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Prepublished online as a Blood First Edition Paper on June 14, 2002; DOI 10.1182/blood-2002-02-0428.
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Blood, 15 October 2002, Vol. 100, No. 8, pp. 3045-3048
BRIEF REPORT
Developmental dissociation of T cells from B, NK, and myeloid
cells revealed by MHC class II-specific chimeric immune receptors
bearing TCR- or FcR- chain signaling domains
Wei Yu Lin and
Margo R. Roberts
From the University of Virginia,
Charlottesville.
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Abstract |
The T-cell receptor  (TCR-) and FcR- chains
play a critical role in mediating signal transduction. We have
previously described HIV glycoprotein 120 (gp120)-specific
chimeric immune receptors (CIRs) in which the extracellular domain of
CD4 is linked to the signaling domain of (CD4) or (CD4).
Such CIRs are efficiently expressed following retroviral transduction
of mature T cells and specifically redirect effector functions toward
HIV-infected targets. In this report, we examine development of CD4-
or CD4-expressing T cells from retrovirally transduced hematopoietic
stem cells following bone marrow transplantation. Although
CD4/-expressing myeloid, NK, and B cells were efficiently
reconstituted, parallel development of CD4/-expressing T cells
was blocked prior to the CD25+CD44+
prothymocyte stage. In contrast, T cells expressing a
signaling-defective CIR were efficiently generated. When major
histocompatibility complex (MHC) class II-deficient mice were used as
transplant recipients, development of CD4/-expressing T cells was
restored. We conclude that CD4/ signaling generated following
engagement of MHC class II selectively arrests T-lineage development.
(Blood. 2002;100:3045-3048)
© 2002 by The American Society of Hematology.
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Introduction |
We have previously described HIV glycoprotein
120 (gp120)-specific chimeric immune receptors (CIRs) in which
the cytoplasmic domain of either the T-cell receptor (TCR-) or
the FcR- chain is linked to the extracellular and transmembrane
domains of the human CD4 receptor.1-5 Mature
CD4-expressing T lymphocytes generated ex vivo by retroviral
transduction are capable of highly efficient and specific cytolysis of
both HIV-infected primary cells and HIVgp120-expressing tumors in
vitro,1,4 and clinical trials involving adoptive transfer
of autologous CD4-transduced T cells in HIV-infected patients have
been undertaken.6,7
In vivo development of CIR-modified T cells from transplanted
hematopoietic stem cells (HSCs) may have some advantages over adoptive
transfer of ex vivo-transduced mature T cells, because the former
bypasses the need for extensive ex vivo cell expansion and may improve
T-cell function in vivo. Our previous studies have shown that severe
combined immunodeficient (SCID) mice (which lack T and B cells) rapidly
reconstitute CD4-expressing myeloid and natural killer (NK) cells
following transplantation with retrovirally transduced syngeneic bone
marrow.3 Furthermore, such SCID mice that
received CD4 transplants are protected from a lethal dose of
HIVgp120-expressing leukemia cells.3 One of the many
potential barriers to implementation of a bone marrow transplantation
(BMT) approach for generating functional CD4 T cells is the impact, if any, of retroviral-driven CIR expression early in hematopoiesis on
T-cell development. The affinity of the human CD4 receptor for murine
and human major histocompatibility complex class II (MHCII) is
insufficient for activation of CD4/-expressing T cells.1,2,8-11 However, the consequences of such
low-affinity interactions on lymphoid development have not been examined.
T cells develop from HSC-derived progenitor cells that migrate to the
thymus. Subsequent thymocyte development is regulated by sequential
expression of the pre-TCR12,13 and the mature   TCR,14,15 both of which are multimeric
complexes that rely on the associated invariant CD3 and chains for
transmitting essential survival, differentiation, and proliferation
signals. The most immature thymocytes reside within the
CD4 CD8CD3 triple-negative
(TN) population, which comprises only about 1% to 2% of total
thymocytes. The TN population itself can be divided into the sequential
subsets TN1 (CD44+25), TN2
(CD44+25+), TN3
(CD4425+), and TN4
(CD4425).12,13 The pre-TCR
permits progression from TN3 through to the
CD4+CD8+ double-positive (DP) stage, whereas
the TCR drives subsequent selection to mature CD4+
and CD8+ single-positive cells. The studies described in
this report were designed to determine the impact, if any, of
CD4/ CIR expression on thymocyte development in the setting of BMT.
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Study design |
Vectors
Retroviral vectors expressing CD4del, CD4, and CD4 CIRs
via an internal phosphoglycerate kinase (pgk) promoter have
been described previously.2,3,5,16
Mice
The MHCII-deficient (MHCII) mice possess a
disruption of the MHC class II Ab gene
(B6.129-Abbtm1; Taconic, Germantown, MD)
and therefore do not express surface MHCII in the C57BL/6 background.
Wild-type C57BL/6 mice (Taconic) were used as MHCII+
recipients in experiments involving donor marrow from
MHCII mice.
Flow cytometry
Antibodies (Pharmingen, San Diego, CA) used to stain
peripheral blood and thymocytes are described in the figure legends. Stained cells were analyzed on a FACScan cytometer (Becton
Dickinson, San Jose, CA).
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Results and discussion |
Bone marrow was isolated from C3H mice and retrovirally
transduced with CD4, CD4, or a signaling-defective CIR (CD4del) as previously described.3,5 Surface CIR expression on
transduced bone marrow cells before infusion is shown in
Figure 1A. Although the percentage
of cells expressing CD4 and CD4 was similar, the mean intensity
of expression was considerably higher for the CD4 and CD4del
receptors as seen in previous studies.5 The transduced
bone marrow was subsequently transplanted into sublethally irradiated
C3H mice via tail-vein injection.
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| Figure 1.
Development of CD4/-expressing T cells, but
not CD4/-expressing myeloid, NK, and B cells is blocked following
BMT.
(A) Bone marrow cells isolated from C3H mice were exposed to
retroviral supernatant encoding CD4, CD4, or CD4del (CD4 ) in
the presence of polybrene for 4 hours. Twenty-four hours later, cells
were stained with antihuman CD4-phycoerythrin (CD4-PE; solid line) or
isotype-matched control monoclonal antibody (dotted line) and analyzed
by fluorescence-activated cell sorting (FACS) to determine the
efficiency of transduction. Histograms for control cells stained with
antihuman CD4-PE or isotype control were indistinguishable (data not
shown). The percentage of human CD4-expressing cells over background is
indicated. (B) The transduced bone marrow cells shown in panel A were
infused into sublethally irradiated C3H mice. Six weeks after
transplantation, peripheral blood was isolated and analyzed by 2-color
flow cytometry using PE-conjugated antihuman CD4 and FITC-conjugated
anti-Gr-1, anti-B220, anti-5E6, or anti-CD3. Forward and side-scatter
properties were used to determine gates for myeloid-specific (Gr-1) and
lymphoid-specific (B220, 5E6, and CD3) markers. The percentage of
lineage-positive cells expressing human CD4 is shown in the top left
hand corner of each dot plot. Control cells stained with antihuman
CD4-PE or isotype control yielded indistinguishable results (data not
shown). Results are representative of at least 15 additional mice. (C)
Thymocytes were isolated from mice that received transplants of CD4
or CD4del (CD4) 6 weeks after transplantation,
stained with allophycocyanin (APC)-conjugated
anti-CD3/anti-CD4/anti-CD8, PE-conjugated anti-CD25, fluorescein
isothiocyanate (FITC)-conjugated anti-CD44, and energy-coupled dye
(ECD)-conjugated antihuman CD4, and analyzed by 4-color flow
cytometry. APC (ie, TN cells) were subsequently analyzed
for CD44 and CD25 expression to define TN subsets. The level of human
CD4 expression (solid line) for CD25+CD44+
(TN2), CD25+CD44 (TN3), and
CD25CD44 (TN4) cells is shown.
APC+ TP (ie,
CD3+CD4+CD8+) thymocytes were also
analyzed for human CD4 expression (solid line). The percentage of
peripheral blood-derived B cells (B220) expressing human CD4 in the
same animals is also shown for comparison. Shaded histograms represent
cells from control animals that received transplants of unmodified bone
marrow in each case. The percentage of TN2, TN3, TN4, TP, or B cells
(B220) expressing human CD4 is indicated in each histogram.
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Peripheral blood was isolated from reconstituted animals at
approximately 6 weeks after transplantation and analyzed by 2-color flow cytometry using monoclonal antibodies against human CD4 and the
following mouse cell lineage markers: GR-1 (granulocytes); B220 (B
cells); 5E6 (NK cells), or CD3 (T cells; Figure 1B). CD4, CD4,
and CD4del were expressed on granulocytes and NK cells at similar
frequencies, as previously described for SCID mice following BMT.5 Although similar levels of expression were observed
in B cells for all 3 receptors, T cells expressing CD4 or CD4
were not detected. In contrast, expression of the signaling-defective CIR, CD4del, was retained on this lineage.
The differential expression of CD4del and CD4/ in mature T
lymphocytes prompted us to examine CIR expression during thymocyte development. Thymocytes were harvested from mice that had received either CD4- or CD4del-transduced bone marrow 6 weeks before, and
TN2, TN3, TN4, and TP (triple-positive)
CD3+CD4+CD8+ populations were
examined for human CD4 expression by flow cytometry as described in
Figure 1C. Low levels of TN1 cells precluded analysis of this
particular subset. Whereas CD4del expression was observed at similarly
high levels at every stage of thymocyte development examined, CD4
expression was not detected at any stage. Similar results were observed
for CD4 (data not shown). In contrast to the T-cell lineage, CD4
expression was detected at comparable levels to that of CD4del in B,
myeloid, and NK cells (Figure 1B-C) in peripheral blood samples
isolated from the same CD4-marrow animal that underwent transplantation.
To determine whether engagement of CD4 or CD4 (CD4/) by
MHCII was indeed responsible for the failure of CD4/-bearing thymocyte development, immune reconstitution was examined in mice lacking MHCII expression. Specifically, bone marrow derived from C57BL/6 mice lacking expression of MHCII (MHCII) was
transduced with CD4, CD4, or CD4del, and transplanted into
syngeneic (MHCII) or congenic (MHCII+)
wild-type recipients. As before, peripheral blood was harvested from
reconstituted mice approximately 6 weeks after transplantation, and B-
and T-cell lineages examined for human CD4 expression by flow cytometry
(Figure 2A). Analysis of peripheral blood
isolated from MHCII+ or MHCII recipients
revealed expression of each of the 3 receptors on B cells (Figure 2A),
myeloid cells, and NK cells (data not shown) as expected. Consistent
with the experiment summarized in Figure 1 using C3H mice, T cells
expressing the CD4del receptor, but not CD4 or CD4, were present
in reconstituted MHCII+ recipients. In striking contrast, T
cells from MHCII recipients expressed either CD4 or
CD4 receptors at levels comparable to those of the CD4del receptor.
Flow cytometric analysis of thymocytes isolated from the
MHCII recipient mice in Figure 2B revealed that
CD4-expressing TN2, TN3, TN4, and TP thymocytes could now be
detected at equivalent frequencies to the CD4del subsets (Figure 2C).
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| Figure 2.
Development of CD4/-expressing thymocytes and T
cells is rescued in MHCII mice.
Bone marrow cells isolated from MHCII mice were exposed
to retroviral supernatant encoding CD4, CD4, or CD4del
(CD4), and then infused into sublethally irradiated
wild-type C57BL/6 (A) or MHCII C57BL/6 (B) mice. Six
weeks after transplantation, peripheral blood was isolated and analyzed
by 2-color flow cytometry using PE-conjugated antihuman CD4 and
FITC-conjugated anti-B220 or anti-CD3. The percentage of lineage
positive cells expressing human CD4 is shown in the top left hand
corner of each dot plot. Control cells stained with antihuman CD4-PE or
isotype controls yielded indistinguishable results (data not shown).
Results are representative of at least 8 additional mice. (C)
Thymocytes were isolated from MHCII mice in panel B,
which had received CD4- or CD4del (CD4)-transduced
bone marrow, and were analyzed by 4-color flow cytometry as described
in the legend to Figure 1. The percentage of human
CD4-expressing cells (solid line) in each TN and TP subset is indicated
in each histogram. Shaded histograms represent cells from control
animals that received transplants of unmodified bone marrow in each
case. ND indicates insufficient cells to analyze.
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In summary, we have shown that development of CD4/-bearing T
cells from transplanted HSCs is arrested prior to the TN2
(CD44+CD25+) stage in normal mice. The
developmental block is specific for the T lineage because myeloid, NK,
and B cells are unaffected. In addition, we have shown that T-lineage
arrest is dependent on the signaling domain of either or (because CD4del-expressing T cells develop normally), and on expression
of the CD4 ligand, MHCII. The process by which HSCs commit to the
T-lymphoid lineage is poorly understood, particularly during adult
steady-state hematopoiesis. Several studies suggest that TN1 cells in
the thymus arise from, or are equivalent to, common lymphocyte
precursors (CLPs) found in bone marrow.17,18 TN1 cells
(and CLPs) have lost myeloid potential, but can still develop into
lymphocytes (T, B, and NK cells).17,18 By the TN2 stage,
however, the potential to develop into B and NK cells has also been
lost. Our current hypothesis is that /-mediated signal
transduction occurs in response to CD4/-MHCII engagement during
the CLP/TN1 TN2 transition. The TN3TN4 transition is
mediated by pre-TCR signaling via the associated immunoreceptor
tyrosine-based activation motif (ITAM)-bearing CD3/
chains.19-23 Presumably, premature or aberrant
ITAM-mediated signal transduction by CD4/ during the
CLP/TN1TN2 transition arrests T-lineage commitment or
differentiation of committed T-cell precursors. This hypothesis is
consistent with recent data from our laboratory showing that CD4 is
expressed at high levels on TN thymocytes when under the
transcriptional control of the CD2 enhancer instead of the
constitutively active pgk promoter of the retroviral vector
(manuscript in preparation). Despite the presence of only a single
ITAM, CD4 is as effective as CD4 in mediating this developmental
defect. The FcR- chain is known to be expressed in normal DN
thymocytes and has been implicated in the development of
certain T-cell subsets.24,25 The mechanisms driving
lineage commitment during the CLP/TN1TN2 transition remain obscure. Studies are now underway to determine the specific step in this complex pathway at which CD4/-induced arrest occurs, and
to further dissect the process of T-lineage determination.
| |
Footnotes |
Submitted February 8, 2002; accepted June 3, 2002.
Prepublished online
as Blood First Edition Paper, June 14, 2002; DOI
10.1182/blood-2002-02-0428.
Supported in part by research funding from Cell Genesys to M.R.R. and
W.Y.L. and from the University of Virginia to M.R.R.
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: Margo R. Roberts, Department of Microbiology,
University of Virginia, PO Box 800734, UVA Health System,
Charlottesville, VA 22908; e-mail: mroberts{at}virginia.edu.
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Abstract
Introduction