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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4453-4463
A Critical Role for CD48 Antigen in Regulating Alloengraftment and
Lymphohematopoietic Recovery After Bone Marrow Transplantation
By
Bruce R. Blazar,
Patricia A. Taylor,
Angela Panoskaltsis-Mortari,
Hideo Yagita,
Jonathan S. Bromberg, and
Daniel A. Vallera
From the University of Minnesota Cancer Center and the Department of
Pediatrics, Division of Bone Marrow Transplantation and the Department
of Therapeutic Radiology-Radiation Oncology, University of Minnesota
Hospital and Cancer Center, Minneapolis, MN; Jutendo University School
of Medicine, Tokyo, Japan; and the University of Michigan, Ann Arbor,
MI.
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ABSTRACT |
The binding of CD2, present on T cells, to its counterreceptor CD48
facilitates adhesion, signaling, alloantigen-induced cytokine production, and cytotoxic T-lymphocyte responses. Because
these T-cell functions have been implicated in graft-versus-host
disease (GVHD) pathogenesis, we have analyzed the effects of the
CD2:CD48 pathway on GVHD mediated by CD4+ and
CD8+ T cells infused into sublethally irradiated
recipients. CD4+ T-cell-mediated, and to a lesser
extent, CD8+ T-cell-mediated GVHD was inhibited by
 CD2 + 48 monoclonal antibody (MoAb) infusion. To assess
the effects of combined MoAb infusion on alloengraftment, two different
alloengraftment bone marrow transplantation (BMT) models were used. In
both, MoAb infusion markedly inhibited alloengraftment and
hematopoietic recovery post-BMT. To determine if the adverse effects on
lymphohematopoiesis in the allogeneic BMT recipients were caused by an
immune or nonimmune mechanism, studies were performed in congenic BMT
recipients to preclude an immune mechanism as the cause for delayed
recovery post-BMT. MoAb infusion resulted in impaired
lymphohematopoietic recovery in congenic BMT recipients and markedly
reduced day 12 colony-forming unit-spleen formation in syngeneic BMT
recipients, consistent with a nonimmune mediated mechanism. Because the
spleen is a site of early hematopoietic recovery post-BMT, studies were performed using adult splenectomized syngeneic BMT recipients. MoAb
infusion delayed recovery in both nonsplenectomized and splenectomized recipients post-BMT, indicating that the delayed hematopoietic recovery
was not the consequence of an abnormal homing pattern of hematopoietic
progenitors to the spleen early post-BMT. CD48 MoAb was necessary
and sufficient for the inhibition of GVHD lethality and delayed
lymphohematopoietic effects of the combined MoAb regimen. CD48 MoAb
was found to induce a profound modulation of CD48 antigen expression on
BM cells, suggesting that the CD48 antigen may have an important
function in hematopoiesis in the BM compartment. Taken together, these
data provide evidence that the CD48 antigen plays a critical role in
regulating hematopoiesis in post-BMT.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CD2, FIRST DESCRIBED as the sheep
erythrocyte receptor, is a 55- to 60-kD glycoprotein expressed on the
surface of T cells, B cells, natural killer (NK) cells, and some
antigen-presenting cells (APC).1-5 The multimeric binding
of murine CD2 to natural ligand facilitates the adhesion between
T cells and APC and delivers a costimulatory signal for
initiating T-cell responses.6-10 The in vivo administration
of CD2 monoclonal antibody (MoAb) has been shown to inhibit
CD4+ and CD8+ T-cell alloresponses, leading to
prolonged allograft survival.9,11,12 CD48, a 45-kD glycosyl
phosphatidylinositol-anchored glycoprotein member of the immunoglobulin
supergene family, is expressed on almost all T cells and B cells, and
is the natural ligand for CD2. CD48 participates in T-cell
activation.10 CD48 MoAb has been shown to inhibit
interleukin-2 (IL-2) production, IL-2 receptor expression,
T-cell proliferation,13 and IL-4 production.14 CD2 + CD48 MoAb have been shown to have synergistic effects in inhibiting alloresponses including T-cell priming to alloantigen, IL-2
production, and T-cell help for allo-cytotoxic T-lymphocyte (allo-CTL)
generation.14
Although the in vivo administration of CD2 + CD48 MoAb has been
shown to result in the indefinite survival of cardiac
allografts,14 the proinflammatory response generated by
cardiac allografting is predominantly local, whereas that of
irradiation used to condition bone marrow transplantation (BMT)
recipients as well as the graft-versus-host disease (GVHD) process
itself is systemic. Therefore, the current study was undertaken to
determine whether CD2 and CD48 MoAb were capable of inhibiting
GVHD lethality. In the process of performing these studies, we
identified a critical role for CD48 in regulating alloengraftment as
well as lymphohematopoietic recovery in both allogeneic and congenic
BMT recipients. These data have significant implications for human BMT
studies.
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MATERIALS AND METHODS |
Mice.
For BMT experiments, B10.BR/SgSnJ (H2k),
B6.C.H2bm1 (termed bm1), B6.C.H2bm12 (termed
bm12), C57BL/6-severe combined immunodeficiency disorder (scid)/(termed
B6-SCID) (H2b), and DBA/1 (H2q) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 (termed
B6) (H2b; CD45.1), B6-Ly 5.2 (B6-CD45.2), and BALB/c
(H2d) mice were purchased from the National Institutes of
Health (Bethesda, MD). Female donor and recipient mice were 8 to 10 weeks old at the time of BMT. All mice were housed in a specific
pathogen-free facility in microisolater cages.
Anti-CD2 + 48 MoAb preparation and administration.
Anti-CD2 MoAb (hybridoma RM2-2, rat IgG) and CD48 (hybridoma HM48-1,
hamster IgG) were generated as described.10,15 Irrelevant rat IgG and irrelevant hamster IgG were purchased from Rockland Laboratories (Gilbertsville, PA). Anti-CD2 and CD48 MoAb were generated from ascites fluid, purified by ammonium sulfate
precipitation, and dialyzed before injection in vivo. Mice were
injected intraperitoneally (IP) with CD2, CD48, or both MoAb (300 µg/dose each) or irrelevant IgG (300 or 600 µg/dose, as
appropriate) on days  1 and +2, and then twice weekly until day
+21 post-BMT. The maximal immune suppressive CD2 MoAb dose is 100 µg/dose administered on days 0 and 1.11
GVHD induction.
For examining the effects of CD4+ or CD8+ T
cells on the recognition of isolated major histocompatibility complex
(MHC) disparities, bm12 or bm1 recipients were sublethally irradiated
(6.0 Gy total body irradiation [TBI] from a 137Cesium
source at a dose rate of 85 cGy/minute) and injected with highly
purified lymph node (LN) CD4+ (1.0 × 105
cells) or CD8+ (1 × 106 cells) B6 T
cells, respectively, as described.16 In other experiments, purified CD4+ LN bm12 T cells (106 cells) were
infused into nonirradiated B6-SCID recipients to assess whether CD2 + 48 MoAb was preventing GVHD lethality by blocking the interaction of
donor T cells with host B cells. These latter experiments were used to
quantify the degree of donor T-cell engraftment of combined MoAb
treatment in a system in which the only effect of treatment would be on
the immune response of donor T cells to host alloantigens. Five to
eight mice per group per experiment were given allogeneic donor T
cells.
To purify LN cells, single-cell suspensions of axillary, mesenteric,
and inguinal LN cells were obtained (as a source of GVHD-causing effector cells) by passing minced LN through a wire mesh and collecting them into RPMI 1640. Cell preparations were depleted of NK cells and
either CD8+ (hybridoma 2.43, rat IgG2b; provided by Dr
David Sachs, Charlestown, MA) or CD4+ (hybridoma GK1.5, rat
IgG2b; provided by Dr Frank Fitch, Chicago IL) T cells by the
appropriate MoAb coating and passaged through a goat -mouse and goat
-rat Ig-coated column (Biotex, Edmonton, Canada). The final
composition of T cells in the donor graft was determined by flow
cytometry and was always found to be 94% CD4+ or
CD8+ T cells. For sublethally irradiated recipients of
donor MHC disparate T cells, hematocrit values were obtained at
periodic intervals as an indicator of the possible BM destructive
effects of infused T cells.16
Allogeneic and congenic engraftment experiments.
For alloengraftment experiments involving BALB/c donors, B6 recipients
were administered 6.0 or 6.5 Gy TBI. BALB/c donor BM was treated with
Thy1.2 (hybridoma 30-H12, rat IgG2b; provided by Dr David Sachs)
plus complement. After treatment, donor cell suspensions were washed,
resuspended, and 107 treated BM cells were injected via the
caudal vein. For studies involving DBA/1 donors, B6 recipients were
administered 6.0 Gy TBI and relevant MoAb as described above. Ten to 15 mice per experiment were analyzed.
For congenic BMT experiments, B6 recipients were administered 8.0 Gy
TBI and cohorts of mice were infused with 0.3, 1.0, or 3.0 × 106 nonmanipulated B6-CD45.2 BM and either CD48 MoAb or
irrelevant IgG (300 µg/dose) IP as described above. Mice were
analyzed for survival and either peripheral blood hematologic recovery
or donor cell engraftment and cellularity in the BM and spleen at
periodic intervals post-BMT, as indicated in the tables and figures.
To determine whether CD48 MoAb altered the homing of hematopoietic
progenitor cells to the spleen and thereby compromised lymphohematopoietic recovery, cohorts of B6 mice were adult
splenectomized 2 weeks before reconstitution with B6-CD45.2 congenic BM
(3 × 106/recipient) and either CD48 or
irrelevant MoAb.
Day 12 colony-forming unit-spleen (CFU-S) formation.
To determine the effect of MoAb on early BM-derived progenitor cell
repopulation in vivo, syngeneic CFU-S formation was assessed. B6
recipients were irradiated with 7.5 Gy TBI (via 137Cesium
source) on day 1 and then administered nonmanipulated B6 BM
cells (105/recipient) on day 0. Mice were injected with
CD2, CD48, and MoAb and/or irrelevant rat IgG as
described above through day 9 post-BMT. Twelve days post-BMT, mice were
killed and spleens were placed into Bouin's solution to facilitate
enumeration of colonies. Eight mice per group were transplanted.
Ex vivo and in vivo effects of CD48 MoAb on lymphohematopoietic
cells in non-BMT controls.
To determine the effect of CD48 MoAb on lymphohematopoiesis in the
BM and spleen of non-BMT controls, B6 mice were administered irrelevant
or CD48 MoAb (300 µg) IP as described above through day 7 post-BMT. On day 9, mice were electively killed for flow cytometric
analysis of BM and spleen.
To assess the effect of CD48 MoAb on modulation or coating of the
CD48 antigen on spleen cells or BM cells, single-cell suspensions of
each were obtained from non-BMT B6 controls and then incubated with
CD48 MoAb (20 µg/mL) for 30 minutes on ice. Cells were washed and
then incubated with goat anti-hamster fluorescein isothiocyanate (FITC)
or CD48-FITC (Pharmingen, San Diego, CA). As a positive control for
splenocyte analysis, CD3 MoAb (hamster 145-2C11; provided by Dr
Jeffrey Bluestone, University of Chicago) was used under the same
conditions.
Flow cytometry.
The T-cell, B-cell, and granulocyte/macrophage constituency of
splenocytes or BM cells was measured using MoAb directed toward CD4 or
CD8, CD45R/B220, and CD11a, respectively. Anti-CD2 and CD48
fluorochromes also were used to quantify changes in the expression of
CD2 or CD48 antigens in MoAb-treated or control mice.
Fluorochrome-labeled MoAbs all were obtained from Pharmingen. Chimerism
of peripheral blood mononuclear cells was analyzed at an earlier time
point (7 weeks post-BMT) and at later time points (days 110 to 145 post-BMT). For quantification of donor alloengraftment, H2d (hybridoma 34-5-8S, mouse IgG2a) or
H2q (hybridoma 66-3.5, mouse IgG; both provided by Dr
David Sachs) were directly conjugated to FITC. For host-cell
quantification, H2b (hybridoma EH144, mouse IgG;
provided by Dr T.V. Rajan, Farmington, CT) was directly conjugated to
phycoerythrin (PE).
For congenic BMT experiments, the expression of CD45 alleleic
determinants on BM and splenocytes was monitored using CD45.1 (clone
104-2, rat IgG2a) and CD45.2 (clone A20-1.7, rat IgG2a; both
provided by Dr U. Hammerling, New York, NY). For all flow cytometry
studies, single-cell suspensions were incubated with 2.4G2 to block Fc
receptors, and then incubated with an optimal concentration of
fluorochrome-labeled MoAb for 45 minutes at 4°C. Cells were washed
three times and resuspended for analysis. An anti-human CD7 (hybridoma
3A1E, mouse IgG; provided by Dr Barton Haynes, Duke University, Durham,
NC) was conjugated to FITC or PE to determine control (background)
staining. Background values were subtracted from values obtained with
relevant antibodies as previously described.17 Flow
cytometry was performed on a FACScalibur (Becton Dickinson, Mountain
View, CA). Ten thousand events (determined by forward- and side-light
scatter) were analyzed.
Statistical analyses.
Group comparisons of continuous data were made by Student's
t-test. Survival data were analyzed by lifetable methods using the
Mantel-Peto-Cox summary of chi-square.18 Actuarial survival rates (the proportion of mice surviving on each day post-BMT) are
shown. Hematologic and chimerism data were analyzed as individual values with data presented as mean ± 1 standard error of the mean (SEM). Probability (P) values <.05 were considered
significant.
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RESULTS |
The in vivo infusion of CD2 + 48 MoAb is partially effective in
delaying CD8+ T-cell-mediated lethality but is highly
effective in preventing CD4+ T-cell-mediated lethality.
To investigate the effect of CD2:CD48 blockade on CD8+
T-cell-mediated GVHD, CD2 + 48 or irrelevant MoAb was administered to sublethally irradiated bm1 recipients of 106 B6
CD8+ T cells (Fig 1A).
Cumulative data pooled from two replicate experiments indicate that
CD2 + 48 MoAb-treated recipients had a significantly (P = .002) higher actuarial survival rate as compared with controls (n = 16/group), although there was only one long-term survivor treated with
combined MoAb. CD8+ T-cell dose titration experiments
indicated that although combined MoAb treatment was associated with a
significant prolongation in time to mortality, none of the recipients
of as few as 3 × 105 CD8+ T cells
survived beyond day 22 posttransfer (data not shown). Therefore,
combined MoAb infusion was only partially and modestly effective in
reducing CD8+ T-cell alloresponses in vivo.
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| Fig 1.
Anti-CD2 + 48 MoAb is ineffective in preventing
GVHD-induced lethality by donor CD8+ T cells infused into
sublethally irradiated recipients. Sublethally irradiated bm1 (A) or
bm12 (B) recipients were administered the indicated number
of highly purified CD8+ or CD4+
B6 LN cells as shown in parentheses on day 0. Mice received either
irrelevant IgG or CD2 + 48 MoAb (300 µg/dose each) IP twice
weekly from days 1 through +21 posttransfer. On the x-axis are the
days posttransfer and on the y-axis is the proportion of mice
surviving. The survival data are plotted. Results from two (A, n = 16/group) or three (B, n = 24/group) replicate experiments with
similar results are pooled. Mice administered CD2 + 48 MoAb had a
significantly (P = .002) higher actuarial survival rate as
compared with controls.
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To determine whether CD2:CD48 blockade could inhibit CD4+
T-cell-mediated GVHD, CD2 + 48 or irrelevant MoAb was administered to sublethally irradiated bm12 recipients of B6 CD4+ T
cells (105/mouse) (Fig 1B). Cumulative data pooled from
three replicate experiments (n = 24/group) indicate that CD2 + 48 MoAb-treated recipients had a significantly (P = .0009) higher
actuarial survival rate as compared with controls (82% v 0%,
respectively). Day 14 mean hematocrit values were significantly
(P = 5.6 × 10 11) higher in CD2 + 48 MoAb-treated recipients as compared with controls (33% v
19%, respectively). Anti-CD2 + 48 MoAb-treated recipients were
not GVHD free as shown by splenic B-cell lymphopenia found on days 82 and 109 posttransfer and confirmed by acute GVHD histology of the lung,
liver, skin, and colon (n = 3 mice per time point each for flow
cytometry and histology). Although only 105
CD4+ T cells were infused on day 0, 0.96 ± 0.41 × 106 and 0.23 ± 0.10 × 106 donor
CD4+ T cells were detected at these time points,
respectively, in the spleen of anti-CD2 + 48 MoAb-treated recipients,
indicating that combined MoAb treatment did not completely prevent
donor T-cell engraftment from occurring. Despite the presence of GVHD in these recipients, the severity of the GVHD response in combined MoAb-treated recipients was insufficient to cause mortality in most
recipients. Together, these data indicate that CD2 + 48 MoAb
treatment impairs donor CD4+ T-cell alloresponses in vivo.
Because CD2 is expressed on 85% of murine B cells15 and
CD48 on virtually all B cells,10 we asked to what extent
the binding of CD2 or CD48 MoAb on host B cells, which can
express MHC class II antigen, was required for the protective effects
of these MoAbs on GVHD. For this purpose, we used B6-SCID mice as
B-cell- (and T-cell-) deficient recipients
(Fig 2). B6-SCID recipients of
106 bm12 CD4+ T cells had a 12% actuarial
survival rate, with most succumbing to GVHD-induced mortality by 3 weeks posttransfer. In contrast, recipients administered CD2 + 48 MoAb had a 100% actuarial survival rate, exceeding their pre-BMT body
weights by 2 months posttransfer (data not shown). Long-term survivors
(n = 4) had a mean of 4.6 × 106 donor
CD4+ T cells present in the spleen on day 78 posttransfer,
indicating at least a 4.6-fold expansion of T cells as compared with
the number infused. In addition, recipients had histological evidence of acute GVHD involving the lung, colon, skin, and liver. Thus, CD2 + 48 MoAb did not completely eliminate the GVHD potential of the
inoculum of 106 donor CD4+ T cells.
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| Fig 2.
The anti-GVHD effect of CD2 + 48 MoAb is not
dependent on interference with T:B cognate interaction. Nonirradiated
B-cell-deficient B6-SCID recipients were administered 106
highly purified bm12 CD4+ LN cells. Mice receiving CD2 + 48 MoAb were treated as in Fig 1. The survival data are plotted.
The days posttransfer are plotted on the x-axis and the proportion of
mice surviving on the y-axis. Mice administered CD2 + 48 MoAb had
a significantly (P = .00038) higher actuarial survival rate
as compared with controls (n = 8 per group).
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Anti-CD48 MoAb inhibits the alloengraftment of T-cell-depleted BM
administered to irradiated recipients.
Because CD2 + 48 MoAb was effective in reducing GVHD lethality and
donor T cells are required for optimal alloengraftment, studies were
performed to determine whether combined MoAb would have a negative
impact on engraftment. B6 recipients were irradiated and then
administered T-cell-depleted BALB/c BM. In this strain combination and
under these conditions, both T cells and NK cells of the host are
involved in graft resistance.19 Anti-CD2 + 48 MoAb impaired
the engraftment of T-cell-depleted BALB/c (H2d) donor BM
in each of three replicate experiments when recipients were analyzed
early (6 to 8 weeks) or late (3.5 to 5 months) post-BMT (Table 1). To determine whether combined
MoAb administration would inhibit engraftment in a system in which only
host T cells were capable of resisting the donor graft,19
B6 recipients were administered T-cell-depleted DBA/1
(H2q, hematopoietic histocompatibility [Hh-1] null) donor
BM. Similar to that observed in the BALB/c  B6 system, CD2 + 48 MoAb markedly reduced the engraftment of donor BM when measured
early (7 weeks) and late (3 months) post-BMT (Table 1). CD48 MoAb
alone was responsible for the impairment of alloengraftment in B6
recipients of T-cell-depleted DBA/1 donor BM grafts.
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Table 1.
Anti-CD2 + 48 MoAb Inhibit
Long-Term Alloengraftment in Irradiated B6 Recipients of
T-Cell-Depleted BALB/c or DBA/1 Donor BM
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In both alloengraftment systems, the combined administration of CD2 + 48 MoAb had no effect on the recovery of neutrophils but did
significantly reduce the absolute lymphocyte counts in the peripheral
blood as measured on day 14 (Table 2). In
the DBA/1 B6 system, total leukocyte and lymphocyte numbers
as well as hematocrit values also were significantly, albeit modestly, lower in the CD2 + 48 MoAb-treated group on day 14. In both
systems, a more pronounced effect on each of these parameters was
evident on day 28 post-BMT, a time when recipients of irrelevant MoAb but not CD2 + 48 MoAb had a more vigorous lymphohematopoietic recovery. On day 28 post-BMT, recipients of combined MoAb had a
recovery inferior to control recipients analyzed on day 14 post-BMT.
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Table 2.
Analysis of Lymphohematopoietic Reconstitution in the PB
of BMT Recipients Administered T-Cell-Depleted Allogeneic BM
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Anti-CD48 MoAb inhibits engraftment of congenic marrow and syngeneic
day 12 CFU-S formation in vivo.
Although CD2 + 48 MoAb infusion inhibited the engraftment of
T-cell-depleted allogeneic BM, the mechanism(s) responsible for the
inhibitory effect are unknown. To discriminate between an immune and
nonimmune mechanism, experiments were performed in a congenic BMT
setting, thereby precluding an alloimmune mechanism (Table 3). Lethally irradiated B6
recipients were administered congeneic BM cells (1.0 or 3.0 × 106) along with irrelevant MoAb or CD48 MoAb. At both
cell doses, CD48 MoAb infusion was associated with a significant
reduction in the day 14 post-BMT mean numbers of peripheral blood
(PB) leukocytes and PB lymphocytes, and at the lower BM
cell dose (106), hematocrit values were significantly,
albeit modestly, reduced as well.
Mice administered B6-CD45.2 BM at a cell dose of 3 × 106 cells/recipient were studied for lymphohematopoietic
reconstitution in the spleen and BM on days 7, 10, 14, 20, (Tables 4 and
5; Figs 3 and
4) and day 47 post-BMT (not shown). In the BM compartment, CD48 antigen is expressed in a trimodal fashion with low, medium, or
high density (Fig 3A) in contrast to CD48+ splenocytes
which uniformly express a medium density of the CD48 antigen (Fig 3B).
CD48 antigen surface expression in the BM (Fig 3A) and spleen (Fig 3B)
was markedly reduced in CD48 MoAb-treated recipients at all time
points examined between days 7 and 20. Anti-CD48 MoAb-treated mice
experienced a delay in the recovery of BM (Table 4 and Fig 4) and
splenic (Table 5) cellularity. Moreover, the total number of BM cells
CD48hi and CD48med were significantly lower and
the numbers of CD48neg BM cells higher in MoAb-treated
recipients (Table 4). Splenic flow cytometric analysis indicated that
MoAb infusion significantly decreased donor cell recovery for at least
10 days post-BMT (Table 5).
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Table 4.
The Effects of CD48 MoAb Infusion on BM
Lymphohematopoietic Reconstitution in Lethally Irradiated
Recipients of Congenic BM Grafts
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Table 5.
The Effects of CD48 MoAb Infusion on Splenic
Lymphohematopoietic Reconstitution in Lethally Irradiated
Recipients of Congenic BM Grafts
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| Fig 3.
The in vivo infusion of CD48 MoAb in irradiated
recipients of congenic BM shifts BM and splenic populations toward a
CD48low or CD48neg phenotype. Single-cell
suspensions of BM (A) or spleen (B) were obtained from mice undergoing
congenic BMT from the experiment shown in Tables 4 and 5 on days 10, 14, and 20 post-BMT as listed. Mice received irrelevant MoAb or CD2 + 48 MoAb according to the regimen listed in Fig 1. Cells were
stained for CD48 expression and then analyzed by fluorescence-activated
cell sorting (FACS). Isotype-matched control MoAb was used to set
gates. For BM (A), CD48 expression was segregated into three antigen
density levels. The percent of cells falling within each antigen
density level is listed. The isotype-matched control MoAb is shown in
the thin line and the CD48 MoAb staining in the heavy line. A
representative overlay histogram from three individually analyzed mice
per group and a non-BMT control analyzed at each time point are shown.
The percentages indicate the percent positive cells in the gate
shown.
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| Fig 4.
Anti-CD2 + 48 MoAb infusion slows the recovery of
lymphohematopoiesis in the BM and splenic compartments in lethally
irradiated recipients of congenic BM grafts. B6 recipients were
lethally irradiated on day 1 and then administered 3.0 × 106 BM from B6-CD45.2 congenic donors on day 0. Mice were
treated with irrelevant IgG or CD2 + 48 MoAb as described in Fig
1. At the indicated time periods listed on the x-axis, three mice per
group were analyzed for reconstitution of the BM compartment as shown
in Table 4. The absolute number of cells is listed on the y-axis. Data,
mean values ± 1 standard deviation of the mean, are depicted.
Comparisons between the groups which were significant (*P < .05; **P < .01) are shown. Representative histograms from
these mice are shown in Fig 3A. These data were replicated in a second
experiment as shown in Table 7.
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To determine whether CD48 MoAb was depleting CD48hi and
CD48med cells, CD48 MoAb or hamster IgG (300 µg/dose)
was injected into B6 mice on days 0, 3, and 7. On day 9, three mice per
group were killed for determination of BM and splenic cellularity and
phenotype. A representative experiment of two experiments that were
performed with similar results is shown in
Table 6. Anti-CD48 MoAb did not reduce the
total number of BM cells or splenocytes and analysis of the T-cell,
B-cell, and myeloid series did not show any adverse effect of CD48
MoAb on these cell types. The only difference in the number and
composition of the BM and splenocyte compartments in these two groups
of mice was in the level of detectable CD48 antigen expression, in that
all mice receiving CD48 MoAb had a marked reduction in the level of
cell surface CD48 antigen expression (Fig 5
and Table 6). Therefore, the infusion of CD48 MoAb did not result in
a physical depletion of CD48+ cells.
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| Fig 5.
The in vivo infusion of CD48 MoAb in non-BMT B6 mice
shifts the BM and splenic populations toward a CD48low or
CD48neg phenotype. Non-BMT B6 control mice received
irrelevant MoAb or CD48 MoAb (300 µg) on days 0, 3, and 7. On day
9, cells were stained for CD48 expression and then analyzed by FACS.
Isotype-matched control MoAb was used to set gates. For BM, CD48
expression was segregated into three antigen density levels. The
percent of cells falling within each antigen density level is listed.
For spleen, the isotype-matched control MoAb is shown in the thin line
and the CD48 MoAb staining in the heavy line. A representative
overlay histogram from three individually analyzed mice per group at
each time point are shown. The percentages indicate the percent of
positive cells in the gate shown. The complete data are tabulated in
Table 6.
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The lack of CD48hi cells in CD48 MoAb-treated mice
could have been caused by MoAb coating or CD48 antigen modulation.
Single-cell suspensions of BM and spleen were incubated with CD48
MoAb (20 µg/mL) for 30 minutes on ice. Cells were washed and then
incubated with goat anti-hamster FITC or CD48-FITC. As a positive
control for splenocyte analysis, CD3 MoAb was used under the same
conditions. The proportion of CD48+ splenocytes
(Fig 6, top) and CD48hi BM
cells (Fig 6, bottom) was markedly reduced after CD48 MoAb incubation (Fig 6) but not CD3 MoAb incubation (data not shown). As with the in vivo experiment, only a low proportion of BM or spleen
cells exposed to CD48 MoAb bound goat anti-hamster IgG FITC (Fig 6,
right-hand panels), in contrast to the 45% binding noted for CD3
MoAb-exposed splenocytes. Collectively, these data would indicate that
CD48 MoAb induces a rapid modulation of the CD48 epiptope and would
exclude depletion of CD48hi or coating of the CD48 antigen
as the primary reason for the lack of CD48hi cells in the
BM and spleen in CD48 MoAb-treated recipients.
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| Fig 6.
Analysis of modulation and coating of the CD48 antigen
induced by the ex vivo incubation of splenocytes or BM cells with
CD48 MoAb. Single-cell suspensions of spleen (top panels) and BM
(bottom panels) from non-BMT B6 control mice were incubated with
CD48 MoAb (CD48aby) (20 µg/mL) for 30 minutes on ice. Cells were
washed and then incubated with goat anti-hamster FITC (anti-H) or
CD48-FITC (CD48). The thin lines are the isotype-specific negative
control MoAb staining and the dark lines are the staining with either
CD48-FITC or anti-hamster FITC. The proportion of CD48hi
BM cells and splenocytes was markedly reduced after CD48 MoAb. Only
a low proportion of BM or spleen cells exposed to CD48 MoAb bound
goat anti-hamster IgG FITC.
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|
To determine whether the negative effects of CD2 + 48 MoAb infusion
on engraftment may be related to in vivo inhibition of BM-derived
progenitor cell growth, day 12 CFU-S formation was quantified in
syngeneic BMT recipients of CD2, CD48, or CD2 + 48 MoAb. The
mean colony number in irrelevant IgG-treated control recipients was 11 versus  4 that were present in recipients of no BM or recipients of BM
plus CD48 MoAb (Fig 7). Moreover, the size of the colonies was much smaller in the latter groups, consistent with a marked reduction in colony cellularity caused by CD48 MoAb
infusion. Cumulatively, the alloengraftment and syngeneic engraftment
studies are most consistent with a negative effect of CD48 MoAb on
hematopoiesis in irradiated, transplanted mice and indicate that
CD48 MoAb affects engraftment through nondepletionary mechanism(s).
View larger version (11K):
[in this window]
[in a new window]
| Fig 7.
The in vivo infusion of CD48 MoAb or CD2 + 48 MoAb in irradiated recipients of syngeneic BM inhibits day 12 CFU-S
formation. B6 recipients were irradiated with 7.5 Gy total body
irradiation via 137Cesium source on day 1 and then
administered 105 B6 BM cells on day 0. Mice (n = 8 per
group) were injected with CD2 MoAb, CD48 MoAb, CD2 + 48 MoAb, or irrelevant IgG, as indicated on the y-axis at a dose of 300 µg each, administered IP on days -1, 2, 6, and 9 post-BMT. Twelve
days post-BMT, mice were killed and spleens were placed into Bouin's
solution to facilitate enumeration of colonies. The mean ± 1 SEM
number of colonies is listed on the x-axis. *P < .001 as
compared with irrelevant IgG control. #Colonies are all
smaller in size than observed in irrelevant MoAb-treated controls.
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|
Because CD48 MoAb administration led to a marked reduction in day 12 CFU-S formation, we considered the possibility that CD48
MoAb-coated BM progenitor cells had an impaired ability to traffic to
the spleen, a major site of early post-BMT hematopoiesis. To exclude
the effect of CD48 MoAb infusion on splenic homing, cohorts of mice
were splenectomized 2 weeks before congenic BMT. At all time points
examined (days 7, 10, and 14), CD48 MoAb administration was
associated with a marked reduction in the absolute number of donor
lymphohematopoietic cells in the BM (Table
7). Both the lymphoid and myeloid lineage cells were affected.
Interestingly, the absolute number of residual host BM cells was
approximately twofold lower in CD48 MoAb-treated recipients,
suggesting that CD48 MoAb had a direct effect on inhibiting the
production of BM hematopoietic cells. Splenectomized mice had similar
responses to CD48 MoAb infusion as nonsplenectomized controls.
Although combined MoAb infusion provided a more pronounced and
consistent delay of donor BM engraftment than in the previous study
(Table 4), in both studies combined MoAb infusion was associated with a
delay in recovery at one or more time periods post-BMT.
View this table:
[in this window]
[in a new window]
|
Table 7.
The Effects of CD48 MoAb Infusion on BM
Lymphohematopoietic Reconstitution in Splenectomized or
Nonsplenectomized Lethally Irradiated Recipients of Congenic BM
Grafts
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|
| |
DISCUSSION |
Although we have shown that CD48 MoAb infusion is highly effective
in inhibiting CD4+ and to a far lesser extent
CD8+ T-cell-mediated GVHD lethality, CD48 MoAb infusion
was associated with a pronounced decrease in the alloengraftment of
T-cell-depleted BM grafts. Because congenic lymphohematopoietic
recovery and syngeneic day 12 CFU-S formation also were impaired by
CD48 MoAb infusion, the adverse effects on engraftment do not seem
to be related to the effects of these MoAbs on the immune system.
Splenectomized recipients treated with aCD48 MoAb also had decreased
donor and host lymphohematopoiesis, indicating that CD48
MoAb-coated donor BM cells were not being redirected into unfavorable
sites for hematopoiesis post-BMT. Thus, the CD48 antigen seems to have
a critical role in regulating lymphohematopoiesis post-BMT. These findings have significant implications for the extrapolation of this
approach to human BMT recipients.
An intriguing and important aspect of our study was the profound
deleterious effect of CD48 MoAb on engraftment. Anti-CD48 MoAb
infusion alone or in combination with CD2 markedly inhibited the
engraftment of two types of allogeneic pan-T-cell-depleted BM in
irradiated B6 recipients. In one strain combination (BALB/c B6), depletion of either NK cells or T cells from the host enhance engraftment, whereas in the other (DBA/1 B6), only host T
cells are involved in graft resistance.19 Because CD48
MoAb binds T cells and NK cells,4-7,10,20-22 it was
possible that engraftment might be enhanced or unimpaired if
CD8+ T cells were the major effectors of graft rejection.
Because donor cell engraftment was suppressed in recipients of CD48
MoAb-containing regimens, we needed to distinguish between an
immunologic effect unique to an allogeneic environment or a primary
hematopoietic effect which would be independent of the effects of these
MoAbs on allorecognition. Experiments performed in non-BMT mice did not
show any evidence of suppressed hematopoiesis under normal physiological conditions. Specifically, as compared with recipients of
irrelevant IgG, non-BMT mice administered CD2, CD48, or both MoAbs and individually studied did not have significant differences in
absolute numbers of BM or splenocytes or the proportion of Mac1+, B220+, CD4+, or
CD8+ cells when analyzed 2 days after discontinuing MoAb
administration.
To uncover a more subtle contribution by CD48 MoAb infusion,
experiments were performed in irradiated, congenic BMT mice experiencing stress hematopoiesis. As compared with recipients of
irrelevant IgG, mice administered CD48 MoAb had a substantial reduction in the absolute number of donor BM-derived cells localized in
the spleen on day 7 post-BMT and in the BM compartment as assessed on
days 10 and 14 post-BMT (few donor cells were detectable in the BM of
either group on day 7 post-BMT). A striking finding was the absence of
CD48hi-expressing cells in the spleen and
CD48hi and CD48med cells in the BM compartment.
Because there was a proportional increase in the number of
CD48neg cells that mostly compensated for the loss of
CD48med/hi cells in the BM, the effects in the BM
compartment seemed to be caused by CD48 modulation as has been
previously described in non-BMT mice administered CD48
MoAb.23 Our in vitro and in vivo results directly show that
CD48 MoAb is a potent modulator of CD48 antigen expression.
Because the absolute number of donor BM-derived cells localized to the
spleen on day 7 post-BMT was markedly reduced, whereas the BM
compartment was relatively intact, we considered the possibility that
the spleen, the major site of early post-BMT hematopoiesis and of
allogeneic graft resistance, was preferentially affected by CD48
MoAb infusion. In syngeneic BMT recipients, the infusion of regimens
containing CD48 MoAb reduced the number of day 12 CFU-S colonies, an
indicator of multipotential progenitor cell engraftment, to the levels
observed in irradiated mice that were not administered any BM cells.
Because donor BM cells are infused under the cover of CD48 MoAb
while recipient hematopoiesis is already occurring in the BM at the
time of MoAb adminstration, it is possible that CD48 MoAb was
affecting the homing and migration of donor BM-derived cells to the
spleen early post-BMT. However, splenectomized recipients of congenic
BM grafts were equally susceptible to the inhibitory effect of CD48
MoAb on BM lymphohematopoiesis. Moreover, CD48 MoAb infusion also
reduced the residual host hematopoiesis. Therefore, CD48 MoAb
infusion did not inhibit hematopoietic recovery and engraftment by
altering the trafficking of intravenously infused donor BM progenitor
cells. Rather, we favor the hypothesis that CD48 MoAb has a direct
effect on lymphohematopoiesis by affecting the necessary signals for
hematopoiesis provided by the host microenvironment.
In addition to our observations on combined MoAb-induced inhibition of
lymphohematopoietic recovery post-BMT, we also have shown that CD2 + 48 MoAb infusion is an effective approach to downregulating GVHD
induced by CD4+ T cells infused into either sublethally
irradiated or nonirradiated SCID MHC class II disparate recipients.
Thus, combined MoAb infusion has a direct immune suppressive effect on
GVHD-induced mortality. Anti-CD48 MoAb infusion was necessary and
sufficient for the GVHD protective effect of the combined MoAb regimen
and the biological effect of CD2 MoAb infusion was only uncovered at
low CD4+ T-cell doses (data not shown). The explanation for
the lower efficacy of this combined MoAb regimen on CD8+
T-cell-mediated GVHD is unknown. The inferior efficacy of combined MoAb in the setting of CD8+ T-cell-mediated GVHD was
unexpected for several reasons. The in vivo administration of CD48
or CD2 MoAb has been shown to inhibit the in vivo priming and in
vitro generation of allospecific CTL.9,11,12,14,23-26
Although CD2 + 48 MoAb inhibits the generation of allospecific CTL,
it is possible that there is a differential effect on the generation or
function of CTL derived from CD4+ as compared with
CD8+ CTL. We do not have any direct evidence to support
this hypothesis. However, it is interesting to note that CD2 ligation
has been shown to upregulate Fas ligand (CD95L).27 Recent
studies by Graubert et al28 have indicated that
CD4+ T cells can use CD95L to cause GVHD lethality in MHC
class II disparate recipients, whereas CD8+ T cells are
unable to use CD95L to mediate GVHD lethality in MHC class I disparate
recipients. Therefore, CD2 + 48 MoAb infusion theoretically may inhibit the development of CD4+
CD95L+ CTL effectors in vivo that are responsible for GVHD
in the B6 bm12 system.
Another possibility is that CD48 MoAb affects lymphokine production
differently in CD8+ and CD4+ T cells and this
difference leads to a more effective inhibition of CD4+
T-cell-mediated lethality as compared with CD8+ T cells.
For example, the lymphokines triggered by phytohemagglutinin but not
concanavalin A are substantially inhibited by the in vivo infusion of
CD48 or CD2 MoAb and mixed lymphocyte reaction
responses are unaffected by either MoAb.10,11,14,23 These
data would indicate that lymphokine responses of some subsets of T
cells are more susceptible than others to MoAb inhibition.
Collectively, these data are most consistent with the interpretation
that there is a qualitative difference between the inhibitory effects
of CD48 MoAb on CD4+ and CD8+
T-cell-mediated GVHD. Regardless of the explanation for the
differential GVHD inhibitory effects of CD2 + 48 MoAb infusion, it
is clear that the suppression of lymphohematopoiesis conferred by this combined MoAb regimen will dampen the enthusiasm for the clinical trials of CD2 + 48 MoAb in humans undergoing GVHD unless
it can be determined that our findings are either species specific or dependent on the particular epitope recognized by the CD48 MoAb we
have studied.
| |
FOOTNOTES |
Submitted May 25, 1998;
accepted July 21, 1998.
Supported in part by National Institues of Health Grants No. R01 AI
34495, R01 HL56067, R29 AI32655, R01 AI41428, P60 AR20557, and P01
AI-35296
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Bruce R. Blazar, MD, Box 109 UMHC, 420 SE
Delaware St, Minneapolis, MN 55455; e-mail: blaza001{at}maroon.tc.umn.edu.
| |
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Top
Abstract
Introduction