By
From the * Division of Molecular Immunology, The National Institute for Medical Research, Mill Hill,
London NW7 1AA, United Kingdom; and Gesellschaft für Biotechnologische Forschung, D-38124
Braunschweig, Germany
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Abstract |
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CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4+ cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8+ cells in thymus organ culture from mice impaired in CD4+ cell development (invariant chain-deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.
Key words: antagonist; major histocompatibility complex; thymocyte differentiation; thymic selection; T cell receptor ![]() |
Introduction |
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he T cell repertoire is shaped in the thymus by positive and negative selection, such that the mature T cell population recognizes foreign antigens and tolerates self-peptides. The involvement of self-peptide in both selection processes has been explained by the avidity model, according to which high avidity interactions between thymocytes and antigen presenting cells lead to deletion of T cells, whereas low avidity interactions induce positive selection (for reviews see references 1, 2).
The selection of CD8+ cells expressing MHC class I-restricted TCRs appears to follow this model. Positive selection of T cells expressing a class I-restricted transgenic TCR was induced in fetal thymic organ culture with low concentrations of antigenic peptide, whereas high concentrations of the same peptide induced negative selection (3, 4). More efficiently, analogues of the antigenic peptide could be used for the induction of positive selection (4, 5). Such analogues are frequently antagonists of mature T cells and were shown to provide a decreased affinity interaction with the TCR (6). The fact that analogues of the antigenic peptide, but rarely unrelated peptides (7), caused positive selection of CD8+ cells indicated a high degree of peptide specificity for selection whenever single peptides were used.
Surprisingly, nobody has reported positive selection of
transgenic class II-restricted cells using similar assays. On
the other hand, mice expressing class II molecules loaded
with a single peptide were used to identify T cells selected
by this single ligand. In H2-M-deficient mice that almost
exclusively display class II-associated invariant chain peptides on their class II molecules (10), as well as in mice
expressing an E peptide in the context with H2-Ab in the
absence of endogenous class II molecules (13), a diverse repertoire was selected, although with less efficiency than
in wild-type mice. This indicated a less stringent peptide
specificity for positive selection of class II-restricted TCRs.
Also, introduction of single peptides into mice by means of
intrathymic injection of an adenovirus vector expressing
such peptides provided evidence that in addition to antigenic peptide and its analogues, some unrelated peptides
could induce positive selection (14).
A disadvantage of these polyclonal, nontransgenic systems is that they do not give information about particular
TCR VV
sequences selected. The fact that none of several different transgenic TCRs was positively selected in
H2-M-deficient and E
/H2-Ab expressing mice (15)
shows that unrelated single peptides are less adequate for
positive selection than one might conclude from a polyclonal repertoire. The only report in which a transgenic class II-restricted TCR system with known TCR V
V
sequences was used to investigate positive selection mediated by antigen-related peptides was by Spain et al. (18).
They showed that the addition of antagonist peptide to
fetal thymic organ culture of class II-restricted thymocytes
inhibits the development of CD4+ cells. However, this
study was performed on a normally selecting background
in which endogenous peptides are presented. Therefore, the inhibition of CD4+ cell development might have been
due to interference of the antagonist peptide with the physiological positively selecting peptide.
To circumvent this potential problem, we used invariant
chain deficient mice (Ii/
) that express reduced levels of
surface MHC class II molecules with greatly increased ability to present exogenously added class II binding peptides
(19, 20). These mice were crossed to the MHC class II-
restricted TCR transgenic mouse line A18 to enable analyses of selection requirements for a particular TCR. This TCR is specific for a peptide derived from the fifth component of complement (21). Neonatal thymic lobes
(NTOC)1 from these mice did not generate mature CD4+
cells, due to an altered repertoire of self-peptides bound to class II that is lacking the endogenous peptide responsible
for positive selection. When antagonist peptide was loaded
exogenously onto class II molecules, CD4+ cell development could not be rescued. Instead, this treatment resulted
in the generation of CD8+ cells. These data address important questions about the interaction of the TCR and its
ligand, leading to CD4 versus CD8 lineage commitment.
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Materials and Methods |
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Mice.
The C5 TCR transgenic mouse line A18 on the Rag1Peptides.
The agonist peptide for the A18 T cell receptor is peptide 106-121 from mouse C5. Two antagonists were generated by replacement of lysine residue 113 with either isoleucine for antagonist 113I (KAntagonist Assay.
Dendritic cells derived from cultures of bone marrow cells with GM-CSF as described (22) were pulsed with 10 nM A18 agonist peptide for 2 h. They were then washed and cultured with different doses of antagonist or control peptide in the presence of spleen T cells from A18 Rag1Thymus Organ Culture.
NTOC were cut in half and placed on nucleopore membranes (0.8 µm pore size; Costar Corp., Cambridge, MA) floating on 1 ml Iscove's modified Dulbecco's medium (Life Technologies, Inc., Paisley, Scotland) supplemented with 10% heat-inactivated FCS, 5 × 10FACS® Analyses and Antibodies.
Expression of cell surface antigens by thymocytes from NTOC was determined by cytofluorometry using a FACScan® and Cell Quest software (Becton Dickinson & Co., Mountain View, CA). Dead cells were excluded by forward and side scatter characteristics. mAbs used were PE-labeled anti-CD4 (H129.19; Boehringer Mannheim, Mannheim, Germany), FITC-labeled anti-CD8Functional Analyses.
Thymocytes from NTOC were plated out in 96-well microtiter plates (5 × 104/well) and treated with 10 µg/ml anti-CD4 (RL172.4), 10 µg/ml anti-CD8 ![]() |
Results |
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Exchange of lysine residue 113 in the C5 peptide
recognized by the A18 TCR results in loss of stimulatory
activity for C5-specific A18 T cells. Two such altered peptides with a change of K I (peptide 113I) or K
V
(peptide 113V) displayed antagonist activity as judged by
their ability to inhibit activation of A18 T cells to the agonist A18 peptide. Both peptides are nonstimulatory for A18
T cells in a range of concentrations from 1 nM to 10 µM
(data not shown). Fig. 1 shows that both 113I and 113V
blocked the IL-2 response of A18 T cells to dendritic cells
prepulsed with agonist peptide, whereas an unrelated H2-Ek binding peptide from hen egg lysozyme (HEL 1-18, 2G7; reference 26) did not influence the response.
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To decrease presentation of endogenous peptides and enable preferential loading of exogenously added peptides onto MHC class II molecules, we
crossed class II-restricted TCR-transgenic A18 mice on the
RAG-1/
background to invariant chain-deficient (Ii
/
)
mice. MHC class II expression in Ii
/
mice has been reported to be reduced but not abolished and the absence of
invariant chain interferes with CD4 T cell development of
many, but not all, specificities (20, 27). Neonatal thymus organ culture of A18 Ii
/
mice showed no differentiation
of mature CD4+ cells (Fig. 2 A, medium), placing A18 in
the former category. The few CD4+ cells that were found
in NTOC displayed an immature phenotype (TCRlo-int
and HSAhi). Thymus cortical epithelial cells, which are responsible for the induction of positive selection of T cells
(28), show a twofold reduction in MHC class II levels (data
not shown). Since we know that the A18 TCR is positively selected in F1 mice with the nonselecting H2-q
background (Barthlott,T., R.J. Wright, and B. Stockinger,
manuscript submitted for publication), we can exclude that
this degree of reduction in MHC class II expression is the
reason for the lack of mature CD4+HSAlo cells in A18 Ii
/
lobes. An alternative and more likely explanation for the
defect in positive selection of A18 cells is an impairment in
the processing of the normally positively selecting peptide
or its proper assembly with class II, due to the absence of Ii.
The greatly increased peptide binding capacity of Ii
/
antigen-presenting cells allowed us to test the effect of exogenously added peptides on positive selection of A18 T cells.
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Antagonist peptides have been shown to be successful
in generating class I-restricted CD8 T cells. We asked
whether modified A18 peptides with antagonist activity
for the nominal C5 peptide would rescue the development of CD4 T cells in NTOC of invariant chain negative A18 thymocytes. However, thymus organ culture in the presence of antagonist peptide did not result in the
generation of mature CD4+ cells at any concentration
tested. Fig. 2 shows antagonist peptide 113V as an example, antagonist 113I behaved in the same way (data not
shown). Instead of expected CD4+ cells, CD8+ cells with
the characteristics of mature T cells (TCRhi HSAlo) were
generated (Fig. 2 A, 1 µM antagonist and 10 µM antagonist). These cells express CD8 heterodimers on the surface (Fig. 3). The increased percentage of CD8+ cells was
reproducibly reflected in a 5-10-fold increase in absolute cell numbers as shown in Fig. 4. These CD8+ cells could
not have been derived by endogenous receptor rearrangements leading to expression of a class I-restricted TCR
because of the absence of RAG-1 protein in those mice.
Therefore, it clearly demonstrated that the antagonist
peptide caused T cells with the class II-restricted A18
TCR to differentiate into the CD8 lineage. The antagonist peptide is presented on MHC class II since anti-class II antibodies block the generation of CD8 cells in NTOC
(Fig. 3). The antigenic C5 peptide was also tested for its
influence on positive selection in NTOC. However, it
mostly led to deletion of A18 thymocytes. Very inefficient selection of CD8 and CD4 cells occurred in some
but not all experiments with C5 peptide at picomolar concentrations (data not shown).
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Thymus
lobes from newborn A18 Ii+/ mice were cultured under
the same conditions as described above for A18 Ii
/
lobes,
to test whether the antagonist would influence normal positive selection. As expected, mature CD4+ TCRhi HSAlo
cells, but no mature (HSAlo) CD8+ cells were generated
from lobes cultured in medium alone (Fig. 2 B, medium). In
contrast, CD4+ cell maturation did not take place in the
presence of antagonist peptide and, as seen with Ii
/
lobes,
CD8+ cell maturation was induced (Fig. 2 B, 1 µM antagonist and 10 µM antagonist). Prevention of CD4 lineage commitment by antagonist peptide confirms data from Spain et
al. (18) showing a block in the differentiation of CD4+ 2B4
TCR transgenic thymocytes in thymus organ culture in the presence of a cytochrome c antagonist variant. Spain et al.
(18) did not report an enhanced differentiation into the CD8
lineage, presumably because their mice were not deficient
for RAG and therefore contained significant numbers of
CD8+ cells in the absence of antagonist peptide.
The CD8+ cells generated with antagonist peptide in NTOC from A18 Ii/
mice displayed a
mature phenotype according to their high expression of the
TCR and downregulation of expression as shown in Fig. 2. To investigate the functional ability of these cells, we cultured them with dendritic cells presenting the C5 agonist
peptide and assayed for IL-2 production. As expected,
given that they contained no mature single positive cells,
thymocytes recovered from NTOC from A18 Ii
/
mice
did not react to C5 peptide (Fig. 5, medium). In contrast, lobes cultured in the presence of antagonist peptide gave
rise to thymocytes responding to C5 peptide (Fig. 5, 1 µM
antagonist and 10 µM antagonist). This response was mediated by the CD8+ population, as depletion of CD8+ cells
before the functional assay ablated C5 reactivity. In contrast, depletion of CD4+ cells did not affect C5 reactivity.
These results confirm the maturity of the CD8+ cells and
also their TCR specificity for C5 peptide. C5 peptide/class II complex recognition by the A18 TCR in the absence of
CD4 was observed previously in CD4-negative T cell hybrids (data not shown).
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Discussion |
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We have demonstrated that antagonist peptide can alter
lineage decision from CD4+ to CD8+ cells. Previously,
positive selection of CD8+ cells was achieved with peptide
analogues of the antigenic peptide or low concentrations of
the nominal antigen presented by the selecting MHC class I
molecule (3). In contrast, positive selection of CD4+ cells
could be induced through coengagement of the TCR/CD3
complex with a variety of thymocyte surface molecules such
as CD2, CD4, CD5, CD8, CD24, CD28, CD49d, CD81,
or TSA-1 (29). In addition, cross-linking of TCR or
CD3
with mAb (34), as well as targeting thymocytes to
thymic cortical epithelium via anti-TCR/CDR-1 hybrid antibodies (31), resulted in the exclusive generation of CD4+
cells even in the absence of MHC molecules. Thus, the signals for CD4 differentiation seemed to be promiscuous in
comparison to signals for CD8 differentiation and it was suggested that development into the CD4 lineage follows a
"default" pathway (32). However, we show here that CD8+
rather than CD4+ cells developed, even without the involvement of either CD8 or class I-specific signals. Instead of a
default model for either lineage, the involvement of distinct
signals seems to be more likely.
In terms of the effect of antagonist peptides, it has been
shown that binding of the TCR to MHC molecules occupied by antagonist peptide results in a higher off rate (35,
36). A shorter interaction time between the TCR and its
ligand might not allow sufficient time for coreceptor binding and consequently for the recruitment of the tyrosine
kinase p56lck (37). Lack of lck recruitment is presumably
more debilitating for CD4 lineage cells since a much larger
fraction of CD4 than CD8 molecules is associated with lck
(38, 39), implying a more prominent role for lck in CD4+
cell development. Therefore, the generation of class II-restricted CD8+ cells in NTOC might be the consequence
of insufficient lck recruitment in the presence of antagonist
peptide (40). In support of this, class II-restricted T cells
choose the CD8 pathway in mice lacking the CD4 molecule (41). Recruitment of lck to the TCR complex was
first implied as a major player in CD4 lineage decision by Itano et al. (42). By introducing a hybrid protein consisting of the extracellular and transmembrane domain of CD8
and the cytoplasmic part of CD4, they could generate a
large number of MHC class I-restricted CD4+ T cells in
transgenic mice. The only known difference to CD8 transgenic mice was more efficient lck recruitment by the CD4 cytoplasmic domain. Basson et al. (43) directed differentiation into the CD8 lineage through TCR engagement independently of MHC specificity by using CD3-specific
F(ab')2 antibodies. The CD3-F(ab')2 reagent was unable to
activate mature T cells and instead resembled an antagonist
peptide in terms of downstream signaling and inhibitory effect on agonist peptide responses. Based on this, they hypothesized that CD8 cell development is favored by antagonist-like signals, which have limited participation of lck
signals. On the other hand, CD4 differentiation would require a stronger lck signal in relation to the TCR signal.
Our results are consistent with these models and demonstrate for the first time the ability of a single peptide to convert CD4/CD8 lineage decision.
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Footnotes |
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Address correspondence to Brigitta Stockinger, Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Phone: 44-181-959-3666; Fax: 44-181-913-8531; E-mail: b-stocki{at}nimr.mrc.ac.uk
Received for publication 21 April 1998 and in revised form 22 June 1998.
Ariane Volkmann's present address is Division of Immunology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720.We thank Rose Zamoyska, Dimitris Kioussis, and David Raulet for critical comments on the manuscript.
Abbreviations used in this paper HSA, heat-stable antigen; NTOC, neonatal thymic lobes.
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