of June 6, 2013.
This information is current as
Hemopoietic Stem Cell Transplantation
Steroid Ablation following Allogeneic
Enhanced Immune Reconstitution by Sex
Ann Chidgey, Marcel R. M. van den Brink and Richard L.
Sutherland,Andrew S. Greenberg, Kartono H. Tjoe, Jayne S.
Eng, Vanessa M. Hubbard, Adam Kochman, Lucy M. Willis,
Muriglan, Maree V. Hammett, Morag K. Milton, Jeffrey M.
Gabrielle L. Goldberg, Önder Alpdogan, Stephanie J.
2007; 178:7473-7484; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 6, 2013
Enhanced Immune Reconstitution by Sex Steroid Ablation
following Allogeneic Hemopoietic Stem Cell Transplantation1
Gabrielle L. Goldberg,2* O¨nder Alpdogan,†Stephanie J. Muriglan,†Maree V. Hammett,*
Morag K. Milton,* Jeffrey M. Eng,†Vanessa M. Hubbard,†Adam Kochman,†Lucy M. Willis,†
Andrew S. Greenberg,†Kartono H. Tjoe,†Jayne S. Sutherland,* Ann Chidgey,*
Marcel R. M. van den Brink,3†and Richard L. Boyd3*
Delayed immune reconstitution in adult recipients of allogeneic hemopoietic stem cell transplantations (HSCT) is related to
age-induced thymic atrophy. Overcoming this paucity of T cell function is a major goal of clinical research but in the context of
allogeneic transplants, any strategy must not exacerbate graft-vs-host disease (GVHD) yet ideally retain graft-vs-tumor (GVT)
effects. We have shown sex steroid ablation reverses thymic atrophy and enhances T cell recovery in aged animals and in congenic
bone marrow (BM) transplant but the latter does not have the complications of allogeneic T cell reactivity. We have examined
whether sex steroid ablation promoted hemopoietic and T cell recovery following allogeneic HSCT and whether this benefit
was negated by enhanced GVHD. BM and thymic cell numbers were significantly increased at 14 and 28 days after HSCT
in castrated mice compared with sham-castrated controls. In the thymus, the numbers of donor-derived thymocytes and
dendritic cells were significantly increased after HSCT and castration; donor-derived BM precursors and developing B cells
were also significantly increased. Importantly, despite restoring T cell function, sex steroid inhibition did not exacerbate the
development of GVHD or ameliorate GVT activity. Finally, IL-7 treatment in combination with castration had an additive
effect on thymic cellularity following HSCT. These results indicate that sex steroid ablation can profoundly enhance thymic
and hemopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.
Immunology, 2007, 178: 7473–7484.
overall survival of transplant recipients, infections (particularly vi-
ral and fungal) remain a major cause of posttransplant morbidity
and mortality. In adults, the increased incidence of infections is
directly related to prolonged immune deficiency. In contrast, chil-
dren generally recover immune capacity within ?4–6 mo after
HSCT (1). The delay in lymphoid recovery in adult recipients is
The Journal of
llogeneic hemopoietic stem cell (HSC)4transplantation
(HSCT) is a potentially curative therapy for a variety of
hemological malignancies. Despite improvements in the
dependent on a variety of factors but seems to be primarily related
to the age-associated progressive decline of naive T cell export
from the thymus (1).
This decrease in T cell output results in a narrowing of the TCR
repertoire and a loss of humoral and cell-mediated immunity in
adults (2). This thymic involution becomes particularly pro-
nounced after puberty, coinciding with an increase in the produc-
tion of sex steroids (3–7).
Because thymocyte export is directly proportional to the cel-
lularity of the thymus (8, 9), age-related thymic atrophy results
in a gradual decrease in recent thymic emigrants (RTEs) (10,
11) and a decrease in the naive to memory T cell ratio (12–14)
resulting in a restricted TCR repertoire in both CD4?and
CD8?T cells (15, 16).
In addition, T cell proliferation in response to nonspecific and
receptor-mediated (CD3/TCR) stimulation is severely compro-
mised with age (17–19). B cell function is also diminished with
age, which is in part, due to the decline in T cell production and
subsequent lack of T cell help. However, there are also significant
age-associated changes inherent to B cell function (20). Despite B
cell numbers remaining relatively constant throughout life due to
tightly regulated homeostatic mechanisms, there is a decrease in ex-
port from the bone marrow (BM) and a subsequent clonal expansion
of peripheral B cells and thus a narrowing of the Ab repertoire (21).
Decreased Ab responses to foreign Ags in the aged are thought
to be primarily due to a decline in T cell help (20, 22). However,
defective class switching (23) and a preferential loss of high-af-
finity Abs may play a role (24).
Collectively, these data strongly align aging to a decline in im-
mune capacity involving both cellular and humoral responsive-
ness. Although these age-related changes appear to be largely be-
nign in healthy individuals, they have a profound impact in
*Department of Pathology and Immunology, Central and Eastern Clinical School,
Monash University, Melbourne, Australia; and†Department of Medicine, Memorial
Sloan-Kettering Cancer Center, New York NY 10021
Received for publication April 27, 2006. Accepted for publication March 21, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants HL69929, HL72412, CA107096, CA33049, and
P20-CA103694 from the National Institutes of Health and awards from the Leukemia
and Lymphoma Society, Emerald Foundation, Ryan Gibson Foundation, Elsa U.
Pardee Foundation, and the Experimental Therapeutics Center of Memorial Sloan-
Kettering Cancer Center funded by Mr. William H. Goodwin and Mrs. Alice Good-
win and the Commonwealth Foundation for Cancer Research. The work in the lab-
oratory of R.L.B. was partially supported by Norwood Immunology.
2Address correspondence and reprint requests to Dr. Gabrielle L. Goldberg at the
current address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New
York, NY. E-mail address: firstname.lastname@example.org
3M.R.M.v.d.B. and R.L.B. contributed equally.
4Abbreviations used in this paper: HSC, hemopoietic stem cell; HSCT, HSC trans-
plantation; RTE, recent thymic emigrant; BM, bone marrow; cx, castrated; GVHD,
graft-versus-host disease; GVT, graft vs tumor; DTH, delayed-type hypersensitivity;
Tg, transgenic; TCD, T cell depleted; DC, dendritic cell; TN, triple negative; DP,
double positive; SP, single positive; HPRT, hypoxanthine phosphoribosyltransferase;
LHRH, luteinizing hormone-releasing hormone.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on June 6, 2013
severely immunodepressed states, such as HIV infection and ther-
apeutic myeloablation and lymphoablation. In such cases, lympho-
cyte recovery is severely retarded with age. The atrophic thymus is
unable to reconstitute CD4?T cells that are lost during HIV in-
fection (25) and CD4?T cells take three to four times longer to
return to normal levels following chemotherapy in postpubertal
We and others have demonstrated that surgical and chemical
castration both delay the onset of, and reverse, age-related thymic
atrophy (3–7, 27, 28). Increases in thymic cellularity, T cell emi-
gration, and peripheral T cell function have been found following
sex steroid ablation in aged mice (27) (29, 30). Castration of aged
mice also results in an increase in IL-7-responsive B cell progen-
itors (including late pro-B cells, pre-B cells, and immature B cells)
and peripheral B cells (31). This increase in circulating B cells is
largely due to an increase in the number of recent BM emigrants
(CD45RlowCD24high) and these cells remain at an elevated level
for up to 54 days after castration (31).
More recently, we have investigated whether the inhibition of
sex steroids can be used to enhance the recovery of the hemopoi-
etic system following HSCT, using congenic transplants as a
model for autologous HSCT. We showed that sex steroid ablation
enhanced thymic reconstitution (27). However, this model does
not have the clinical complications of allogeneic HSCT, where the
recipients need to balance the polarized effects of GVHD and GVT
as well the increased susceptibility to posttransplant infections.
These problems coupled with the regular posttransplant immuno-
deficiency represent major challenges in the clinic. In this initial
study, we also showed preliminary data that chemical castration,
using an luteinizing hormone-releasing hormone agonist, increased
overall thymocyte number in allogeneic HSCT. However, we did
not examine the nature or extent of the T cell or BM recovery and,
most importantly, the issues critical to allogeneic HSCT: whether
the impact of renewed T cell function, which could also include
donor-derived T cells, exacerbated graft-vs-host disease (GVHD)
yet retained graft vs tumor (GVT). Therefore, the present study
addressed in detail the impact of sex steroid ablation on immune
and hemopoietic reconstitution and the levels of GVHD and GVT
in the context of allo-HSCT as well as in vitro and in vivo T cell
We found a remarkable increase in T and B cell reconstitution
without an exacerbation of GVHD or loss of GVT activity in mice
castrated (cx) before HSCT. Proliferation, cytotoxicity, and de-
layed-type hypersensitivity (DTH) assays were used to determine
the function of the lymphocytes produced. IL-7?/?and KGF?/?
mice and RT-PCR for several growth factors were used as a means
to elucidate the mechanisms by which this enhanced reconstitution
Materials and Methods
Anti-murine CD16/CD32 FcR block (2.4G2) and all of the following flu-
orochrome-labeled Abs against murine Ags were obtained from BD
Pharmingen: Ly-9.1 (30C7), CD127 (IL-7R) (A7R34), TER119 (TER-119)
CD3 (145-2C11), CD4 (RM4-5), CD8?.2 (53-5.8), TCR-? (H57-597),
CD45R/B220 (RA3-6B2), CD43 (S7), IgM-FITC (R6-60.2), CD11b (M1/
70), Ly-6G (Gr-1) (RB6-8C5), c-kit (2B8), Sca-1 (D7), CD11c (HL3), I-Ak
(11-5.2); isotype controls: rat IgG2a-k (R35-95), rat IgG2a-l (B39-4), rat
IgG2b-(A95-1), rat IgG1-k (R3-34), hamster IgG-group 1-k (A19-3), ham-
ster IgG group 2-l (Ha4/8), and 2.4G2 anti-FcR (FcR blocking). Strepta-
vidin-FITC, PerCP-PE also were obtained from BD Pharmingen.
Recombinant human IL-7 was provided by Dr. M. Morre (Cytheris,
Vanves, France). It has been confirmed that the proliferative effect of hu-
man recombinant IL-7 is equal to murine IL-7 (32). Tissue-culture medium
consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS,
100 U/ml penicillin, 100 ?g/ml streptomycin, and 2 mM L-glutamine (as
well as 50 mM 2-ME for the culture of cells and proliferation assays).
Mice and HSCT
Male C57BL/6J (B6, H-2b), C3FeB6F1/J([B6 ? C3H]F1; H-2b/k), B10.BR
(H-2k), B6D2F1/J (H-2b/d), CBA/J (H-2k), BALB/c (H-2d) B6;129-
Fgftm1Efu (KGF?/?) mice were obtained from The Jackson Laboratory
and used in experiments when they were between 8 and 12 wk of age.
IL-7?/?mice (BALB/c) were provided by Dr. B. Rich (Harvard Institute
of Medicine, Boston, MA). KGF?/?and IL-7?/?mice were used between
4 and 7 mo of age. RAG2p-GFP transgenic (Tg) mice (FVB background:
H2q) were provided by M. Nussenzweig, (Rockefeller University, New
York, NY). HSCT protocols were approved by the Memorial Sloan-Ket-
tering Cancer Center Institutional Animal Care and Use Committee. The
BM cells were removed aseptically from femurs and tibias. Donor BM was
depleted of T cells by incubation with anti-Thy-1.2 for 40 min at 4°C
followed by incubation with Low-TOX-M rabbit complement (Cedarlane
Laboratories) for 40 min at 37°C. Splenic T cells (for GVHD experiments)
were obtained by purification over a nylon wool column. Cells (5 ? 106
BM cells with or without splenic T cells and tumor cells) were resuspended
in DMEM (Invitrogen Life Technologies) and transplanted by tail vein
infusion (0.25-ml total volume) into lethally irradiated recipients on day 0.
Before transplantation, on day 0, recipients received 1300 cGy total body
irradiation (137Cs source) as split dose with 3 h between doses (to reduce
gastrointestinal toxicity). Mice were housed in sterilized microisolator
cages and received normal chow and autoclaved hyperchlorinated drinking
water (pH 3.0). Cell lines A20 and P815 were obtained from American
Type Culture Collection.
Mice were anesthetized and a small scrotal incision was made to reveal the
testes. These were sutured and removed along with surrounding fatty tis-
sue. The wound was closed using surgical staples. Sham castration required
the same surgical procedure, except for the removal of the testes. Castra-
tion was performed 1 day before BM transplant for both immune recon-
stitution and GVHD/GVT studies.
Flow cytometric analysis
BM cells, splenocytes, or thymocytes were washed in FACS buffer
(PBS/2% BSA/0.1% azide) and 1–2 ? 106cells were incubated for 30 min
at 4°C with CD16/CD32 FcR block. Cells were then incubated for 30 min
at 4°C with primary Abs and washed twice with FACS buffer. Where
necessary, cells were incubated with conjugated streptavidin for a further
30 min at 4°C. The stained cells were resuspended in FACS buffer and
analyzed on a FACSCalibur flow cytometer (BD Biosciences) with
For one-way MLRs, splenocytes for transplanted mice (4 ? 105cells/well)
were incubated for 5 days with irradiated (2000 cGy) BALB/c splenocytes
as stimulators (2 ? 105cells/well) in 96-well plates. For pan T cell acti-
vation, splenocytes (4 ? 105cells/well) were stimulated with anti-CD3
(145-2c11) and anti-CD28 (37.51) (2.5 ?g/ml as a final concentration of
each) for 4 days. In both assays, cultures were pulsed during the final 18 h
with 1 ?Ci/well [3H]thymidine and DNA was harvested on a Top Count
Harvester (Packard Biosciences). Stimulation indices were calculated as
the ratio of stimulated cells (cpm) over unstimulated cells (cpm).
51Cr release assays
Target cells were labeled with 100 ?Ci51Cr at 2 ? 106cells/ml for 2 h at
37°C and 5% CO2. After three washes, labeled targets were plated at 2.5 ?
103cells/well in U-bottom plates (Costar). Splenocytes cultured with irra-
diated BALB/c splenocytes (1:2 ratio) for 5 days were added at various E:T
ratios in a final volume of 200 ?l to 4–6 wells and incubated for 4 h at
37°C and 5% CO2. Subsequently, 35 ?l of supernatant was removed from
each well and counted in a gamma counter (Packard Biosciences) to de-
termine experimental release. Spontaneous release was obtained from wells
receiving target cells and medium only; total release was obtained from
wells receiving 5% Triton X-100. Percent cytotoxicity was calculated by
the following formula: percent toxicity ? 100 ? ((experimental release ?
spontaneous release)/(total release ? spontaneous release)).
Detection of alloreactive T cell clones with intracellular IFN-?
Briefly, splenocytes were incubated for 12–15 h (for secondary allogeneic
stimulation with T cell-depleted (TCD), irradiated stimulator cells) with
7474ENHANCED IMMUNE RECONSTITUTION AFTER ALLOGENEIC HSCT
by guest on June 6, 2013
Brefeldin A (10 ?g/ml), harvested, washed, stained with primary (surface)
fluorochrome (FITC, PerCP, and allophycocyanin)-conjugated Abs, fixed,
and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen), and
subsequently stained with anti-IFN-? PE. FACS analysis was conducted by
gating for the designated populations. Flow cytometer and software were
used as mentioned below.
Sham-cx and cx mice were sensitized day 42 after allogeneic HSCT by tail
vein injection with 200 ?l of 0.01% sheep RBC (Colorado Serum) in PBS.
Sensitized animals were challenged at day 46 in the right hind footpad with
50 ?l of 20% sheep RBC suspension while the left hind footpad received
the same volume of PBS solution as a control. Forty-eight hours later,
footpad swelling was measured with a dial-thickness gauge (Mitutoyo).
The magnitude of the response was determined by subtracting measure-
ments of PBS-injected left footpads from the experimental right ones.
Assessment of GVHD
Two models of GVHD were used: C57BL/6J (H-2b) into C3FeB6F1/J
(H-2b/k)—a major mismatch model and B10.BR (H-2k) into CBA/J
(H-2k)—a minor mismatch model. Three different T cell doses were used
in the minor mismatch model: 0.1 ? 106, 0.5 ? 106, and 1 ? 106. The
severity of GVHD was assessed with a clinical GVHD scoring system as
first described by Cooke et al. (33). Briefly, ear-tagged animals in coded
cages were individually scored every week for five clinical parameters on
a scale from 0 to 2: weight loss, posture, activity, fur, and skin. A clinical
GVHD index was generated by summation of the five criteria scores (0–
10). Survival was monitored daily. Animals with scores of 5 or more were
considered moribund and were humanely killed.
Assessment of GVT-P815 (H-2d) mastocytoma induction and
assessment of mastocytomic death vs death from GVHD
B6D2F1/J recipients received 1 ? 103P815 (H-2d) cells i.v. on day 0 of
allogeneic HSCT (5 ? 106TCD BM cells and 5 ? 105T cells of C57BL/6
origin). Survival was monitored daily and the cause of death after HSCT
was determined by necropsy by our veterinary pathologist Dr. H. T.
Nguyen (Cornell University, New York, NY) as previously described.
Briefly, death from leukemia was characterized by hepatosplenomegaly
and the presence of mastocytoma cells in liver and spleen on microscopic
examination, whereas death from GVHD was defined as the absence of
hepatosplenomegaly and leukemic cells in liver and spleen, and the pres-
ence of clinical symptoms of GVHD as assessed by our clinical GVHD
scoring system at the time of death.
Administration of IL-7
IL-7 was either given from days 0 to 13 or 21 to 27 i.p. at 10 ?g/day for
immune reconstitution studies. PBS was injected into control mice at the
same time points.
Thymic stromal cell isolation
Thymic stromal cells were isolated as described in Ref. 34. Briefly, thymic
tissue from at least 10 mice/treatment group was digested in 0.125% (w/v)
collagenase D (Roche Applied Sciences), then trypsin (Sigma-Aldrich) and
0.1% (w/v) DNase (Roche Applied Sciences) in RPMI 1640. Cells were
incubated with anti-CD45 microbeads and depleted of CD45?cells using
an autoMACS (Miltenyi Biotec).
Total cellular RNA from whole BM and CD45?thymic stromal cells was
reverse-transcribed using Superscript II reverse transcriptase (Invitrogen
Life Technologies). cDNA was PCR-amplified for 35 cycles (94°C for
30 s; 56°C for 30 s; 72°C for 60 s) with PCR Master Mix (Promega).
HPRT: 5?-CACAGGACTAGAACACCTGC-3? and 5?-GCTGGTGAA
TGCACTTGCAGGAGCGCAC-3? and KGF: 5?-GCCTTGTCACGACCT
GTTTC-3? and 5?-AGTTCACACTCGTAGCCGTTTG-3?. IL-7: 5?-GCCT
GTCACATCATCTGAGTGC-3? and 5?-TGAACCAGTAGATTCTTGGA
Enzymic digestion of IL-7?/?thymi
IL-7?/?mice, having a marked reduction in thymocyte development, con-
tain a large proportion of CD45?thymic stromal cells and hence each
thymus was subjected to enzymic digestion in 0.125% (w/v) collagenase/
dispase (Roche Applied Sciences) with 0.1% (w/v) DNase, to release most
of the stromal and lymphoid cells. This allowed for the accurate calculation
of total cellularity. Stromal cells were identified as being CD45?.
All values are expressed as mean ? SEM. The Mantel-Cox log-rank test
was used for survival data and all other statistical analysis was performed
with the nonparametric, unpaired Mann-Whitney U test. A p value of
?0.05 was considered statistically significant.
Castration increases BM cellularity, HSC numbers, and B cell
Male CBA mice were cx 1 day before allo-HSCT. There were sig-
nificantly more cells in the BM (16 ? 106? 1.4 ? 106) of cx mice,
compared with the sham-cx controls (9.5 ? 106? 3.0 ? 105) as early
as 14 days after HSCT (Fig. 1A). These numbers remained elevated in
HSCT recipients. Eight- to 12-wk-old male CBA mice were sham-cx or cx
and transplanted with 5 ? 106B10.BR TCD BM cells. A, BM cellularity.
B and C, Lineage mixture comprised CD3, CD8, CD4, CD45R, Gr1,
CD11b, NK1.1, and TER119 donor-derived LSK (Ly9.1?Lin?c-kit?Sca-
1?) (B) number and donor-derived CLP (Ly9.1?Lin?c-kitlowSca-1lowIL-
7R??) (C) number donor-derived B cell precursor number: pro-B cells
(Ly9.1?B220/CD45R?CD43?IgM?) (D); pre-B cells (Ly9.1?B220/
CD45R?CD43?IgM?), and immature B cells (Ly9.1?B220/CD45R?
CD43?IgM?). ?, Age-matched, untreated controls; u, sham-cx mice; f,
cx mice. ?, p ? 0.05 and each group contained four to five animals.
Castration enhances reconstitution of the BM in allogeneic
7475The Journal of Immunology
by guest on June 6, 2013
cx mice at day 28 (BM: 22 ? 106? 4.0 ? 106vs 14 ? 106? 2.2 ?
106). The cx mice had begun to approach pretransplant levels at this
time point. By day 42, there was no longer a difference between cx
and sham-cx mice with respect to BM cellularity.
Several studies have shown that sex steroids inhibit the prolif-
eration and/or differentiation of early hemopoietic precursors (35–
37). Therefore, the impact of castration on donor-derived LSK
(Lineage?Sca-1?c-kit?) numbers in the allogeneic HSCT setting
was investigated. The number of donor-derived LSK (Ly9.1?Lin?
Sca-1?c-kit?) was not significantly different 14 days after allo-HSCT
(Fig. 1B). By day 28, there were more Ly9.1?Lin?Sca-1?c-kit?do-
nor-derived LSKs in the BM of cx mice compared with the sham-cx
controls. Donor-derived LSK numbers in both treatment groups were
derived HSC number 42 days after allo-HSCT (Fig. 1B).
c-kitlowSca-1lowIL-7R??, were also significantly increased in cx
mice 14 and 28 days after allo-HSCT (Fig. 1C). This finding is in
agreement with earlier studies that suggest that lymphoid progen-
itors are steroid sensitive (36, 37).
In our analysis of B cell recovery, three stages in B cell
development were distinguished: pro-B cells (CD45R?CD43?
IgM?), pre-B cells (CD45R?CD43?IgM?), and immature B
cells (CD45R?CD43?IgM?). As early as 14 days after alloge-
neic HSCT, pre-B cell numbers in the BM of cx mice had reached
pretransplant levels (5.5 ? 106? 1.7 ? 106) and were significantly
higher than the sham-cx controls (2.08 ? 106? 5.0 ? 104) (Fig.
1D). At day 28, again there were significantly more pre-B cells
(sham-cx: 3.1 ? 106? 3.7 ? 105c.f. cx: 6.6 ? 106? 6.6 ? 105)
and immature B cells (sham-cx: 1.3 ? 106? 2.6 ? 105c.f. cx:
3.0 ? 106? 3.4 ? 105) in the BM of cx mice (Fig. 1D).
Castration before allo-HSCT results in an increase in thymic
cellularity as well as thymocyte and dendritic cell (DC) numbers
At the early time point of day 14, thymic cellularity is increased in
cx mice (55.4 ? 106? 1.8 ? 106) compared with sham-cx control
(25 ? 106? 2.6 ? 106) (Fig. 2A). These numbers remained sig-
nificantly elevated in cx mice 28 days after HSCT (72 ? 106?
5.9 ? 106vs 45 ? 106? 2.9 ? 106). By day 42, there was no
longer a significant difference between cx and sham-cx mice with
respect to thymic cellularity.
Donor-derived thymocytes (Ly9.1?) were divided into devel-
opmental stages on the basis of expression of CD3, CD4, and CD8:
donor-derived thymocyte and DC num-
ber are significantly increased follow-
ing allo-HSCT and castration. Eight-
to 12-wk-old male CBA mice were
sham-cx or cx and transplanted with
5 ? 106B10.BR TCD BM cells. A,
Thymic cellularity. B, Donor-derived
TN cells (Ly9.1?CD3?CD4?CD8?).
C, Donor-derived DP cells (Ly9.1?
CD4?CD8?). D, Donor-derived ma-
ture CD4 SP cells (Ly9.1?CD3?CD4?
CD8?). E, Donor-derived mature CD8
SP cells (Ly9.1?CD3?CD4?CD8?).
F, Host-derived DCs (Ly9.1?CD11c?
MHCIIhigh). G, Donor-derived DCs
(Ly9.1?CD11c?MHCIIhigh). ?, Age-
matched, untreated controls; u, sham-
cx mice; f, cx mice. ?, p ? 0.05;
??, (p ? 0.01) and each group con-
tained four to five animals.
Thymic cellularity and
7476ENHANCED IMMUNE RECONSTITUTION AFTER ALLOGENEIC HSCT
by guest on June 6, 2013
triple negative (TN) (CD3?CD4?CD8?), double positive (DP)
(CD4?CD8?), single-positive CD4 (SP CD4) CD3?CD4?CD8?,
and SP CD8 (SP CD8)-CD3?CD4?CD8?(Fig. 2, B–E). There
were no differences between sham-cx and cx mice when compar-
ing the proportions of the different thymocyte subsets (data not
shown). At day 14, the donor-derived thymocytes in both groups
were predominantly TNs and DPs (data not shown). However, as
early as 14 days after allo-HSCT, there were significantly more
donor-derived TN, DP, SP CD4 and SP CD8 thymocytes in cx
mice compared with sham-cx controls (Figs. 2, B–E). Twenty-
eight days after HSCT, DP and CD4 SP cell numbers remain sig-
nificantly elevated in the cx group. By day 42, all thymocyte sub-
sets were equivalent in sham-cx and cx mice.
Host and donor-derived DCs are thought to play integral roles in
the avoidance of self and graft rejection, respectively (38). Both host
and donor-derived DCs in the thymus were significantly increased in
the cx mice 14 and 28 days after allo-HSCT (Fig. 2, F and G).
Splenic cellularity is increased with more donor-derived
peripheral T and B cells 28 days after castration and allo-HSCT
Splenic cellularity in the cx mice was significantly elevated above
sham-cx spleen cell numbers 28 days after allo-HSCT (253 ? 106?
28.4 ? 106vs 126 ? 106? 13.9 ? 106) (Fig. 1C). The cx mice
had begun to approach pretransplant cellularities by day 28.
Again, this may have been because the recipients were young
mice and they had active posttransplant lymphopoiesis which
facilitated their recovery: the time required to generate normal
cellularity in the primary and secondary lymphoid tissues in the
sham-cx mice, however, was markedly delayed compared with
The increase in BM B cells and their precursors translated to a
significant increase in the number of immature B cells in the
spleens of cx mice, 28 days after HSCT (sham-cx: 64.9 ? 106?
6.4 ? 106c.f. cx: 112.0 ? 106? 10.0 ? 106) (Fig. 3B). These
ripheral T and B cell reconstitution in allogeneic
HSCT recipients. A–C, Eight- to 12-wk-old male
CBA mice were sham-cx or cx and transplanted with
5 ? 106B10.BR TCD BM cells. A, Splenic cellular-
ity. B, Donor-derived B cell number based on the
expression of CD45R/B220, IgM, and CD43. Pro-
B cells (Ly9.1?B220/CD45R?CD43?IgM?), pre-B
cells (Ly9.1?B220/CD45R?CD43?IgM?), and im-
mature B cells (Ly9.1?B220/CD45R?CD43?IgM?).
C, Donor CD4 T cells were Ly9.1?CD3?CD4?CD8?
and donor CD8 T cells were Ly9.1?CD3?CD4?
CD8?. ?, Age-matched, untreated controls; u, sham-
cx mice; f, cx mice. ?, p ? 0.05, ??, p ? 0.01 and
each group contained four to five animals. D and E,
Eight- to 12-wk-old male BALB/c mice were
sham-cx or cx and transplanted with 5 ? 10?6TCD
RAG2p-GFP Tg BM cells. D, Ly9.1?CD3?GFP?
RTEs in blood (per milliliter). E, Ly9.1?CD3?GFP?
splenic RTEs day 42 post allogeneic HSCT. ?, p ?
0.05 and each group contained five to seven animals.
Castration enhances donor-derived pe-
7477 The Journal of Immunology
by guest on June 6, 2013
results are in agreement with previous studies that suggest
that castration enhances B cell production and export from
the BM (31).
The increase in thymocyte numbers in cx mice translated to a
significant increase in the number of donor-derived mature CD4?
and CD8?T cells in the spleens of cx mice compared with the
sham-cx controls at day 28 (Fig. 3C).
RTEs are a distinct population of naive, immature, peripheral T
cells that have recently left the thymus (9, 39). In this study, we
used mice that have a GFP transgene driven by the MLR 2
(RAG-2) promoter to identify RTEs. These mice, which have been
used previously to identify RTEs (40), begin to express high levels
of GFP at the CD4?CD8?double-negative stage of thymocyte
development. GFP and RAG2 expression remain high throughout
the CD4?CD8?DP stage of development and although RAG2
expression decreases with the SP transition, these cells remain pos-
itive for GFP through SP maturation and export. Peripheral T cells
contain GFPhigh, GFPlow, and GFP?populations, of which Bour-
salian et al. (40) have shown that the GFPhighare the most recent
In this study, we found an increase in donor-derived (Ly9.1?
GFP?CD3?) RTEs in the blood at days 28 and 42 (Fig. 3D) and
spleen at day 42 (Fig. 3E) after HSCT in cx mice compared with
sham-cx controls. The difference reached significance in the spleen
at day 42 (Fig. 3E).
On a per cell basis, there is no significant functional differ-
ence between T cells from sham-cx and cx mice. To determine
the functional potential of peripheral T cells in cx mice after
allogeneic HSCT, a series of in vitro assay were performed. The
proliferative capacity of the splenic T cells was tested in two
sham-cx or cx and transplanted with 5 ? 106B10.BR TCD BM cells. Forty-two days after transplantation, T cell functionality was assessed. A,
Castration has no effect on the proliferative capability of T cells after allogeneic HSCT. Splenocytes were obtained from sham-cx (n ? 5) and cx
(n ? 5) for proliferation assays and were cultured for 4 days with anti-CD3 and anti-CD28 (5 ?g plate-bound) and [3H]thymidine was added during
the final 18 h of culture. B, Alloreactive T cell proliferation. Splenic T cells (4 ? 105cells/well) were incubated with irradiated (20 Gy) BALB/c
splenic stimulator cells (2 ? 105cells/well) in 96-well plates for 5 days and [3H]thymidine was added during the final 20 h of culture. Each group
contained five animals. C, Cytolytic activity of donor-derived T cells. Splenocytes were harvested from the transplanted sham-cx or cx mice
(described in Fig. 1) and cultures (2 ? 106cells/well) for 5 days in 24-well plates with irradiated (20 Gy) BALB/C (third-party) splenic stimulator
cells (1 ? 106cells/well). Cytotoxicity was determined against A20 (a BALB/c B cell lymphoma cell line) in a51Cr release assay. D and E,
Intracellular IFN-? expression of alloreactive T cells. Splenic B6 T cells were harvested on day 42 from sham-cx or cx recipients as described above
and incubated with irradiated (20 Gy) (BALB/c, third party) splenic stimulator cells in 24-well plates for 5 days. Cells were harvested, and
restimulated with TCD, irradiated (20 Gy) (BALB/c or B10.BR internal biological control) splenic stimulator cells for 16 h. Brefeldin A (10 mg/ml)
was added after the first hour of incubation. Intracellular IFN-? expression in donor-derived CD3?CD8?cells was measured by flow cytometric
analysis. Representative plots are shown in D and graphically represented as the percentage of donor-derived CD8?T cells that express IFN-? in
E. F, Measurement of T cell functionality by DTH assay. DTH assay (see Materials and Methods) was performed at week 6 following allogeneic
HSCT in sham-cx and cx mice, and the swelling was measured by subtracting left hind footpad swell from the right hind one.
Castration does not alter the function of donor-derived T cells following allogeneic HSCT. Eight- to 12-wk-old male CBA mice were
7478 ENHANCED IMMUNE RECONSTITUTION AFTER ALLOGENEIC HSCT
by guest on June 6, 2013
ways: anti-CD3/anti-CD28 cross-linking (Fig. 4A) and in a
third-party MLR (using irradiated BALB/c splenocytes as stim-
ulators) (Fig. 4B). There was no significant difference in the
proliferative capacity of peripheral T cells when comparing
sham-cx and cx mice in either of these settings. Forty-two days
after allogeneic HSCT, splenocytes were cultured with irradi-
ated BALB/c splenocytes (third party) for 5 days. Following 5
days of allogeneic stimulation, the vast majority of cells in cul-
ture were CD8?T cells. Half these cells were used in a CTL
(51Cr release) assay to determine the cytotoxicity of splenocytes
from sham-cx and cx mice. Splenocytes were tested for their
ability to kill51Cr-loaded A20 (BALB/c B cell lymphoma tu-
mor cell line) cells at different E:T ratios (Fig. 4C). There was
no significant difference between sham-cx and cx mice with
respect to cytotoxicity. The other half of the cells cultured for
5 days were restimulated overnight with either third-party
(BALB/c) or syngeneic (B10.BR) irradiated splenocytes and
brefeldin A to determine IFN-? production. Fig. 4D shows
IFN-? production by donor-derived CD8?splenic T cells fol-
lowing BALB/c primary stimulation and either BALB/c or
B10.BR secondary stimulation (control). This is represented
graphically in Fig. 4E. There was no significant difference in the
proportion of IFN-?-producing donor-derived CD8?when
comparing sham-cx and cx mice. To assess immune function in
vivo, a DTH assay was used whereby 42 days after castration
and allogeneic HSCT mice were sensitized with sheep RBCs.
On day 46, they were challenged and 24 and 48 h later, footpad
swelling was determined. The DTH response was enhanced
48 h after challenge when mice were cx at the time of allo-
HSCT compared with sham-cx controls (Fig. 4F). Collectively,
these functional assays demonstrate that the T cells in cx re-
cipients are comparable on a per cell basis with T cells from
sham-cx recipients and are capable of responding to novel Ags
with intact proliferation, cytotoxicity, and cytokine production.
However, the significantly more rapid T cell numerical recon-
stitution in cx recipients translates to an enhanced DTH re-
sponse even at 42 days after transplant suggesting a persistence
of the castration-mediated effects.
Castration prior to allogeneic HSCT does not exacerbate GVHD
and maintains GVT activity
Both GVHD and GVT are mediated, primarily, by alloreactive
donor-derived T cells, which are transferred with the allograft.
Any treatment used to enhance immune reconstitution has the
potential to exacerbate GVHD or, conversely, decrease GVT
To assess the effects of castration on GVT activity, we in-
jected the mastocytoma cell line P815 (H-2d) into B6D2F1/J
recipients at the time of transplant. Animals that died during the
experiment were autopsied and the cause of mortality (tumor vs
GVHD) was determined. Mortality due to mastocytoma re-
mained unchanged following castration (six of nine mice) when
compared with sham-cx controls (five of eight mice). This sug-
gests that castration does not diminish GVT response following
HSCT (Fig. 5A).
To establish that castration does not have a stimulatory effect on
alloreactive T cells of donor origin, GVHD was induced by the
addition of allogeneic donor T cells to the allograft. Two models
were used: a major mismatch model (C57BL/6J (H-2b) into
C3FeB6F1/J(H-2b/k)) (Fig. 5B) and a minor mismatch model
(B10.BR (H-2k) into CBA/J (H-2k) (Fig. 5, C–E). Three different
T cell doses were used in the minor mismatch model: 0.1 ? 106
(Fig. 5C), 0.5 ? 106(Fig. 5D), and 1 ? 106(Fig. 5E). There was
no significant difference in morbidity or mortality due to GVHD
when comparing cx and sham-cx mice (Fig. 5, B–E).
IL-7 and castration have an additive effect following
We and others have previously shown that IL-7 treatment can in-
crease the number of T and B cells in otherwise untreated animals
and can also enhance lymphoid recovery following severe immu-
nodepletion (32, 41–43). IL-7 is known to increase T cell numbers
through increased thymic activity as well as peripheral expansion
(44). We therefore assessed the effects of IL-7 administration in
combination with castration following allogeneic HSCT. Fourteen
days after treatment there were significantly more cells in the
thymi of cx mice and those given the combined treatment (cas-
tration and IL-7 administration). At this early time point, there
was no difference seen between the PBS-treated, sham-cx con-
trols and the IL-7-treated, sham-cx mice. There was also no
significant difference seen between the cx group and those re-
ceiving the combined treatment, suggesting that it is only the
effects of castration acting 14 days after allo-HSCT, IL-7 treat-
ment, and castration (Fig. 6A). At a later time point, day 28, the
cellularity of the thymi in both the castration alone group and
the IL-7 alone group is significantly higher than the control
crease GVT activity in allogeneic HSCT recipients. For A–E: Solid line and
filled circle is a TCD-BM-only (no T cells) control group; solid line and
outlined squares is a TCD-BM cx control group; solid line and filled tri-
angle is the sham-cx treated group; and dotted line and outlined triangle is
the cx-treated group. Control groups, n ? 4; test groups, n ? 8–10; sur-
vival is depicted as a Kaplan-Meier curve. A, GVT: Lethally irradiated,
B6D2F1/J recipients received P815 (H-2d) cells (1 ? 103), C57/BL6 TCD
BM cells (5 ? 106) and C57/BL6 T cells (5 ? 105). B, Major mismatch
GVHD model: lethally irradiated (B6 ? C3H)F1recipients received trans-
plants with B6 TCD BM cells (5 ? 106) plus splenic T cells (0.5 ? 106).
C–E, Minor mismatch GVHD model: lethally irradiated CBA recipients
received transplants with B10.BR TCD BM cells (5 ? 106) and a range
of T cell doses: C, 0.1 ? 106T cells; D, 0.5 ? 106T cells; E, 0.1 ? 106
Castration administration does not aggravate GVHD or de-
7479 The Journal of Immunology
by guest on June 6, 2013
have an additive effect in the thymus follow-
ing allogeneic HSCT. Eight- to 12-wk-old
male CBA mice were sham-cx or cx and
transplanted with 5 ? 106B10.BR TCD BM
cells. Recipient organs were harvested on day
14 (A) received in addition 10 ?g/day IL-7 or
PBS (control) by i.p. injection from days 0 to
13. Recipients killed on day 28 (B) received
10 ?g/day IL-7 or PBS from days 21 to 28.
Thymic cellularity was calculated from total
cell counts. ?, p ? 0.05, represents a signif-
icant increase in cell number in the cx group
compared with the sham-cx control. Control
indicates, sham-cx, PBS injected; CX: cx and
PBS injected; IL-7: sham-cx and IL-7 inject-
ed; and IL-7 and CX: cx and IL-7 injected. C,
Semiquantitative RT-PCR was performed on
whole BM and CD45?thymic stroma (at
least 10 mice/treatment group) 14 days after
allogeneic HSCT and castration. After HPRT
equilibration templates from cx and sham-cx
mice were compared for the expression of
TGF?1and KGF. These results have been
confirmed using BM and thymic stromal
template from a second experiment. IL-7 re-
mained undetectable in the BM and thymus
for up to 28 days after HSCT. D, KGF?/?
and IL-7?/?mice (n ? 6–8) were sham-cx
or cx and 14 days later thymic, BM, and
splenic cellularity were analyzed.
7480 ENHANCED IMMUNE RECONSTITUTION AFTER ALLOGENEIC HSCT
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group. In addition, the combination of IL-7 treatment and cas-
tration had an additive effect on thymic cellularity at day 28
after allogeneic HSCT (Fig. 6B).
Semiquantitative RT-PCR for IL-7, TGF-?1, and KGF reveals
an increase in KGF and a decrease in TGF-?1following
allogeneic HSCT and castration
RT-PCR analysis of whole BM cells revealed undetectable levels
of IL-7 transcript in both sham-cx and cx mice as late as 42 days
after allogeneic HSCT (Fig. 6C). When template from control,
untransplanted mice were used IL-7 was detected (Fig. 6C).
TGF?1and KGF are known to be key mediators of hemopoiesis.
Using 4-fold serial dilutions of template from at least 10 mice per
treatment group, templates were hypoxanthine phosphoribosyl-
transferase (HPRT) equilibrated and there appeared to be a de-
crease in TGF?1and an increase in KGF 14 days after castration
and allo-HSCT (Fig. 6C). These results are reproducible with sam-
ples from within the same experiment and samples from a second
independent experiment. CD45?thymic stromal cell TGF?1and
KGF levels were also tested. There was no visible difference in
either growth factor when comparing template from sham-cx and
Changes that occur following castration were seen in KGF?/?
mice but not IL-7?/?mice
To further study the possible mechanisms behind the enhanced
immune reconstitution following castration, KGF?/?and IL-7?/?
mice (4–6 mo old n ? 6–8) were cx and 14 days later, thymus,
spleen, and BM were analyzed (Fig. 6D). TGF?1
not be analyzed because they die prepubertally (45). Thymic cel-
lularity was significantly (p ? 0.01) increased when comparing
sham-cx and cx KGF?/?mice. Although no differences were seen
in the total cellularity of the BM and spleen at this early time point,
changes were seen in the B cell compartment of the BM, as seen
previously in wild-type mice (31) (data not shown). Due to the fact
that a large proportion of cells in the thymi of IL-7?/?mice are
CD45?stromal cells, enzymic digestion was used to obtain a sin-
gle-cell suspension when using these mice. By doing this, many
more cells are released into suspension which accounts for the
slightly larger thymic cellularity seen in this experiment compared
with previous literature (46). No differences were seen in the
thymi, spleen, or BM of IL-7?/?mice when comparing cx mice
and sham-cx controls (Fig. 6D). Taking into account the variability
within these experiments, it would appear that KGF is not oblig-
atory for castration-induced immune recovery, while IL-7 may
play a role.
Recipients of an allogeneic HSCT experience a prolonged period
of immune deficiency, which is often associated with life-threat-
ening infections. With increasing age of the recipient, the risk of
infection increases as does the time it takes for full immunological
reconstitution. The period of immunodeficiency following HSCT
can be ?1 year and recent long-term studies demonstrated a
decrease in TCR excision circle?CD4?T cells in older HSCT
patients compared with their donors (47, 48). This suggests that
thymic damage and the subsequent decline in T cell production
may be more prolonged than once thought. The majority of
post-HSCT infections are associated with a lack of CD4?pe-
ripheral T cells (49).
It is widely accepted that there is an association between the
clinical outcomes of HSCT and the number of transplanted cells
(50–53). Transplantation of an insufficient number of progeni-
tor cells may lead to delayed and reduced immune reconstitu-
tion and an increase in transplant associated morbidity and mor-
tality (50–53). In this study, we have shown that there are
significantly more donor-derived lineage?Sca-1?c-kit?LSKs
and CLPs when castration is performed before allo-HSCT. This
finding may allow for a decrease in the number of cells required
for a viable transplant.
Thymic production of naive T cells of a diverse TCR repertoire
is essential for the establishment of normal T cell function follow-
ing allo-HSCT (54–56). In an earlier study, we presented prelim-
inary data that sex steroid ablation induced by agonist LHRH in-
creased overall thymocyte number (27), however, we did not
examine the nature or function of these T cells, nor the impact on
GVHD and GVT. We also did not examine any effects on BM
recovery in allogeneic. Therefore, in the present series of experi-
ments, castration of mice before allo-HSCT has been shown to
reverse thymic damage caused by the conditioning regime and to
enhance thymic reconstitution following allo-HSCT. The in-
creased thymopoiesis was reflected across all thymocyte subsets.
At the earliest time point, the donor-derived thymocytes were pre-
dominately TNs and DPs. The more mature donor-derived SP cells
followed at days 28 and 42. At the later time points, in both the
sham-cx and cx groups, the proportion of donor-derived thymo-
cytes was equivalent to that of an untreated thymus suggesting
normal thymopoiesis and a lack of evidence for pathological T
cells in the postcastration setting. The changes observed in the
thymus translated to an increase in donor-derived peripheral T
cells and we have shown that this increase was at least in part due
to an increase in thymic export.
T cell-mediated immune responses are known to be diminished
for an extended period following allo-HSCT. In this study, we
demonstrated that when tested, in vitro T cell function did not
differ on a per cell basis when comparing cx and sham-cx mice
following allo-HSCT. Using DTH as a measure of in vivo T cell
function, castration before allo-HSCT lead to a stronger DTH (T
cell-mediated) response than that seen in control animals, suggest-
ing that sex steroid ablation may enhance T cell function following
HSCT in a persistent manner.
Although B cell reconstitution following HSCT is compara-
tively fast, functional deficiencies in these cells are present for
extended periods after transplantation (57). In this study, we
have shown that castration before allo-HSCT results in an in-
crease in donor-derived B cell lymphopoiesis and subsequent
increase in peripheral B cell numbers (also donor derived).
Hence, as for T cells, sex steroid ablation leads to accelerated
normalization of B cell numbers which may lead to an increase
in B cell function.
DCs are the key mediators of negative selection in the thymus
(58, 59) and in a transplant setting have been implicated in induc-
ing graft acceptance by presenting alloantigens in the thymus after
transplantation, deleting newly arising donor-specific T cells. For
example, donor-derived cells in the thymi of MHC class I-mis-
matched recipients mediate deletion of donor-reactive cells (60). It
has also been shown that thymus-derived DCs injected i.v. traffic
to the host thymus (61), however, whether this occurs physiolog-
ically is unclear. Also, intrathymic injection of host cells pulsed
with alloantigen, donor cells, or donor-soluble peptides increases
graft acceptance (62–65). In the current study, castration signifi-
cantly increased the number of host and donor-derived DCs in the
thymus following allogeneic HSCT. It is therefore possible that
castration, used in conjunction with hemopoietic stem cell and
solid organ transplantation, may increase graft acceptance. This is
particularly relevant in the clinic where the vast majority of trans-
plants are performed on older adults in whom thymic function is
7481The Journal of Immunology
by guest on June 6, 2013
minimal and hence HSC uptake for chimera formation greatly
Although they very likely act via intermediate cell types, both
estrogen and testosterone can directly affect the differentiation and
proliferation of HSCs (35–37). Estrogen directly inhibits the pro-
liferation and differentiation of HSCs as well as some lymphoid
precursor subsets (36, 37). HSCs express functional estrogen re-
ceptors (ERs) and estrogen administration decreases the number of
Lin?c-kit?Sca-1?HSCs (35, 37). Thurmond et al. (35) suggest
that the transition between c-kit?Sca-1?precursors and the more
mature subsets (c-kit?Sca-1?and c-kit?Sca-1?) is blocked when
ER? is present in the hemopoietic cells of the BM (35). ERs are
also present on BM stromal cells (66, 67), suggesting that es-
trogen may also have an effect on the production of growth
factors by the stroma, which in turn affects HSC proliferation
RT-PCR of the BM in the present study provided evidence for
an increase in KGF and a decrease in TGF-?—both potential mol-
ecules involved in the castration—induced enhanced BM function.
Use of KGF?/?and IL-7?/?mice demonstrated that both of these
may be required, yet there is also a wide body of evidence impli-
Batard et al. (68) have demonstrated that physiological con-
centrations of TGF-?1inhibit the proliferation and differentia-
tion of HSCs in vitro. As a corollary, disruption of TGF-? sig-
naling in HSCs (via the transient expression of a mutant type II
receptor) enhances survival and proliferation of these cells (69).
These findings are entirely consistent with the possibility that
the increased number of HSCs seen 28 days after allogeneic
HSCT and castration may in fact be due to a decrease in the
production of TGF-? by BM stromal cells, as indicated from the
Several studies have shown that sex steroid ablation, be it by
surgical or chemical castration, of male mice increases both BM
and splenic B cell numbers (31, 70–72). Olsen et al. (73) have
demonstrated that androgens enhance the production of TGF-?1by
stromal cells within the BM, which in turn suppresses B cell devel-
opment (73). In addition, neutralization of TGF-?1in vitro reverses B
cell suppression by dihydrotestosterone (73). It is therefore possible
that in our setting of sex steroid ablation, the opposite is occurring. A
enhancing B lymphopoiesis.
Sex steroid ablation reverses age-related thymic atrophy (3–7).
What remains to be fully understood is the mechanism by which
this occurs. Using transfer experiments with wild-type and testic-
ular feminization (tfm) mice, which have a point mutation in the
androgen receptor, Olsen et al. (74) have shown that it is the pres-
ence of a functional androgen receptor on the thymic epithelium
but not on the thymocytes that is essential for age-related thymic
involution and the subsequent regeneration via sex steroid
Although the molecular mechanisms for thymic involution
and age-related B cell defects (and their subsequent reversal)
remain unclear there are several potential candidates. Thymic
IL-7 levels decline with age (2, 75, 76), but it is unclear whether
this is due to a decrease in the number of cells that produce IL-7
or a decrease in the ability of the existing cells to produce the
cytokine. IL-7 treatment of old mice can reverse age-related
increases in thymic apoptosis and enhance thymopoiesis (77).
Stem cell factor and M-CSF mRNA expression is also de-
creased in the mouse thymus with age (75). Sempowski et al.
(78) have monitored mRNA steady-state levels in aging humans
and shown a significant increase in leukemia inhibitory factor,
oncostatin M, IL-6, and stem cell factor mRNA.
The above studies suggest that it is unlikely that castration
affects a single growth factor and it is more likely that the
response is multifactorial. Our experiments with castration of
IL-7?/?mice (Fig. 6B) suggest that increased production of
IL-7 is an important component of the castration effect. How-
ever, we observed an additive effect on thymic cellularity when
recipients were treated with both high-dose IL-7 and castration,
which would suggest that castration provides more thymopoi-
etic effects than increased IL-7 levels alone. The current study
has clearly demonstrated that sex steroid blockade has a pro-
found positive effect on immune reconstitution following my-
eloablation and allo-HSCT. HSC and B and T cell progenitor
recovery was enhanced leading to increased T and B cell pro-
duction in the primary lymphoid tissues and a subsequent in-
crease in donor-derived peripheral lymphocytes. Furthermore,
in vivo T cell function, as tested by DTH, is augmented. This
provides an important platform for increasing the efficiency of
engraftment and posttransplant strategies that depend on an in-
tact hemopoietic system.
When developing immune-enhancing treatments in the setting
of allo-HSCT it is essential to also address the unique dichotomy
of GVHD and the GVT response. In this study, we have estab-
lished that while sex steroid ablation enhanced in vivo T cell func-
tion, GVT activity was maintained and, importantly, GVHD was
Collectively, these results suggest that transient sex steroid ab-
lation (using, for example, LHRH agonists or antagonists) could be
developed as a prophylactic therapy to enhance posttransplant im-
mune reconstitution in allogeneic HSCT.
Richard Boyd, Ann Chidgey, Jayne Sutherland and Gabrielle Goldberg are
named as inventors on patents pertaining to this work.
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