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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-02-0486.
CHEMOKINES
From the Partners AIDS Research Center and MGH Cancer
Center, Harvard Medical School, Massachusetts General Hospital, Boston;
and Indiana University Cancer Research Institute, Indiana University
School of Medicine, Indianapolis.
T-lymphocyte depletion of bone marrow grafts compromises
engraftment, suggesting a facilitating mechanism provided by the T
cells that has been shown to associate with CD8+ but not
CD4+ T cells. Explanations for this phenomenon have focused
on immune targeting of residual host cells or cytokine production. We
provide evidence for an alternative mechanism based on cooperative
effects on cell motility. We observed that engraftment of
CD34+ cells in a Bone marrow grafts are depleted of T cells to
reduce graft-versus- host disease (GVHD), as a by-product of tumor cell
purging or as a result of hematopoietic stem/progenitor cell selection for the introduction of genetic constructs.1,2 However,
when compared with unmanipulated grafts, T cell-depleted autologous and allogeneic transplants result in lower levels of engraftment during
the first 6 months after transplantation.3,4 This implies that T cells facilitate optimal engraftment of
CD34+ cells and has been supported by clinical trials in
humans demonstrating that the presence of CD8+ cells but
not CD4+ cells results in an increased level of engraftment
in the allogeneic transplant setting.5 Studies of
allogeneic transplants in mice have also shown that CD8+
cells facilitate engraftment6-8; however, the exact
phenotype of these cells remains controversial. The mechanism of action by which CD8+ cells facilitate engraftment is generally
attributed to the elimination of residual immune cells of the
recipient.9 However, stem cells transplanted into
multi-immunodeficient hosts similarly benefit from accessory cells,
suggesting the possibility of other roles often assumed to be that of
cytokine production.10,11
The mechanisms by which hematopoietic stem cells home and engraft have
been defined to a limited extent. Mice engineered to be deficient in
either the chemokine receptor CXCR4 or its ligand, stromal derived
factor-1 Using in vivo and in vitro model systems, we assessed the interaction
of CD8+ and CD34+ cells resulting in the
clinically observed facilitating effect of the lymphoid population. We
demonstrate correlation between in vivo and in vitro systems to define
a CD8+ cell population capable of augmenting
CD34+ engraftment. We define that a secreted product of
CD8+ cells is not required; rather, altered CXCR4 signaling
in CD34+ cells mediated by CD8+ cells
associates with augmentation of CD34+ migration, homing,
and engraftment. These data demonstrate a unique cell-cell
cooperativity affecting chemotaxis and provide a novel mechanism for
lymphoid augmentation of bone marrow transplantation.
Cell purification
Flow cytometry
Engraftment of  2-microglobulin-deficient nonobese diabetic/severe
combined immunodeficient (2m /
NOD/SCID) mice (Jackson Laboratories, Bar Harbor, ME), which had been
sublethally irradiated (3.5 Gy) 24 hours previously. After 6 to 8 weeks, the mice were killed with CO2, and the bone marrow and spleen were removed. The bone marrow was flushed with fully
supplemented Iscove medium, and the spleen was mechanically disaggregated. The level of engraftment of the mice was calculated by
flow cytometry using antibodies toward human CD45, CD3, CD19, CD33, and CD34.
In vivo homing Selected CD34+ cells (1 × 105 to 4 × 105) were labeled with 0.1 µM carboxyfluorescein diacetate (CFDA)-succinimidyl ester (SE) (Molecular Probes, Eugene, OR) according to the manufacturer's instructions and incubated either alone or with an equal number of CD4+ or CD8+ cells for 2 to 3 hours. Cells were then injected into the tail vein of 6- to 8-week-old female littermate2m/ NOD/SCID mice or SCID mice (bred at
Massachusetts General Hospital), which had been sublethally irradiated
(3.5 Gy) 24 hours previously. Mice were then killed after 9 hours, and
the levels of human cells were measured in the bone marrow and spleen
through the detection of CFDA-SE+ cells by flow cytometry.
Chemotaxis assay Chemotaxis assays were performed by plating 3 × 104 bone marrow endothelial cells (BMECs) (kindly provided by Dr Joao Ascensao, University of Nevada School of Medicine, Reno) in a Transwell (5-µm pore size) (Corning-Costar, New York, NY) and incubating at 37°C for 3 days or until confluent. Purified CD34+ cells (1 × 104 to 2 × 104) with or without an equal number of purified CD4+ or CD8+ cells in fully supplemented Iscove medium were then added to the upper well. Chemotaxis toward 300 ng/mL SDF-1 (PeproTech, Rocky Hill, NY) was
allowed to continue for 4 hours at 37°C/5% CO2 in a
humidified atmosphere. Cells were harvested from the lower well,
counted with a hemocytometer, and the relative numbers of the different
cell types calculated by flow cytometry. To block secretion of factors
from or movement of the CD8+ cells, they were treated with
3.5 µM brefeldin A (Sigma Chemical, St Louis, MO) for 4 hours at
37°C or 10 µg/mL cytochalasin D (Sigma) for 15 minutes at 37°C.
To assess the integrity of the endothelial barrier, we used a labeled
protein as a surrogate as has been described by
others.25-27 FITC-conjugated albumin (Molecular Probes) was added to the upper well at a concentration of 0.2 mM, and chemotaxis assays were performed as described above. The level of
fluorescence was then measured in the lower well using a CytoFluor II
plate reader (Perspective Biosystems, Framingham, MA).
Time-lapse video microscopy BMEC cells were plated onto 12-well plates and allowed to grow until confluent. CD34+ and CFDA-SE-labeled CD8+ cells were then added on the cell line and incubated for 30 minutes at 37°C to allow the cells to settle upon the monolayer. Images (both light and fluorescence) were acquired every minute using an inverted microscope with a Spot RT Color (version 3.0.3) camera (Diagnostic Instruments, Sterling Heights, MI) and UniBlitz shutter driver (model VMM-D1) (Vincent Associates, Rochester, NY). Images were first acquired using IPLab software (version 3.5.2) (Scanalytics, Fairfax, VA) and then converted into time-lapse sequences with Scion Image software (Scion, Frederick, MD).Calcium flux Selected CD34+ cells were incubated alone or with either CD4+ or CD8+ cells for 2 to 3 hours following which they were incubated with 3 µg/mL Indo-1 (Molecular Probes) for 45 minutes at 37°C. Cells were then stained for CD34, CD4, or CD8 for 15 minutes on ice. Calcium flux was measured by a ratio of 400:40 (short) to 510:20 (long) wavelengths with UV light from a He-Cad laser (325 nm) on an LSR cytometer (Becton Dickinson) after the addition of 1 µg/mL SDF-1. Calcium flux was measured specifically
in the CD34+ cell populations using FlowJo software (Tree
Star, Stanford, CA).
Measurement of tyrosine phosphorylation Selected CD34+ cells were incubated alone or with either CD4+ or CD8+ cells for 2 to 3 hours. SDF-1 was then added to the cells at a concentration of 1 µg/mL.
At 0, 1, 2, and 3 minutes following the addition of SDF-1, the cells
were permeabilized with Fix & Perm Cell Permeabilization Kit (Caltag
Laboratories, Burlingame, CA) and then stained for CD34, CD4, CD8, and
antiphosphotyrosine (Upstate Biotechnology, Lake Placid, NY) or
phospho-FAK (Becton Dickinson). Cells were then analyzed on a
FACSCalibur cytometer using CellQuest software.
Statistical analysis Paired comparisons were carried out using the Student t test.
CD8+ cells enhance the engraftment of CD34+
cells in 2m/ NOD/SCID
mouse model. To assess the impact of mixed cell populations, we
titrated down the dose of infused cells reasoning that above a certain
threshold no further augmentation would be observable. Rather than the
customary 3 × 105 CD34+ cells, we employed
3 × 104 CD34+ cells preincubated and
undergoing transplantation with either no cells or autologous
CD4+ or CD8+ cells at a 1:1 ratio into
sublethally irradiated mice and evaluated for engraftment after 6 to 8 weeks. Stem cells admixed with CD4+ cells consistently
engrafted less well than those admixed with CD8+ cells, and
those admixed with CD8+ cells engrafted with a mean 10-fold
increase over control CD34+ cells (P = .029)
(Figure 1A).
CD8+ cells enhance the in vivo homing of CD34+ cells to the bone marrow To assess the mechanism of enhancement of engraftment by CD8+ cells, homing to the bone marrow of CD34+ cells mixed with CD4+ or CD8+ cells was studied using direct examination of fluorescently labeled cells in vivo. CD8+ cells significantly enhanced the homing to the bone marrow of CFDA-SE-labeled CD34+ cells (P = .006) (Figure 1B) compared with control CD34+ cells alone. This augmentation of homing was specific to the CD8+ subset of cells. Incubation of CD34+ cells with CD4+ cells led to a decrease in bone marrow homing (P = .045). To determine if this enhancement of CD34+ localization was distinct for the bone marrow environment, homing to the spleen was evaluated (Figure 1C). Consistent with that seen in marrow, increased CD34+ homing was noted when CD8+ cells were added (P = .014), though no inhibition or a trend toward increased homing was observed with addition of CD4+ cells (P = .255).CD8+ cells enhance the transmigration of CD34+ cells To study the mechanism of the enhancement of in vivo homing, an in vitro transmigration model of homing was used. In these experiments, an SDF-1 concentration of 300 ng/mL was used as the chemotactic agent.
This concentration of SDF-1 has been originally defined by Aiuti and
colleagues as a strong stimulus for CD34+ cell
chemotaxis18 and within the range of 200 ng/mL to 600 ng/mL as reported by others.28-30 To better mimic the
cellular context of the bone marrow, transmigrations were carried out
with adherent bone marrow endothelial cells. The immortalized
primary BMEC line was used to standardize the cell environment. This
cell line is derived from adult human bone marrow endothelial cells yet
retains the characteristics of primary cells.31 Due to
concerns about potential effects of immune activation in the mixed cell experiments, we used autologous cells only and did not mix cord blood-derived cells from different donors, thereby constraining the
number of cells possible to use in each well. Although small numbers of
cells (1 × 104 to 2 × 104 cells per well)
were used, these numbers are now commonly used by others in chemotaxis
analyses.32,33 In this assay system, minimal random
migration (chemokinesis) was observed either with or without the
addition of T cells (approximately 5% of the CD34+ cells
transmigrating to the lower chamber). Chemotaxis of CD34+
cells over a 4-hour period is shown in Figure 1D. The kinetics of
transmigration demonstrated that the addition of CD8+ cells
increased the transmigration of CD34+ cells at the 3- and
4-hour time points. The addition of CD4+ cells to the
transmigration assay had no effect on CD34+ cell
transmigration. The kinetics of transmigration of CD34+
plus CD8+ or CD4+ cells paralleled the kinetics
of transmigration for either lymphocyte subset alone (data not shown),
suggesting that lymphoid migration kinetics dictated the effect on
CD34+ cells.
To test whether the enhancement of CD34+ transmigration was
due to a factor secreted from the CD8+ cells,
CD8+-conditioned medium was added to the upper
well of the transmigration chamber. This had no effect on the
CD34+ cell transmigration (data not shown). However,
because the direct interaction of the cells could lead to the secretion
of a factor from the cells, we next pretreated CD8+ cells
with the secretory pathway inhibitor brefeldin A and tested their
effect on CD34+ cell transmigration. In these experiments,
significant enhancement of transmigration of the CD34+
cells was observed upon addition of CD8+ cells
(P = .0002) (Figure 2A).
Treatment of the same cells with brefeldin A did not result in any
discernable alteration of the enhancing effect (P = .0027)
(Figure 2B). There was no significant difference between the levels of
enhancement of CD34+ cell transmigration shown by untreated
CD8+ cells or those that had been pretreated with brefeldin
A (P = .64), arguing against a secreted factor from the
CD8+ cells contributing to CD34+ cell
localization.
Enhancement of CD34+ cell transmigration is dependent upon cytoskeletal rearrangement in CD8+ cells To test whether cytoskeletal rearrangements within the CD8+ population of cells was a necessary component for the enhancement of transmigration, the CD8+ cells were pretreated with cytochalasin D, an inhibitor of actin polymerization. Cytochalasin D is well defined as an inhibitor of the cytoskeletal relationships within a cell necessary for chemotaxis34 and indeed diminished chemotaxis of the CD8+ cells to a level of 12.8% as compared with the untreated CD8+ cells (data not shown). Cytochalasin D-induced inhibition of CD8+ cell chemotaxis abrogated their enhancing effect on CD34+ cell transmigration and actually decreased CD34+ cell transmigration (P = .0009) (Figure 2C). To rule out the possibility that the cytochalasin D may have leached out of the CD8+ cells and inhibited CD34+ cells directly, we also treated CD4+ cells with cytochalasin D. This treatment had no effect on the level of transmigration of the CD34+ cells (data not shown). An alternative method of impairing cytoskeletal-mediated events is irradiation.35 We observed decreased CD8+ cell chemotaxis (to 8%) and abrogation of the CD8+ cell effect on CD34+ migration (migration of CD34+ cells alone vs migration of CD34+ cells plus irradiated CD8+ cells, P = .636) following pretreatment of CD8+ cells (data not shown). Together, these data demonstrate that CD8+ cell cytoskeletal rearrangements were required for the enhancement of CD34+ cell transmigration; the basis for the more profound effects of cytochalasin D is not clear but may reflect inhibitory products released by cytochalasin D-treated CD8+ cells.Cytoskeletal rearrangements within CD8+ cells are required for the enhancement of CD34+ cell homing to the bone marrow in vivo To determine whether the same mechanism by which CD8+ cells enhanced in vitro CD34+ cell transmigration also applied in the in vivo setting, we treated the CD8+ cells with brefeldin A or cytochalasin D before performing in vivo homing assays. Treatment of the CD8+ cells with brefeldin A continued to result in an augmentation of CD34+ cell homing to the bone marrow (P = .008) (Figure 3). Treatment of CD8+ cells with cytochalasin D led to a reduction in the number of CD34+ cells homing to the bone marrow (P = .014). These results parallel the in vitro transmigration setting, suggesting that similar mechanisms are involved in both systems.
CD8+ cells do not alter permeability of the endothelial barrier Because treatment of CD8+ cells abrogated any chemotactic ability of these cells, we investigated the possibility that CD8+ cell transmigration may alter the endothelial barrier, leading to enhancement of CD34+ cell transmigration. We first tested whether the CD8+ cells disrupted the integrity of the endothelial layer by placing FITC-conjugated albumin in the upper chamber of the Transwell. We did not observe any significant differences in the level of protein that passed across the BMEC cells in the setting of CD34+ cells with or without CD8+ cells. These data argue against a fundamental change in the endothelial barrier that may allow increased transmigration of the CD34+ cells (Figure 4). Using time-lapse video microscopy over a period of 2 to 4 hours, we then observed mixtures of differentially fluorescently labeled CD34+ and CD8+ cells on the BMEC cell line. Both CD34+ and CD8+ cells transmigrated through the BMEC layer but did not comigrate in tandem across the endothelial layer (Videos 1-3 in the online supplemental materials). Therefore, enhancement of CD34+ transmigration does not require cell-cell contact during the migration process itself.
CD8+ cells affect SDF-1 . The Ca++ flux response for CD34+
cells was unaltered whether incubated alone or with either
CD4+ or CD8+ cells (Figure
5A). Therefore, neither CD4+
nor CD8+ cells appear to affect the intracellular calcium
shifts downstream of SDF-1 interaction with CXCR4. However,
measurements of the level of phosphorylated tyrosine residues within
the CD34+ cells following SDF-1 stimulation demonstrated
an increase only in those cells that had been incubated with
CD8+ cells (Figure 5B). Examination of a specific component
of the CXCR4 signaling pathway that may be envisioned to alter
transmigration, focal adhesion kinase,36,37 demonstrated
differences in the level of phosphorylation of this protein (increase
in mean fluorescence intensity from 50.69 to 63.77) (Figure 5C).
Baseline phospho-FAK mean fluorescence intensity levels were increased
by the coincubation of CD34+ and CD8+ cells
(from 427 to 537), indicating altered FAK activation in CD34+ cells by the presence of CD8+ cells. The
level of phospho-FAK decreased with SDF-1 stimulation in the setting
of CD34+ cells coincubated with CD8+ cells in
contrast to CD34+ cells alone where phospho-FAK increased.
These differences indicate alteration of the constitutive regulation of
FAK and in the CXCR4 signaling cascade in CD34+ cells
induced by the presence of CD8+ cells. The specific
biochemical basis for the changes in protein phosphorylation was not
further defined.
It is known that lymphocytes augment the recovery of hematopoiesis following stem cell transplantation, yet the contribution by T cells to stem cell function has remained an area of controversy.3,4 Leading hypotheses have focused on infused immune effector cells abrogating residual host rejection phenomena5 or providing cytokines that augment stem cell proliferation.10,11 The data presented here provide an alternative model in which CD8+ T cells contribute to stem/progenitor cell localization through augmentation of the entry to the bone marrow microenvironment, an essential part of establishing bone marrow hematopoiesis. The model we propose is one in which events between cotransplanted cells influence the efficiency of localization and engraftment in hematopoietic tissues. However, these early events after transplantation do not preclude later, distinct immunologically mediated phenomena. Indeed, collaborative data generated in the setting of cotransplanted T cells lacking cytotoxic effector mechanisms suggest an additional role for cytolytic interactions (G.B.A. and D.T.S., manuscript submitted). The relationship of cell movement to hematopoiesis was first suggested
by Aiuti and colleagues, who identified the chemokine receptor CXCR4 on
the surface of CD34+ cells and demonstrated the ability of
the cognate ligand, SDF-1 We explored the relationship of engraftment and homing using in vivo chimeric models of human hematopoiesis. The presence of CD8+ cells led to an increase in the engraftment of the CD34+ cells, recapitulating previously published reports on the effects of CD8+ cells on engraftment.5-8 Analysis of the mechanism of this enhancement demonstrated that CD8+ cells but not CD4+ cells enhanced the homing of CD34+ cells to the bone marrow. Comparison of this effect in another hematopoietic organ, the spleen, also showed an enhancing effect mediated by CD8+ cells. However, in this case the CD4+ cells actually had no effect on the level of homing. These results are not entirely comparable with the effect seen in the bone marrow and may imply that differing mechanisms exist for the homing of cells to the bone marrow and spleen.38,39 Homing involves at least 3 steps, including loose or rolling interaction of cells with vascular endothelium, firm adhesion to the vessel or sinus, and diapedesis through the endothelial layer. Although any of these processes may affect in vivo homing, we noted reasonable correlation between our in vivo observations and in vitro transmigration in which only limited adhesive interactions may be necessary. To at least approximate cellular interactions in the low flow marrow sinus, we used an endothelial-coated Transwell system. Whether this system requires anything beyond cell crawling phenomena is not defined, but based on the videomicroscopic imaging of cell movement in culture (Video 1) it appears that adhesive interactions between CD34+ cells and endothelial cells are ongoing and dynamic. The stimulus we used for cell migration was SDF-1 The basis for a CD8+ cell effect on CD34+ cells
was unexpectedly not due to a secreted cell product but, rather,
required cell-cell contact and a dynamic cytoskeleton of the
CD8+ cells. Given that the interaction was cytoskeleton-
and time-sensitive (preincubation of CD8+ and
CD34+ cells was necessary), it may be that cell crosstalk
involved clustering of cell surface proteins on the CD8+
cells to enable the changes in CD34+ cell function. That
CD34+ cell physiology was altered is best evident in the
changed profile of FAK phosphorylation under resting conditions when
preincubated with CD8+ cells. Basal levels of phospho-FAK
were increased following cell-cell contact, and less pronounced
phosphotyrosine increases were seen after cell-cell contact and
SDF-1 Whether the CD8+ cells affecting CD34+
function bear the phenotype of the "facilitator cell" defined by
various groups was not tested here. Those investigators have identified
that CD8+ (T-cell receptor-negative or -positive),
CD2+, CD5+, and CD3 The data presented here demonstrate cell-cell cooperativity in chemotaxis where one cell type modulates the responsiveness of a different cell type to a chemokinetic stimulus. This adds an additional dimension to chemokine activity where the cellular context in which a chemokine stimulus is received influences the phenotype of response. Depending upon the cellular milieu, a highly specific effect may be envisioned that may be physiologically significant if the model proposed here for stem cell engraftment is representative. Varying the composition of the cellular context may be a mechanism for achieving highly modulable, combinatorial effects in vivo. Understanding the role of chemotaxis in physiologic settings may therefore require analysis of chemokines on complex mixtures of cells as well as individual cell types. The potential to exploit such interactions therapeutically is evident in the bone marrow transplantation setting and may be instructive for future efforts to enhance cell targeting in vivo to bone marrow and other tissue types.
The authors thank Cory Johnson and Dr Donna Wall of St Louis University for the invaluable provision of umbilical cord blood. We also thank Drs Richard H. Evans, Tao Cheng, and Kenneth S. Cohen for their valued input.
Submitted February 15, 2002; accepted July 3, 2002.
Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-02-0486.
Supported by the National Institutes of Health (D.T.S., M.C.P.), the Richard Saltonstall Charitable Foundation (D.T.S.), and the American Foundation for AIDS Research (M.C.P.).
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David T. Scadden, Massachusetts General Hospital, Harvard Medical School, Building 149, 13th St (Room 5212), Boston, MA 02129; e-mail: scadden.david{at}mgh.harvard.edu.
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Top
Abstract
Introduction
represents
experiment (expt) 1; 
, expt 2; 
, expt 3; ×, expt 4; 
, expt 5;
and 
, expt 6.
+ cells were
able to enhance engraftment without inducing
GVHD.