Cross-Linking the TCR Complex Induces Apoptosis in CD4
Cross-Linking the TCR Complex Induces Apoptosis in CD4/8/ Thymocytes in the Presence of Cyclosporin A
HEATHER L. POETSCHKE 0
DAVID B. KLUG 0
DAWN WALKER 0
ELLEN R. RICHIE 0
0 The University
Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD4/8 double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.
Apoptosis; cyclosporin A; negative selection; thymocytes
INTRODUCTION
Intrathymic T-cell development is the consequence
of an orderly series of molecular events initiated by
contact between bone marrow derived precursors
and thymic stromal cells. Discrete stages of the
T-cell developmental process are defined
phenotypically by expression of CD4 and CD8 coreceptors
and the TCR/CD3 complex. Productive
rearrangement of the TCR chain locus is a prerequisite for
transition of immature CD4-8- double-negative (DN)
cells to the CD4/8 double-positive (DP) stage via a
CD4-81 intermediate
(Nikolic-Zugic and Bevan,
1988; Mombaerts et al., 1992; Dudley et al., 1994)
.
Subsequent TCR0 gene rearrangement within the
DP subset results in low-level expression of 0TCR/
CD3 complexes
(Kearse et al., 1994)
. The process of
thymic selection operates on these TCR1 DP cells to
shape the final MHC-restricted TCR repertoire
*Corresponding author.
(Robey and Fowlkes, 1994)
. Positive selection refers
to the maturation of thymocytes expression TCRs
that mediate low-affinity interactions with
selfpeptides presented by self-MHC molecules
(AshtonRickardt et al., 1994; Hogquist et al., 1994; von
Boehmer, 1994)
. The minor fraction of DP
thymocytes that undergoes positive selection
differentiates to either CD4-8 (MHC class I restricted)
or CD4/8 (MHC class II restricted) mature
singlepositive (SP) cells that express high levels of TCR/
CD3 complexes. In contrast, DP thymocytes
expressing TCRs with high affinity for
self-MHC/selfpeptide complexes undergo clonal deletion, a
process referred to as negative selection
(Fowlkes et al.,
1988; Kisielow et al., 1988; Ashton-Rickardt et al.,
1994)
. Because most DP thymocytes fail to express
TCRs that interact with self-MHC molecules, they
are neither positively nor negatively selected. These
"neglected" DP thymocytes have a short life span
(~3.5 days) in vivo and undergo apoptosis when
cultured in vitro
(Robey and Fowlkes, 1994;
Kishimoto et al., 1995)
. Recently, direct evidence for
thymocyte apoptosis in situ was obtained using a
highly sensitive technique to detect DNA strand
breaks, i.e., terminal deoxynucelotidyl transferase
(TdT)-mediated labeling of nicked DNA with dUTP
biotin (TUNEL)
(Surh and Sprent, 1994)
.
Although negatively selected and neglected
thymocytes undergo internucleosomal DNA
fragmentation and morphological changes consistent
with apoptosis, the signal transduction pathways
and the role of APCs in promoting apoptosis are not
yet resolved. Several groups have demonstrated that
DP thymocytes expressing a transgenic TCR are
depleted when cultured in the presence of antigen
and APCs expressing appropriate MHC molecules
(Swat et al., 1991; Iwabuchi et al., 1992; Vasquez et al.,
1992)
. Cellular depletion also has been induced by
antibody-mediated ligation of the TCR/CD3
complex. Anti-CD3 administered in vivo or in fetal thymic
organ cultures results in thymocyte death associated
with DNA degradation
(Smith et al., 1989; Shi et al.,
1991)
. However, there are conflicting reports
concerning the requirement for APCs in
anti-CD3induced apoptosis in vitro. Although some studies
conclude that antibody-mediated TCR cross-linking
alone is sufficient to stimulate DNA fragmentation
and/or cell death
(McConkey et al., 1989a; Carlow
et al., 1992; Migita et al., 1994)
, other groups (...truncated)