphopenic environment can undergo
prethymic progenitors are unlikely to cause GVHD, as proliferation while maintaining a mainly naive pheno-
long as the recipient thymus maintains the ability to type.5 Such homeostatic proliferation differs from acti-
select out undesirable T cells. However, the develop- vation-induced proliferation in which naive T cells rec-
ment of an immune repertoire from prethymic progeni- ognize foreign antigens and proliferate in response to
tors may be delayed by ontogenic factors such as com- IL-2. In homeostatic proliferation, the stimuli recog-
mitment to the T lymphoid lineage and the time nor- nized by the TCR are the same self antigens that nor-
mally required for development of large numbers of mally induce positive selection in the thymus. Like
diverse T cells. Between 7 to 9 weeks of gestation, he- thymopoiesis, homeostatic proliferation also requires
matopoietically derived cells enter the thymic anlage, IL-7, although the source is probably extrathymic, not
which forms from mesenchyme and endoderm of the TEC. Homeostatic proliferation has also been described
III-IV pharyngeal pouches. Alloreactive T cells are for memory T lymphocytes with evidence that IL-15 is
present by week 14–15 and antigen-specific T lympho- critical for the expansion of these cells.
cytes appear later in the second trimester. Recapitula- The appearance of T lymphocytes after HSCT is
tion of fetal ontogeny provides a framework for under- conventionally seen as evidence of de novo generation
standing the development of lymphoid cells after HSCT. of T lymphocytes from progenitor cells. However, as
The generation of T lymphocytes post-HSCT requires previously noted, transplantation of stem cell products
events similar to those observed in normal immune de- that have been extensively depleted of mature T lym-
velopment, e.g., lymphoid commitment, thymic mi- phocytes in order to prevent GVHD results in signifi-
gration and entry, and thymic differentiation. It is likely cant delays in immune reconstitution. Therefore, it is
that the time period required for such development is likely that much of what has been attributed to T cell
similar to the time normally required for prenatal T generation in the first few months after transplant is the
364 American Society of Hematology
result of adoptive transfer of post-thymic cells. Such ence may be the age of the HSC—a few months for
cells may undergo homeostatic proliferation in the UCBC versus decades for adult HSC. There have been
lymphopenic environment of the host, or IL-2 driven some studies of the aging process in hematopoietic pro-
proliferation if activated by exposure to alloantigens or genitors, but there is little direct evidence that HSC
nominal antigens. Analyses of the length of time re- from adult donors have decreased capacity to contrib-
quired for de novo generation of T lymphocytes after ute to lymphocyte development compared to other pro-
HSCT are best performed in patients receiving highly genitors.
purified progenitors are depleted of all post-thymic T
lymphocytes. The Placenta as Unique Microenvironment
That Influences UCBT
UCBC HSC Besides intrinsic differences, HSC in UCBC have had a
HSC derived from UCBC differ from those of adult different set of microenvironmental exposures compared
marrow or PBSCT in several ways. The most notable to those of adult marrow or PBSC. All HSC sources
difference is quantitative—the cell dose in UCBT is are influenced by the microenvironment from which
significantly less than the HSC dose in adult sources they are derived. An example of differences between
such as PBSC or marrow. While low cell dose has been sources are some of the observed changes in HSC cell
associated with failure of hematopoietic engraftment, cycle status, gene expression and adhesive and invasive
HSC dose may also influence immune reconstitution. properties induced by mobilization procedures used to
In a murine congenic transplant model, increasing the generate PBSC, e.g., G-CSF.
dose of phenotypic HSC administered resulted in greater The placenta is a complex organ that regulates ma-
thymic cellularity post-transplant, as well as greater ternal-fetal interactions. Many cytokines that can influ-
numbers of mature T lymphocytes derived from the ence lymphohematopoietic development, e.g., G-CSF,
transplanted cells.6 Thus, one of the limiting factors for c-kit ligand (stem cell factor [SCF]), GM-CSF, IL-15,
T lymphocyte reconstitution after HSCT, besides mi- and others, are produced by the placenta. Production of
croenvironmental defects, may be the number of pro- G-CSF by the placenta may be especially relevant to
genitor cells entering the thymus and contributing to UCBT (Figure 2). G-CSF is produced both by the
thymopoiesis. Limited numbers of HSC in UCBC com- maternal decidua and the fetal chorionic villi8 and en-
pared to marrow or PBSC products may delay or de- ters the fetal circulation by a process that does not re-
crease thymic regeneration. quire a functional G-CSF receptor. G-CSF from the
There are also qualitative differences between mother probably does not enter the fetal circulation as
UCBC and other HSC sources (Table 5). Comparisons administration of recombinant human G-CSF (rhG-
of CD34+ CD38– phenotypic HSC derived from UCBC CSF) to pregnant macaques did not result in detectable
versus adult marrow have shown that UCBC contain a rhG-CSF in the fetuses. The function of placental G-
higher frequency of LTCICs and a higher percentage CSF production is unknown; however, it may serve as
of cycling cells.7 UCBC-derived HSC also had greater an immunoregulator that protects the mother and fetus
cloning efficiency and generative capacity. Thus, the from each other’s allogeneic immune systems. G-CSF
HSC in UCBC may be both more primitive and more inhibits the ability of placental mononuclear cells to
capable of regenerating hematopoiesis in the recipient. mediate cytotoxicity against allogeneic targets includ-
The contrasting biological properties may represent in- ing choriocarcinoma cells. The production of G-CSF
trinsic or environmental differences in the UCBC and and other cytokines capable of HSC mobilization, e.g.,
adult marrow HSC. The most likely intrinsic differ- SCF, is likely to explain the high percentage of HSC in
Table 5. Properties of umbilical cord blood (UCB) cell populations relevant to immune responsiveness.
Hematopoietic Stem Cells Dendritic Cells T Lymphocytes
Decreased number Decreased immune activity Decreased number
Increased cell cycling Decreased co-stimulation Naive phenotype
Increased generative capacity Decreased IL-12 production Resistance to activation
More immature? Decreased cytokine expression
Skewing to Th2 responses (decreased IL-2, INF-γ production)
Abbreviations: IL, interleukin; INF, interferon
Hematology 2004 365
besides the logistical problem of trans-
planting the intrathymic ETP popula-
tion, it is unclear if such cells could
home and engraft after transplantation.
Murine CLP can be transplanted into
irradiated hosts and contribute to post-
transplant lymphopoiesis. Co-transplan-
tation of CLP with HSC is able to more
rapidly restore immune function than
transplantation of HSC alone, and can
result in increased protection of mice
from experimental challenge with mu-
rine CMV.9 Although human cells with
CLP-like features can be found abun-
dantly in UCBC (personal communica-
tion, Gay Crooks), rigorous compari-
son of the numbers and functional prop-
erties of CLP in UCBC versus marrow
or PBSC still need to be performed.
Figure 2. Placental granulocyte colony-stimulating factor (G-CSF)
production affects umbilical cord blood cells (UCBC).
UCBC Post-Thymic T Cells
G-CSF produced in the placenta may affect both fetal HSC and dendritic cells.
Placental G-CSF may induce mobilization of the fetal HSC, resulting in significant Studies over the last 20 years have dem-
numbers of circulating HSC among UCBC, which declines rapidly after delivery. onstrated differences between neonatal
G-CSF may also suppress the immunological potency of fetal dendritic cells by T lymphocytes and those of adults
interfering with IL-12 production and co-stimulatory activity. As a result, the
immune responsiveness of fetal T lymphocytes is attenuated and skewed away (Table 5). The major notable difference
from Th1 responses. between the T lymphocytes in UCBC
and those derived from adult marrow
or mobilized PBSC is the maturational
UCBC and their rapid disappearance from the neonate’s status. Because the fetus is exposed to few foreign anti-
circulation after birth. In contrast to mobilization in- gens, the T lymphocytes in UCBC are almost exclu-
duced by a short period of monotherapy with G-CSF sively naive. Naive T lymphocytes express a phenotype
before PBSCT, the HSC in UCBC have been exposed that is identifiable as CD45RA+ CD45R0– and CD62L+.
continually to a complex mixture of cytokines that likely As individuals age, there is an increase in the frequency
affect their behavior, e.g., induce a high rate of cell of T cells that have differentiated into a memory phe-
cycling,8 or alter the homing and invasive properties notype, CD45RA– CD45R0+ and CD62L–/low as a result
needed for entry of progenitors into the thymus. of antigenic exposure. Memory cells are more readily
activated by antigen than naive T cells and may be able
UCBC and Lymphoid Progenitors to respond to weaker co-stimulatory signals through
Little is known about the numbers and functional prop- the B7-CD28 pathway. The predominant naive pheno-
erties of circulating lymphoid progenitors present in type of T lymphocytes in UCBC may contribute to the
UCBC compared to other sources. Commitment to lym- reduced alloreactivity observed after UCBT.
phoid differentiation by donor HSC could be a rate- Investigations of neonatal T cell function have been
limiting step in the ability to repopulate the recipient driven by interest in the increased susceptibility to se-
immune system after HSCT. At present, there is some vere viral infections in neonates. For example, new-
controversy regarding the nature of intermediate lym- borns infected with HSV have higher rates of dissemi-
phoid progenitors. The murine common lymphoid pro- nation (sepsis, meningoencephalitis) than adults with
genitor (CLP) has been described as a phenotypic popu- primary infection, and increased severity of some viral
lation in the marrow that can contribute to T, NK, and infections such as enteroviruses is observed until at least
B lymphopoiesis, but it is not clear if CLP generate all several months of age. Investigations of neonatal helper
thymocytes. There is some evidence that early thymic T lymphocytes have demonstrated decreased produc-
progenitors (ETP) in the thymus can arise in a CLP- tion of Th1 cytokines needed for cytolytic and anti-
independent manner. At present, CLP are the only lym- viral responses, compared to adult T lymphocytes.10
phoid progenitor that are relevant to transplantation; Specifically, the activation of the interferon-γ (IFN-γ )
366 American Society of Hematology
gene is markedly reduced in neonatal T lymphocytes, functions of DC. Recent data on DC generation after
because of decreased induction of transcription factors, HSCT suggests that G-CSF administration significantly
e.g., NF-AT, which are required for activation of the decreases the ability to produce IL-12.11 The decrease
IFN-γ gene after TCR stimulation. A central question in IL-12 production after post-HSCT G-CSF adminis-
is whether such differences are intrinsic to UCBC ver- tration may be relevant to UCBT. As discussed above,
sus adult T lymphocytes. Interpretation of the differ- cells in the fetal circulation are probably constantly
ences has been confused by comparison of signaling exposed to placental G-CSF. Furthermore, many clini-
properties of UCBC T lymphocytes to unfractionated cal UCBT protocols have used post-transplant G-CSF
adult T lymphocytes. Since adult T lymphocytes are administration to accelerate the development of granu-
comprised of both naive and memory T cells, while locytes. The routine administration of G-CSF, com-
UCBC are almost exclusively naive T cells, ascribed bined with intrinsic defects in DC function, may de-
differences between them may simply reflect differ- crease the generation of Th1 responses after UCBT.
ences between the stringent activation requirements of The expected consequences of such an effect would be
naive helper T lymphocytes and the more easily acti- decreased capacity to control opportunistic viral and
vated memory cells. Only comparisons between UCBC fungal infections.
and purified naive T lymphocytes from adult blood can
be used to evaluate whether UCBC T cells have unique Anticipated and Empiric Properties of UCBT
properties different from adult peripheral blood naive Based on the progenitor and immunological properties
T lymphocytes. Regardless of the reason, T lympho- of UCBC, certain predictions can be made regarding
cytes from UCBC appear to be less capable of mediat- the expected immune reconstitution after UCBT. Com-
ing Th1 cytotoxic responses than those derived from pared to the detailed information available about the
adult sources. recovery of marrow function and the decreased risks of
The decreased capacity for Th1 responses has both severe GVHD, there are few systematic data on lym-
positive and negative implications for HSCT. The de- phoid recovery, and even less on such properties as an-
creased absolute numbers and decreased responsiveness tigenic repertoire and effector function. As discussed
of UCBC T lymphocytes contribute to the decreased above, immune function after HSCT can be derived
alloreactivity and greater tolerance for MHC disparity from either the adoptive transfer of preformed mature
observed in clinical UBCBT. However, the decreased T lymphocytes in the stem cell product or from the
responsiveness probably means that there is also less development in the host of donor-derived lymphocytes
adoptive immunity derived from UCBC T lymphocytes. from the donor HSC or other prethymic populations,
There are fewer T cells overall, and the number of e.g., CLP.2 The adoptively transferred T cells would be
memory cells capable of mediating recall responses to expected to represent most of the lymphocytes seen in
antigen is extremely limited. Any adoptive immunity the first few months after transplant, but are more likely
must be derived from the naive T lymphocytes present to be both impermanent and narrow in repertoire. Adop-
in the UCBC product. As discussed above, naive T lym- tively transferred T cells after UCBT are largely naive
phocytes are difficult to activate, rendering them less cells which will be less responsive to specific antigen
capable of responses to pathogens. These properties of than the memory cells in adult sources and probably
the mature T lymphocytes in UCBC would predict that less able to mediate Th1 effector functions than mar-
antiviral and fungal immunity may be decreased after row or PBSC. The HSC and pre-thymic progenitors
UCBT than after BMT or PBSCT. are expected to produce a long-lasting, broad immune
repertoire but would be slow to develop. The increased
UCBC Dendritic Cells generative capacity of UCBC HSC might predict more
Besides intrinsic differences in T lymphocytes, there rapid engraftment kinetics, but would be expected to
may be differences in antigen presenting cells (APC) be offset by the decreased dose of transplanted HSC in
derived from UCBC versus adult sources (Table 5). the average UCBT. Analyses of UCBT recipients have
Analyses of dendritic cells (DC) in UCBC have shown demonstrated slow de novo generation of T lympho-
that both so-called myeloid and plasmacytoid DC are cytes in the first year after transplant; however, by 2
present, although there may be a decreased proportion years post-UCBT recipients have evidence of both a
of myeloid DC compared to adult peripheral blood. broad repertoire and thymic function.3,12,13 It is unclear
The myeloid dendritic cells are thought to promote Th1 if there are differences in the kinetics of post-transplant
responses by producing IL-12. Studies of the DC de- lymphopoiesis in UCBT versus BMT. More rapid re-
rived from UCBC have indicated that there may be in- covery in children versus adults and nonmyeloablative
trinsic defects in production of IL-12 as well as other than myeloablative conditioning suggests that damage
Hematology 2004 367
to the thymic microenvironment as a result of aging or virus infections after UCBT versus BMT or PBSCT
cytotoxic therapies may be important determinants of need to analyze at risk populations, not the total num-
post-HSCT immune reconstitution.12 ber of recipients. For several infections, notably CMV
Although several studies have demonstrated the im- and HHV-6, there is evidence that at-risk recipients may
portance of UCBC CD34+ cell dose for likelihood of have greater rate of progression to symptomatic infec-
hematopoietic engraftment and engraftment kinetics, tion, with decreased evidence of viral control, although
analyses of the engraftment kinetics of lymphoid popu- this is not a universal finding.17-19 In the comparison of
lations are more limited. In a study from Case Western UCBT with MUD transplants at Case Western Univer-
University, unrelated donor UCBT was compared to sity, the overall risk of infection, including bacterial
the matched unrelated donor marrow transplantation.14 infection, correlated with the degree of lymphopenia.17
UCBT led to decreased rate of appearance of lymphoid The risk of early bacterial infection in UCBT recipi-
populations in the first 3 months after transplantation. ents may reflect differences in duration of neutropenia,
In the subsequent year, the absolute lymphocyte count not just lymphocyte numbers or function.16
of the UCBT recipients exceeded that of the MUD re- The available information about immune reconsti-
cipients. Indeed, the mean ALC in the UCBT group tution after clinical UCBT is quite limited. In order to
was normal by day 200 post-transplant but remained better understand both the kinetics and quality of im-
abnormally low in the MUD recipients during the en- mune reconstitution after UCBT, analyses of specific
tire first year. Since the T lymphocytes seen in the first lymphoid populations, immunological repertoire, mi-
100 days post-transplant are likely to be derived from togen and antigen-driven proliferation and function, and
adoptively transferred mature T cells, the decreased responses to test neo-antigens, e.g., keyhole limpet
lymphocyte count in the first 3 months after UCBT hemocyanin, are needed. Ideally, such studies would be
would be expected as a result of lower amount of such multi-institutional but use similar degrees of matching,
cells in UCBC products. The increased numbers of lym- CD34+ cell dose, pretransplant conditioning, support-
phocytes observed later could reflect greater capacity ive care and post-transplant immune suppression to con-
of the UCBC progenitors to produce lymphocytes de trol for the variables of GVHD, conditioning related
novo. However, this is uncertain: an alternative expla- microenvironmental damage and progenitor cell dose
nation could be that the increased GVHD observed in that likely have an impact on immune reconstitution.
MUD recipients led to more severe lymphopenia than At this time, the lack of direct comparisons of immune
that seen after UCBT. reconstitution hinders the ability to make decisions re-
The differences in UCBC T cells from adult sources garding the optimal source of HSC for transplantation.
would be expected to translate into UCBT recipients In a patient with an active viral infection, e.g., CMV or
having less GVHD but also less protection from viral a history of Epstein-Barr virus lymphoproliferative dis-
infections such as CMV or EBV-LPD. Overall, the risk ease (EBV-LPD), unrelated marrow from an immune
of infectious death in studies of UCBT may be higher donor may be better than UCBT. However, potential
than that of transplant from other sources, but large, HSCT recipients may have limited choices regarding
multi-institutional, randomized studies have not been potential HSC sources, and frequently the ability to rap-
done, and there is a bias toward use of UCBT in sicker idly perform transplant may trump any immunological
patients who cannot await a MUD BMT.15-19 In a non- disadvantages of UCBT.
randomized study of children with ALL, the incidence
of viral infections was greater after UCBT than after Strategies to Overcome the Problems
unrelated BMT.16 Complicating the analysis of actual of Immune Reconstitution After UCBT
infectious risk for some infections like herpes viruses is A paradox of UCBT is that some of the associated im-
the difference in potential source of virus between munological problems are causally related to the prop-
UCBT and either BMT or PBSCT. CMV- or EBV-in- erties that make UCBT attractive in the first place. For
fected B cells can be derived from either the donor or example, the decreased alloreactivity that has been clini-
recipient in BMT or PBSCT. In UCBT, CMV- or EBV- cally observed in UCBT reflects the quantitative and
infected cells would be expected to exclusively arise functional abnormalities that make adoptive transfer of
from the recipient. Assuming that CMV-negative blood antiviral immunity so unlikely immediately after UCBT.
products are administered to CMV-negative recipients, Efforts to improve immune reconstitution after any form
then only CMV-positive recipients of UCBT are at sig- of HSCT have been directed at two somewhat different
nificant risk of CMV disease, while CMV-negative BMT questions—accelerating the rate of development of func-
or PBSCT recipients could be infected through their tional immunity, especially that required to control cer-
seropositive donor. Therefore, comparisons of herpes tain early infections, e.g., CMV; or altering the long-
368 American Society of Hematology
term qualitative and functional defects such as not been reproducibly observed in larger animal mod-
hypogammaglobulinemia that contribute to morbidity els and IL-7 has additional effects on mature T cells
and mortality over the first decade after transplant. The that could limit its usefulness in allogeneic HSCT. Be-
first goal has been more pressing because of the high sides promoting the development of thymocytes, IL-7
impact of opportunistic infection on transplant-related treatment also increases the expansion of mature T lym-
mortality. Strategies in BMT or PBSCT designed to phocytes by promoting their proliferation and survival.
increase the adoptive transfer of antigen-specific T cells These effects have led to increased GVHD in some pre-
directed against important pathogens such as CMV or clinical experiments but have not been observed by oth-
EBV have used co-transplantation of cloned T cells that ers. Experimental usage of IL-7 in clinical allogeneic
have been expanded in vitro. The application of such transplants should probably be initially restricted to T
strategies to UCBT would require either cloned cells cell–depleted grafts, not UCBT, until more is known
that are derived from the host or a third party, or tech- about both efficacy and promotion of GVHD.
niques for in vitro priming of the naive T cells in the Another strategy to address the problem of mi-
UCBC graft. Nonspecific activation of the T cells in croenvironmental damage has been to decrease the tox-
the UCBC product would likely increase the risk of icity of the conditioning regimen. Besides decreasing
GVHD. the intensity of conditioning, another approach is the
Besides the manipulation of the preformed T cells administration of a biological response modifier, re-
in a graft, other strategies that have been proposed for combinant keratinocyte growth factor (KGF). KGF is
accelerating immune reconstitution have been aimed at a mesenchymally derived member of the fibroblastic
increasing the rate at which prethymic cells contribute growth factor family which interacts with an epithe-
to the mature T lymphocyte compartment, either by lial-specific receptor. Administration of KGF in both
increasing the number of transplanted lymphoid pro- preclinical experimental models and clinical trials has
genitors or by accelerating their development in vivo. demonstrated decreased regimen-related toxicities such
One potential strategy that has been demonstrated in as oral mucositis, but also less GVHD. KGF protects
murine models is the co-transplantation of committed TEC in murine transplant and aging models, thereby
progenitors, such as CLP.9 Administration of 3000 CLP increasing thymopoietic capacity and improving both
in addition to 500 HSC in lethally irradiated mice pro- the immediate rate of lymphocyte recovery and me-
vided significant protection from experimental infec- dium-term (3–4 months) immune reconstitution. The
tion with murine CMV, compared to HSC alone. Im- clinical relevance of protection of murine thymopoiesis
pressively, small numbers of CLP provide levels of pro- by KGF needs to be determined.
tection from MCMV that are comparable to that of mil-
lions of post-thymic cells. The technical difficulty for Summary
UCBT would be obtaining sufficient CLP to affect re- Immune reconstitution after UCBT is promoted by the
constitution, e.g., via ex vivo expansion. The use of increased generative capacity of UCBC HSC, and the
ligands for the Notch receptors that regulate both pro- decreased GVHD, but is hampered by fewer HSC, de-
liferation and commitment to lymphoid lineages might creased adoptively transferred antigen-specific T cells,
be useful for in vitro generation of prethymic progeni- and functional defects in the T cells. Clinical studies to
tors that could be used to augment those present in the understand the relative importance of each of these bio-
UCBC product.20 Based on the data of Chen et al, expan- logical properties to outcome are needed. At this time,
sion techniques to increase the number of transplanted it is difficult to use relative differences in immune re-
HSC might be expected to increase thymic output.8 constitution between HSC sources as a basis for choos-
Other strategies have focused on ways to increase ing the mode of HSCT, although BMT might be pref-
the in vivo development of T lymphocytes in the re- erential to UCBT for a recipient with an active viral
cipient after transplantation. These studies have been infection. Some but not all strategies aimed at improv-
driven by the hypothesis that the thymic microenviron- ing immune reconstitution after HSCT in general may
ment of HSCT recipients lacks sufficient capacity to be relevant to addressing the immunological and infec-
efficiently produce new T lymphocytes from prethymic tious problems observed after UCBT.
progenitors. Our group has emphasized that defects in
TEC numbers or function caused by aging, cytotoxic
therapies, or GVHD lead to a relative lack of intrathymic
IL-7 needed for thymopoiesis. Systemic administration
of IL-7 in murine models can dramatically improve
thymic output after transplantation, but the effects have
Hematology 2004 369
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