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*
Department of Cellular Biology, University of Georgia, Athens, GA 30602;
Department of Pathology, Dana-Farber Cancer Institute, Boston, MA 02115; and
Department of Pathology, University of Massachusetts Medical Center, Worcester, MA 01655
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immature T cells that differentiate into CD4+
helper cells express MHC class II-restricted receptors, whereas those
that differentiate into CD8+ cytotoxic T cells express MHC
class I-restricted T cells. It is generally thought that recognition of
the MHC class I molecule by both the TCR and CD8 molecules will provide
appropriate signals giving rise to CD8+ mature T cells.
Likewise, recognition of the class II molecule by TCRs in conjunction
with the CD4 molecule will give rise to CD4 helper T cells. The signals
that arise from the coreceptor alone are not sufficient for lineage
commitment, since cells do not mature in TCR-
mutant mice (3). What
remains unclear is how the signal delivered by the TCR alone or in
conjuction with each of the coreceptors can give rise to two distinct T
cell subsets. It also remains unclear at what stage of development the
thymocytes commit to CD8 or CD4 lineage and what, if any, additional
signals are required for lineage commitment to CD4 and CD8 T cells.
A striking property of the mature T cell repertoire is the high frequency of reactivity to allogeneic MHC molecules (4, 5). Approximately 1 to 10% of mature T cells are alloreactive, and this repertoire overlaps with the Ag-specific T cell repertoire. It has been hypothesized that alloreactive T cells recognize MHC molecules independently of peptides (6). More recently, it was proposed that much of this reactivity is peptide dependent but may not be peptide specific (7). In addition, there are some examples of alloreactive T cells that corecognize specific peptides complexed with allo-MHC (8). It is unclear whether alloreactivity is a consequence of selecting TCRs that recognize self MHC and therefore may be more likely to cross-react with allogenic MHC molecule or if the initial TCR repertoire has an inherent reactivity to the MHC molecule. It is hypothesized that the initial TCR is biased in its reactivity to MHC molecules (9). Some recent studies in which a c-ovalbumin peptide covalently linked to I-Ab was expressed in transgenic mice observed a high frequency of alloreactive T cells (10). More recently, an in vitro system was developed in which maturation of CD4+ cells was induced in fetal thymic organ culture (FTOC)5 with anti-TCR + anti-CD4 Abs (11). Furthermore, T-T hybridomas were generated from the CD4+ thymocytes that mature in FTOC from MHC class I- and II-deficient mice in the presence of anti-TCR + anti-CD4 Abs. Similar frequencies of MHC reactivity were observed in the preselected repertoire and in the mature T cell repertoire in the thymus (11).
We were able to examine the initial T cell repertoire directly based on
a chance observation in a Ly-6A.2 transgenic mouse. We have previously
generated transgenic mice with the Ly-6A.2 gene under the control of
the human CD2 enhancer. The Ly-6A.2 transgene is highly expressed on
all T cells in the thymus. This dysregulated expression of Ly-6A.2
causes a substantial but incomplete block in T cell development, which
occurs at the stage when Ly-6A.2 expression is normally turned off
(12). In the present study, we crossed the Ly-6A.2 transgene with mice
that lack MHC class I and class II molecules. In control
(nontransgenic) MHC-deficient mice, mature CD4 and CD8 T cells failed
to develop as expected, due to an absence of MHC-dependent positive
selection (13, 14). Surprisingly, we found that only CD4 T cells, but
not CD8 T cells, mature in the absence of MHC molecules in the Ly-6A.2
transgenic mice. The CD4+CD8- T cells that
develop in the absence of MHC in Tg+MHC- mice
are similar to the CD4+ cells that mature in normal mice
and represent the initial T cell repertoire that exists before positive
and negative selection. In this report, we describe the characteristics
of the CD4+ cells that mature in the absence of MHC in
Ly-6A.2 transgenic mice, and we also determine the reactivity of these
cells to MHC molecules to provide direct evidence of MHC reactivity of
unselected T cells in an adult mouse. More importantly, these studies
indicate that overexpression of Ly-6A.2 can initiate signaling into
developing T cells that normally use their Ag-specific ß
TCR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ly-6A.2 transgenic and MHC class I x II-deficient mice that were used in this study have been previously described (13). The mice expressing the Ly-6A.2 transgene were backcrossed to MHC class I- x II- mice for six to eight generations.
Flow cytometry
Cells were stained for immunofluorescence as described
previously (15). One x 106 thymocytes,
Tris-NH4Cl-treated spleen cells or lymphocytes purified
from peripheral blood were incubated with various Abs followed by
appropriate fluorochrome-conjugated second-step reagents. Cells (5 or
10 x 103) were analyzed on a FACScan flow cytometer
(Becton Dickinson, Mountain View, CA). Reagents used for this analysis
were: phycoerythrin-conjugated anti-CD4 (YTS 191.1.2, Life
Technologies), FITC-conjugated anti-CD8 (53-6.7, Life
Technologies), FITC-anti-heat-stable Ag (HSA; PharMingen, San
Diego, CA), biotin anti-Vß, biotin-anti-CD44,
biotin-anti-CD40 ligand, biotin-anti-Ly-6A/E,
biotin-anti-TCR-ß (PharMingen), anti-Ly-6A.2 (3E7) (15),
anti-Ly-6A.2 (3A7) (16), streptavidin-Red 613 and streptavidin-Red
670 (Life Technologies, Arlington Heights, IL), and goat anti-rat
IgG-FITC (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Anti-IAd/b/q (M5/114) and anti-class I (M1/42) were
obtained from American Type Culture Collection, Manassas, VA.
T-T hybridomas
T cells from spleen were cultured with anti-CD3 Abs, and
after 2 days of incubation the blasts were separated by Ficoll-Hypaque
separation. The CD4+ blast cells were obtained by
panning on anti-mouse Ig-coated plates after incubating the cells
with anti-CD4 (GK1.5). Purification of cells by panning usually
gave 
80 to 90% pure cell population in our hands. The
CD4+ cells were fused to BW5147 ß-negative (17), and
hypoxanthine-aminopterin-thymidine (HAT)-resistant cells were selected
per standard protocols. Plating efficiency was >80%.
Cell culture
Microcultures were set up as described before (18) in 96-well flat-bottom plates (Corning Glass, Corning, New York) in a final volume of 200 µl of culture medium consisting of RPMI 1640 supplemented with 20 mM HEPES, 2 mM L-glutamine, 1 mM nonessential amino acids (Irvine Scientific, Irvine, CA), 10% heat-inactivated FCS (Sigma Chemicals, St. Louis, MO), 0.25 µg/ml of fungibact (Life Technologies), and 5 x 10-5 M 2-ME; or in some experiments, cultures were set up in 24-well plates (Corning Glass) in a final volume of 1 ml in the presence or absence of PMA and calcium ionophore (Sigma Chemicals). The precise culture conditions are given in the applicable figure legend.
Reactivity to MHC was examined by culturing 5 x 105 or 1 x 105 hybrid cells with 5 x 105 gamma-irradiated splenic stimulator cells in a 200-µl culture medium. After 24 h, 100 µl from these cultures was harvested and evaluated for IL-2 content by incubating it with the IL-2-dependent cell line HT-2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ly-6A.2Tg+ mice were bred with MHC class
I- and class II- double deficient mice (in
which the Aßb gene and ß2-microglobulin
were disrupted by homologous recombination), and their transgenic
progeny were backcrossed with MHC I- x II-
mutant mice for several generations. The progeny obtained from these
breedings that lacked the expression of MHC class I and II molecules,
with or without expression of the Ly-6A.2 transgene, were used for
analysis. As shown in Table I
, the total
number of cells recovered from the thymi of
Tg+MHC- mice was similar to the number of
cells observed in Tg+MHC+ mice and is markedly
reduced compared with Tg-MHC+ mice. The
Tg+MHC- animals also have a reduction in the
development of T cells beyond the CD4-CD8-
stage as compared with Tg+MHC+ mice. These
observations indicate that interactions of TCRs with MHC molecules at
the CD4+CD8+ cell stage does not contribute to
the block in T cell development in Ly-6A.2 transgenic mice.
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A and
Table I
). In contrast, CD4+CD8- thymocytes
were almost completely absent (<0.5%) in the
Tg-MHC- littermates, as reported previously
(13, 14). Approximately 10 to 15% of the thymocytes were
CD4+CD8- in Tg-MHC+
mice, which is similar to the 7 to 10% of these cells found in
Tg+MHC+ mice (Ref. 12 and Fig. 1A). Furthermore, the
CD4+CD8- cells that appear in the
Tg+MHC- thymus are different from the
transitional cells described in MHC class II- mice,
because these latter cells only partially down-regulate their CD8
coreceptor (19, 20). It is noteworthy that expression of the Ly-6A.2
transgene had no effect on the maturation of
CD4-CD8+ T cells in Ly-6A.2 Tg+
MHC- mice (Table I and Fig. 1A). These
data indicate that development and maturation of
CD4+CD8- T cells are selectively supported in
the thymus of Ly-6A.2 transgenic mice in the absence of cell surface
MHC molecules.
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In the normal thymus, mature T cells up-regulate their expression
of TCR after being positively selected. Therefore, to determine whether
the CD4+CD8- thymocytes in Ly-6A.2
transgenic, MHC- mice are phenotypically mature, we
examined their expression of TCR. Thymocytes were stained with
fluorochrome-conjugated mAbs directed against CD4, CD8, and TCR
molecules in three-color immunofluorescence. As shown in Figure 1B, the CD4+ thymocytes that appear in
Tg+MHC- thymi have up-regulated the surface
expression of TCR. Furthermore, this increase in the expression of TCR
is similar to that seen in CD4+ thymocytes from normal
mice. Very few cells showing TCRintermediate/high were
observed in Tg-MHC- mice, which is consistent
with other published reports (Fig. 1A and 13 . Our
results indicate that TCR expression in
CD4+CD8- cells from normal mice was similar to
that in cells that mature in Ly-6A.2 transgenic mice in the absence
of MHC.
Down-regulation of HSA on CD4+CD8-TCRhigh thymocytes in Tg+MHC- mice
Another molecule expressed on the cell surface of thymocytes in
which down-regulation correlates with cells undergoing positive
selection is the HSA. This molecule is highly expressed on
CD4+CD8+TCRlow/intermediate
thymocytes, but its expression is low on the majority of
TCRhigh thymocytes that are also either
CD4+CD8- or CD4-CD8+
(21, 22). Therefore, we examined the expression of HSA in normal
(Tg-MHC+), Tg-MHC-,
and Tg+MHC- mice by staining with
anti-TCR-ß and anti-HSA Abs. Figure 2 shows that the expression of HSA is
down-regulated in TCRhigh cells in the
Tg+MHC- thymus. This reduction in HSA
expression was similar to that observed in TCRhigh cells in
the normal (MHC+) thymus (Fig. 2). Moreover, the high level
of HSA expression on CD4+CD8+ thymocytes from
Tg+MHC-, Tg-MHC-
animals is comparable. These results suggest that the Ly-6A.2
overexpressed on CD4+CD8+ thymocytes delivers a
signal(s) that is MHC+ peptide independent but results in the selection
of
CD4+CD8-TCRhighHSAlow
mature thymocytes.
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We next examined whether the positively selected
CD4+CD8- cells are exported from the
thymus and populate the peripheral lymphoid organs. We analyzed the
spleen and lymph nodes of 4- to 8-wk-old mice for the expression of CD4
and CD8 molecules. Figure 3A
shows that CD4+CD8- T cells account for 50.2%
(range, 35–60% in different experiments) of cells in lymph nodes of
Tg+MHC- mice and for 49.6% (range, 50–70%)
of lymph node cells in normal MHC+ mice. In contrast, this
subpopulation made up only 8.4% (range, 4–8%) of lymph node cells in
mice lacking MHC class I and II molecules. Moreover, the level of
expression of CD4 on lymph node T cells from
Tg+MHC- and control
(Tg-MHC+) mice was comparable but higher than
that observed in Tg-MHC- mice. The level of
expression of the TCR on CD4+CD8- cells from
Tg+MHC- lymph node and spleen was also similar
to that on CD4+CD8- cells in normal mice (data
not shown). These results indicate that the T cells that mature in
Tg+MHC- mice migrate to the periphery and
accumulate in lymph nodes (Fig. 3A) as well as in the
spleen (data not shown). In contrast, very few
CD4-CD8+ cells were detected in the spleen and
lymph nodes of Tg+MHC- and
Tg-MHC- mice, which is consistent with the
observation that there are few of these cells in the thymi of mice
lacking MHC molecules. These observations reinforce our conclusion that
overexpression of Ly-6A.2 results in the maturation of CD4+
and not CD8+ cells in the absence of MHC molecules.
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It has been reported previously that a majority of mature naive,
CD4+CD8- lymph node T cells from normal
mice express low levels of the CD44 molecule (14). Therefore, we
examined the expression of CD44 molecules on CD4+ cells in
lymph nodes obtained from Tg+MHC- mice. Figure 3B shows that in these mice a majority of CD4+
lymph node cells were CD44low. Their expression of CD44
molecules is similar to that on CD4+ T cells from lymph
nodes of normal mice. In contrast, the small number of
CD4+CD8- cells in class I and II
double-deficient mice express high levels of CD44 (Fig. 3), as reported
earlier (23). These results indicate that
CD4+CD8- cells in
Tg+MHC- mice have a mature phenotype.
The CD4+CD8-TCRhigh cells from Ly-6A.2 Tg+MHC- mice up-regulate the CD40 ligand upon stimulation
CD4+CD8- T cells that are selected
by MHC class II molecules help B cell function in normal mice. The
helper activity is mediated in part by the interaction of the CD40
molecule (expressed on B cells) with the CD40 ligand (expressed on
activated CD4+CD8- T cells) (24). Since the
Ly-6A.2 transgene supports positive selection of
CD4+CD8- cells, we next sought to investigate
whether these lymphocytes cells have the ability to express the ligand
for CD40. The CD40 ligand is expressed preferentially on
CD4+CD8- T cells on stimulation with PMA and
calcium ionophore (23). Lymphoid cells from the spleen (Fig. 4) and lymph nodes (data not shown) of
Tg+MHC- mice were exposed to the combination
of PMA and calcium ionophore for 4 to 5 h and analyzed for the
expression of CD4, CD8, and CD40 ligand. Figure 4 shows that a majority
of CD4+CD8- (Fig. 4B) and
not CD4-CD8+ (Fig. 4A)
splenic cells from Ly-6A.2 transgenic MHC- mice expressed
the CD40 ligand. Similar expression was also observed on
CD4+CD8- cells from the spleen (Fig. 4B) and lymph nodes (data not shown) of normal mice.
In contrast, the CD40 ligand was not up-regulated on the small number
of CD4+CD8- cells that are observed in
Tg-MHC- lymph nodes. These results indicate
that the CD4+CD8- cells that selectively
mature in the Ly-6A.2 transgenic and MHC- thymus respond
to stimulation in a manner similar to normal
CD4+CD8- Th cells and that they are
functionally mature.
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Another property of mature T cells selected in the normal thymus
is their responsiveness to stimulation through the TCR/CD3 complex by
proliferation. In contrast, immature
CD4+CD8+ thymocytes do not proliferate
under similar conditions (25). To determine whether the Tg+
CD4+CD8- thymocytes that have matured in the
absence of MHC molecules are immunoresponsive, we examined whether they
responded to stimulation with anti-CD3 Abs. As shown in Figure 5, thymocytes from
Tg+MHC- mice proliferated under these culture
conditions. Similar results were obtained when Con A was used as
mitogen (data not shown). The magnitude of these responses was even
greater than that observed with Tg-MHC+
thymocytes (data not shown). In contrast, proliferation was not
observed in cultures with Tg-MHC- thymocytes
(Fig. 5). These results further indicate that T cells that mature in
Ly-6A.2 transgenic mice in the absence of MHC molecules are
functionally competent.
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A body of data indicates that negative and positive selection in
the thymus occurs at the CD4+CD8+ cell
stage, and therefore the Vß repertoire of most
CD4+CD8+ cells is an unselected one. To examine
whether the Vß repertoire is unselected, we compared the Vß
repertoires of CD4+ that mature in
Ly-6A.2Tg+MHC- mice with the double-positive
(DP) thymocytes from the same mouse. Figure 6 shows that these two repertoires are
similar. Moreover, this Vß repertoire is also similar to one observed
in DP thymocytes from the Tg-MHC- mice. This
later observation supports the idea that the CD4+ cells
that mature in Ly-6Tg+ mice in the absence of MHC is
unaltered. Analysis of the Vß (Vß3, -5, -8, -9, -10, and -11)
repertoire in the CD4+ and CD4+CD8+
subsets in the C57BL/6 parent mice indicated no differences except
positive selection of Vß8 CD4+ T cells (data not shown).
The absence of clonal deletion may be due to the lack of I-E expression
in C57BL/6, which is known to present MTV-8, -9, and -17, resulting in
efficient deletion of specific Vß-expressing T cells (26, 27).
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CD4+ T cells from Tg+MHC- mice show high reactivity to many MHC molecules
To determine whether the preselected T cells are predisposed to
recognize MHC molecules, we first tested the reactivity of these cells
to irradiated spleen cells derived from H-2b,
H-2k mice. Lymph node cells from
Tg+MHC- mice show high levels of
alloreactivity (response to APC from CBA mice) as well as reactivity to
self MHC (I-Ab-expressing APC, the same as in the MHC
mutant mice) (Fig. 7). These responses
are blocked by the M5/114 (anti-I-Ab,
-I-Ad, -I-Ed, -I-Ek) Ab, and no
response is elicited by APC from mice that lack MHC molecules (Fig. 7, right panel) nor is this reactivity blocked by
irrelevant isotype matched Ab (data not shown). Reactivity of the
CD4+ cells that mature independently of MHC was also
observed with APC derived from mice of the H-2d,
H-2s, and H-2q haplotype, albeit to varying
degrees (data not shown). These data demonstrate that lymph node cells
from Ly-6Tg+MHC- mice respond to MHC
molecules. Similar results were also obtained with T cells from the
thymi of these mice (data not shown). These results suggest that the
preselected repertoire is evolutionarily biased to recognize MHC
molecules.
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Figure 7 indicates that CD4+ cells that mature in
the absence of MHC molecules in Ly-6A.2 transgenic mice show reactivity
to a number of MHC molecules. To test the frequency of this reactivity,
we stimulated T cells from Ly-6Tg+MHC-,
Ly-6Tg-MHC+ (C57BL/6) mice with soluble
anti-CD3 Ab and purified CD4+ cells and fused them with
TCR--negative TCR-ß-negative BW5147 (17). Hybridomas that tested
positive for CD4 expression were further tested for reactivity with
different MHC. Each hybridoma was stimulated with APCs from three
different strains. An average of 12 to 15% of the T cells from
Ly-6Tg+MHC- mice reacted to each of the
MHC-expressing APCs tested (Table II). In
contrast, 10 to 12% of the T cells from
Tg-MHC+ mice reacted with two different MHC
tested, whereas about 8% of the hybrids from
Ly-6A.2Tg+MHC- mice reacted with self MHC
(I-Ab) and about 4% of the hybrids from the normal mice
reacted with self MHC. These results indicate that TCRs on
CD4+ cells that mature in the absence of MHC in Ly-6A.2
transgenic mice recognize many MHC molecules tested at high
frequencies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was originally undertaken to understand the
mechanisms involved in the inhibition of T cell development in Ly-6A.2
transgenic mice. To that end, we conclude that TCR interactions with
the MHC molecules do not contribute to this block in development (Table I). These observations are not surprising because we now know that the
blockade of maturation in the Ly-6A.2 transgenic mice occurs at a stage
of development when the TCR is not expressed (12). However, this was
not known when these experiments were initiated. Nevertheless, these
experiments led us to the unexpected observation that ectopic
expression of Ly-6A.2 molecule on CD4+CD8+
thymocytes allowed maturation of CD4+CD8-
cells independently of MHC-peptide complexes. This later observation
provides another reason that Ly-6A.2 may be extinguished on
CD4+CD8+ cells. These results raise the
possibility that another Ly-6 family member, which is endogenously
expressed on CD4+CD8+ cells, might play a role
in positive selection.
The CD4+CD8- cells that appear in the thymus and periphery of Tg+MHC- mice appear to be similar to the CD4+ mature cells by a number of phenotypic criteria. They have down-regulated CD8 and HSA molecules and up-regulated TCR proteins. The CD4+CD8- cells that mature in the thymi of Ly-6A.2+MHC- mice also express the CD40 ligand on cell stimulation and proliferate upon cross-linking of the TCR/CD3 complex. The proliferative responses of Tg+MHC- thymocytes were similar to the response of Tg+MHC+ thymocytes (data not shown) and were, in all cases, not less than those observed for thymocytes from normal (Tg-MHC+) mice. In contrast, the thymocytes from the Tg-MHC- do not proliferate after cross-linking of their TCRs, which is consistent with a previous report (14). Furthermore, the CD4+CD8- T cell emigrate from the thymus to peripheral lymphoid organs.
Previous reports have demonstrated that overexpression of CD8 can promote the selection of CD4+ cells in the absence of class II molecules (23), and Bcl-2 expression allows maturation of CD8+ cells in the absence of a class I molecule (29). More recently, preferential maturation of CD8 cells in Notch 1 transgenic mice was observed (30). The Notch-mediated maturation of CD8+ cells was not observed in the absence of both class I and II molecules. The Ly-6 transgenic mouse is the first example that we are aware of in which CD4+ cells appear in the absence of both MHC class I and II molecules and therefore provides a unique opportunity to study thymic selection.
Why does overexpression of the Ly-6A.2 transgene result in maturation of CD4+ cells?
Maturation of CD4+ cells in the absence of MHC expression is surprising. Our experiments also indicate that CD4+ cells do not mature in Ly-6A.2Tg+ MHC- mice if the expression is low (fivefold; data not shown). It is possible that this maturation is driven by the interaction of overexpressed Ly-6A.2 on thymocytes with its ligand in the thymus. This notion is consistent with our recent observation that indicates the presence of intrathymic Ly-6A.2 ligand (31). This interaction may either result in signals that mimic the TCR signaling or use the TCR/CD3 complex to signal into the cell. The rationale for this hypothesis is as follows. First, cross-linking Ly-6A.2 proteins on T cells with Abs causes cell activation and secretion of cytokines (16). Second, Ly-6A.2 binds to several key protein tyrosine kinases, e.g., p56lck and p59fyn, that are critical for signaling through TCR (32). Third, mutations or antisense oligonucleotides that decrease Ly-6A.2 expression also diminish T cell responsiveness (33, 34, 35). Reciprocally, a loss of TCR/CD3 expression also results in a lack of immune responsiveness through Ly-6A.2 (36, 37). More recently, Ly-6A null mice have been generated and have altered the proliferative responses of mature T cells (38). In these mice, lack of Ly-6A.2 expression results in higher proliferation of splenic T cells to anti-CD3 Abs, mitogens, and alloantigens. Taken together, these results indicate that Ly-6A.2 is able to regulate signaling negatively or positively through the TCR/CD3 complex. Why Ly-6A.2 expression has opposite effects on signaling through the TCR/CD3 is unclear.
It is interesting to note that signaling through Ly-6A.2 results in the maturation of only CD4+ cells and not CD8+ cells. One possibility is that Ly-6A.2 molecules provide a signal that favors the maturation of CD4+ cells as opposed to CD8 cells. Alternatively, this signaling may simply allow progression down the default pathway of maturation for the maturation of CD4+ cells (39). This interpretation is consistent with the recent observation that maturation of CD8 cells needs additional lineage-specific signals, which may be delivered by the activated Notch-1 molecule upon its interaction with its ligand (30). We propose that the signals to activate the Notch or its ligand or other CD8 lineage-specific signals are not delivered in Ly-6A.2Tg+MHC- mice. We hope that additional experiments with Ly-6A.2Tg+MHC- mice will provide insights into the mechanism of lineage commitment.
The mature CD4 T cell repertoire truly represents the initial T cell repertoire
Since overexpression of Ly-6A.2 results in the maturation of CD4+ cells in the absence of MHC class I and II molecules, it is likely that these cells represent the initial unselected T cell repertoire. This contention is supported by the observation that the Vß repertoire of the CD4+ cells and CD4+CD8+ cells from the Ly-6A.2Tg+MHC- mice are similar. Furthermore, this similarity extends to the repertoire of the CD4+CD8+ DP cells from Tg-MHC- mice.
It is very unlikely that CD4+ cells selected in the thymus of Tg+MHC- mice are selected by H2-O, an atypical MHC class II molecule, for the following reasons: 1) H-2O is primarily expressed intracellularly (40); 2) Abs to I-A and I-E block the reactivity of these cells to I-A- and I-E-expressing APCs, and these Abs are not known to cross-react with H2-O (L. Karlsson, unpublished observations). Therefore, these results strongly suggest that CD4+ cells are not selected by the H-2O protein. It is also unlikely that CD4+ cells are selected by the class I heavy chain on the surface of cells in b2-microglobulin knock-out mice, since the reactivity of these cells to MHC is inhibited by anti-class II Abs. Taken together, these observations strongly argue that CD4+ cells that mature in the absence of MHC class I and II molecules represent the T cell repertoire that exists before thymic selection.
MHC reactivity of the initial T cell repertoire
The phenomenon of alloreactivity is not well understood. Our
results suggest that a higher frequency of alloreactive T cells exists
before thymic selection than previously predicted. Approximately 12 to
15% of the hybrids from the
Ly-6A.2Tg+MHC- mice reacted with
different MHC molecules. This is similar to the 10 to 12% of the
hybrids from the normal mice that showed this reactivity. These results
would suggest that alloreactivity is inherent in the preselected
repertoire, and it is not a consequence of selection of the receptor
that reacts with self MHC and peptide. These results are consistent
with the recent observations that have analyzed the reactivity of
CD4+ cells that mature in the FTOC of MHC-
mice in the presence of a combination of anti-TCR and anti-CD4
Abs (11) or by bispecific CD3/CD4 Abs (41). The percentage of
reactivity to H-2b was lower than with H-2d and
H-2k and probably reflects reactivity to only I-A molecules
(since I-E is not expressed). Approximately 4% of the hybrids derived
from C57BL/6 mice reacted with APCs from the same mouse strain (Table II). This reactivity may reflect T cells that respond to syngeneic
APCs, described as a phenomenon of syngeneic MLR (SMLR) (42, 43, 44).
It has become increasingly clear that negative selection plays a crucial role in shaping the T cell repertoire. The degree to which the clonal deletion may impact the T cell repertoire varies from 5 to 50% (13, 44). CD4+ cells that mature in the Ly-6A.2 transgenic mice react with syngeneic stimulators at a higher frequency, indicating that the T cell repertoire in these mice will include T cells that are normally deleted in the the thymus. Analysis of peripheral repertoire in Ly-6A.2Tg+MHC- mice will provide clues about the nature of self-reactive T cells that normally do not exit the thymus.
In summary, our results indicate that CD4+ cells that appear in the thymi of Ly-6A.2 transgenic mice in the absence of MHC molecules are phenotypically like mature T cells. These cells represent the preselected T cell repertoire that has high reactivity to MHC molecules. These later results indicate that the initial, preselected T cell repertoire has an evolutionary bias in its reactivity to MHC protein.
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Acknowledgments |
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Footnotes |
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2 Present address: Division of Hematology and Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.
3 Present address: Department of Immunology, Saint Mary’s Hospital Medical School, Norfolk Place, London W2 1P6, U.K.
4 Address correspondence and reprint requests to Dr. Anil Bamezai, 615 Biologic Sciences Bldg., University of Georgia, Athens, GA 30602. E-mail address:
5 Abbreviations used in this paper: FTOC, fetal thymic organ culture; Tg, transgenic; HSA, heat-stable Ag; DP, double positive.
Received for publication December 10, 1997. Accepted for publication March 3, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. C. Henderson, M. M. Kamdar, and A. Bamezai Ly-6A.2 Expression Regulates Antigen-Specific CD4+ T Cell Proliferation and Cytokine Production J. Immunol., January 1, 2002; 168(1): 118 - 126. [Abstract] [Full Text] [PDF]
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D. L. Pflugh, S. E. Maher, and A. L. M. Bothwell Ly-6I, a New Member of the Murine Ly-6 Superfamily with a Distinct Pattern of Expression J. Immunol., July 1, 2000; 165(1): 313 - 321. [Abstract] [Full Text] [PDF]
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